Chapter 5 High-Pressure Stopped-Flow Kinetic Studies of Nnos

Probing the dynamics and conformational landscape

of neuronal nitric oxide synthase

A thesis submitted to the University of Manchester for the degree of

Doctor of Philosophy in the Faculty of Life Sciences

2013

Anna Sobolewska-Stawiarz

Contents

Contents ...... 2 List of Figures ...... 6 List of Tables ...... 10 Abstract ...... 12 Declaration ...... 13 Copyright Statement ...... 13 Acknowledgements ...... 14 List of Abbreviations ...... 15 List of Amino Acids Abbreviations ...... 17

CHAPTER 1. INTRODUCTION ...... 18 1.1 Nitric oxide synthase ...... 19 1.1.1 Discovery and isoforms ...... 19 1.1.2 The reaction ...... 23 1.1.3 Structure, function and dynamics ...... 27 1.1.3.1 Flavoproteins and flavin cofactors...... 27 1.1.3.2 Reductase domain ...... 30 1.1.3.3 Oxygenase domain...... 36 1.1.3.4 Function of tetrahydrobiopterin ...... 38 1.1.3.5 Role of calmodulin in control of electron transfer...... 43 1.2 Enzymes related to NOS ...... 47 1.2.1 Cytochrome P450 reductase ...... 47 1.2.2 Cytochrome P450 ...... 49 1.2.3 Flavocytochrome P450 BM3 ...... 52

1.2.4 Flavocytochrome b2 ...... 52 1.2.5 Cellobiose dehydrogenase ...... 53 1.2.6 Flavohemoglobin ...... 54 1.2.7 Methionine synthase reductase ...... 54 1.2.8 Sulphite reductase ...... 57 1.2.9 Novel reductase 1 ...... 57

1.3 Nitric oxide and the importance of NOS ...... 58 1.3.1 Nitric oxide – cardiovascular physiology concepts ...... 59 1.3.2 Nitric oxide and the nervous system ...... 60 1.3.3 Role of NO in the immune system ...... 61 1.3.4 Side effects of uncontrolled nitric oxide production ...... 62 1.4 Overview of EPR spectroscopy ...... 65 1.4.1 Physical origin of the EPR signal ...... 66 1.4.2 PELDOR spectroscopy ...... 73 1.5 Aims of the project ...... 82

CHAPTER 2. MATERIALS AND METHODS ...... 85 2.1 Materials ...... 86 2.2 Methods ...... 88 2.2.1 Recombinant DNA technique ...... 88 2.2.1.1 Site-directed mutagenesis of FL R1400E nNOS ...... 88 2.2.1.2 DNA transformation of cells ...... 90 2.2.1.2.1 NovaBlue E. coli competent cells ...... 90 2.2.1.2.2 BL21(DE3) E. coli competent cells ...... 90 2.2.1.3 Preparation of DNA ...... 90 2.2.1.4 Enzymic DNA modifications ...... 91 2.2.1.4.1 Restriction digestion of pCRNNR ...... 91 2.2.1.4.2 Restriction digestion of pCWORI ...... 91 2.2.1.5 Agarose gel electrophoresis ...... 92 2.2.2 Isolation of nNOS proteins ...... 92

2.2.2.1 nNOSred expression and purification ...... 92 2.2.2.2 FL nNOS expression and purification ...... 94 2.2.3 Methods of protein analysis ...... 97 2.2.3.1 Determination of protein concentration ...... 97

2.2.3.1.1 nNOSred...... 97 2.2.3.1.2 FL nNOS ...... 97 2.2.3.2 SDS-PAGE ...... 97 2.2.3.3 HPLC identification and quantification of FAD and FMN ...... 98 2.2.3.4 Steady state kinetics ...... 99

2.2.3.4.1 Cytochrome c reduction ...... 100 2.2.3.4.2 NADPH oxidation ...... 100 2.2.3.4.3 FL nNOS activity - calmodulin dependence ...... 101 2.2.3.5 Hydrostatic pressure stopped-flow kinetics ...... 101 2.2.3.5.1 Flavin reduction ...... 102 2.2.3.5.2 NADPH oxidation ...... 103 2.2.3.5.3 NO synthesis ...... 103 2.2.3.6 Redox potentiometry ...... 105 2.2.3.7 Electron paramagnetic resonance spectroscopy (EPR) ...... 106 2.2.3.8 Protein mass spectroscopy ...... 107

CHAPTER 3. CHARACTERISATION OF THE nNOS REDUCTASE DOMAIN ...... 108 3.1 Introduction ...... 109 3.2 Results ...... 111

3.2.1 Overexpression and purification of WT and R1400E nNOSred ...... 111 3.2.2 Steady-state kinetics of cytochrome c reductase activity of WT and R1400E

nNOSred ...... 118 3.2.3 EPR redox titration ...... 122 3.2.4 EPR Spectroscopic studies ...... 127

3.2.4.1 CW-EPR studies of nNOSred ...... 127

3.2.4.2 PELDOR studies of nNOSred ...... 133 3.3 Discussion ...... 140

CHAPTER 4. CHARACTERISATION OF FULL LENGTH nNOS ...... 147 4.1 Introduction ...... 148 4.2 Results ...... 149 4.2.1 Overexpression and purification of FL WT and R1400E nNOS ...... 149 4.2.2 Protein mass spectroscopy ...... 156 4.2.3 HPLC identification and quantification of FAD and FMN ...... 158 4.2.4 Steady-state kinetics of cytochrome c reductase activity of FL WT and R1400E nNOS ...... 163 4.2.5 NADPH oxidation ...... 167 4.2.6 FL nNOS activity - calmodulin dependence ...... 169

4.2.7 EPR redox titration ...... 171 4.2.8 PELDOR spectroscopy ...... 176 4.3 Discussion ...... 181

CHAPTER 5. HIGH-PRESSURE STOPPED-FLOW KINETIC STUDIES OF nNOS ...... 189 5.1 Introduction ...... 190 5.2 Results ...... 193 5.2.1 NADPH oxidation ...... 193 5.2.2 Flavin reduction ...... 197

5.2.2.1 The single turnover flavin reduction in WT and R1400E nNOSred ...... 198 5.2.2.2 The single turnover flavin reduction in FL WT and R1400E nNOS .... 207 5.2.3 NO synthesis ...... 217 5.3 Discussion ...... 226

CHAPTER 6. FINAL CONCLUSIONS AND FUTURE PERSPECTIVES .... 233

REFERENCES ...... 244

Word Count: The total word count (including figure legends and references) is 59,402.

List of Figures

Figure 1.1 Block gene alignment of nitric oxide synthase isoforms and CPR .... 21 Figure 1.2 Reaction catalysed by NOS ...... 24 Figure 1.3 Domain arrangement and electron flow in the NOS dimer ...... 25 Figure 1.4 Mechanism of NO synthesis by NOS ...... 26 Figure 1.5 Representation of the structures of FMN and FAD in different oxidation states, with the absorption maxima indicated ...... 29 Figure 1.6 Representation of the position of an aromatic residue F1395 within rat

nNOSred...... 32

Figure 1.7 Structure of rat nNOSred ...... 35 Figure 1.8 Chemical structures of heme ...... 36

Figure 1.9 Structure of rat nNOSoxy ...... 37 Figure 1.10 Role of tetrahydrobiopterin in eNOS activity regulation in vascular disease...... 40 Figure 1.11 Structural arrangements of the heme and tetrahydrobiopterin in nNOS

nNOSoxy dimer ...... 41 Figure 1.12 Chemical structures of 5,6,7,8-tetrahydrobiopterin and 7,8- dihydrobipterin ...... 43 Figure 1.13 Ca2+-calmodulin complex formation ...... 45 Figure 1.14 Structure of rat CPR ...... 48 Figure 1.15 Catalytic cycle for cytochrome P450 ...... 51 Figure 1.16 Role of methionine synthase reductase (MSR) ...... 56 Figure 1.17 Side effects of nitric oxide release ...... 63 Figure 1.18 Zeeman splitting effect ...... 69 Figure 1.19 Idealised EPR spectrum of a free electron ...... 72

Figure 1.20 A collection of spins in an external magnetic field, B0 ...... 75 Figure 1.21 Common pulses and the populations of quantum states in comparison to the thermal equilibrium ...... 76 Figure 1.22 The magnetic behaviour during a Hahn echo as a function of pulse position ...... 78 Figure 1.23 The four-pulse PELDOR sequence ...... 79 Figure 1.24 The dipolar coupling dependence on the interspin distance (r) and the angle (θ) to the external magnetic field ...... 81

Figure 3.1 Map of the vector pCRNNR ...... 112 Figure 3.2 DNA gel of the restriction enzyme digests carried out on vector pCRNNR ...... 113 + Figure 3.3 UV-visible absorbance spectrum of purified WT rat nNOSred ...... 116 + Figure 3.4 12% SDS-PAGE gel showing the WT rat nNOSred ...... 116 + Figure 3.5 UV-visible absorbance spectrum of purified R1400E rat nNOSred 117 + Figure 3.6 12% SDS-PAGE gel showing the R1400E rat nNOSred ...... 117 + - Figure 3.7 Cytochrome c reductase activities of WT nNOSred and WT nNOSred ...... 119 + Figure 3.8 Cytochrome c reductase activities of R1400E nNOSred and R1400E - nNOSred ...... 120 Figure 3.9 UV-visible absorbance spectra collected during anaerobic chemical

titration of WT rat nNOSred ...... 125 Figure 3.10 UV-visible absorbance spectra collected during anaerobic chemical

titration of R1400E rat nNOSred ...... 126

Figure 3.11 An example of an EPR spectrum for WT rat nNOSred in the presence of CaM ...... 128

Figure 3.12 EPR spectra of WT rat nNOSred ...... 129

Figure 3.13 EPR spectra of R1400E rat nNOSred ...... 130 Figure 3.14 Fourier transforms of the X-band 4 pulse ELDOR echo decays

produced by two electron reduced (dithionite) WT nNOSred in the presence and absence of CaM and/or NADP+ ...... 136 Figure 3.15 Fourier transforms of the X-band 4 pulse ELDOR echo decays

produced by two electron reduced (dithionite) R1400E WT nNOSred in the presence of CaM and presence or absence of NADP+ ...... 137 Figure 4.1 Vector map of pCWORI ...... 150 Figure 4.2 DNA gel of the restriction enzyme digests carried out on vector pCWORI ...... 151 Figure 4.3 UV-visible absorbance spectra of purified FL WT rat nNOS- ...... 152 Figure 4.4 12% SDS-PAGE gel showing the FL WT rat nNOS+ and FL WT rat nNOS- ...... 153 Figure 4.5 UV-visible absorbance spectra of purified FL R1400E rat nNOS- .. 154 Figure 4.6 12% SDS-PAGE gel showing the FL R1400E rat nNOS+ ...... 155

Figure 4.7 12% SDS-PAGE gel showing the FL WT rat nNOS+ and FL WT rat nNOS- ...... 156 Figure 4.8 An example of flavin identification, quantification and saturation HPLC data recorded for FL WT nNOS- ...... 159 Figure 4.9 FAD calibration curve determined by HPLC ...... 160 Figure 4.10 FMN calibration curve determined by HPLC ...... 161 Figure 4.11 Cytochrome c reductase activities of FL WT nNOS+ and FL WT nNOS- ...... 164 Figure 4.12 Cytochrome c reductase activities of FL R1400E nNOS+ and FL R1400E nNOS- ...... 165 Figure 4.13 UV-visible absorbance visualisation of NADPH oxidation activity of FL WT nNOS+ ...... 168 Figure 4.14 FL WT nNOS+ activity – calmodulin dependence ...... 1670 Figure 4.15 Relative integrals of the flavosemiquinone spectrum from CW EPR collected for FL WT nNOS ...... 173 Figure 4.16 Relative integrals of the flavosemiquinone spectrum from CW EPR collected for FL WT nNOS CaM- form in the presence of ADP or NADP+ ...... 174 Figure 4.17 Fourier transforms of the X-band 4 pulse PELDOR echo decays produced by two electron reduced nNOS ...... 179 Figure 5.1 The steady-state rates of NADPH oxidation as a function of hydrostatic pressure for the FL WT nNOS in the presence and absence of CaM ...... 195 Figure 5.2 The steady-state rates of NADPH oxidation as a function of hydrostatic pressure for the FL R1400E nNOS in the presence and absence of CaM ...... 196

Figure 5.3 Examples of flavin reduction transients observed for the WT nNOSred in the presence and absence of CaM ...... 201 Figure 5.4 Observed rate constants for flavin reduction as a function of

hydrostatic pressure for the WT nNOSred in the presence of CaM .. 202 Figure 5.5 Observed rate constants for flavin reduction as a function of

hydrostatic pressure for the WT nNOSred in the absence of CaM .... 203 Figure 5.6 Examples of flavin reduction transients observed for the R1400E

nNOSred in the presence and absence of CaM ...... 204

Figure 5.7 Observed rate constants for flavin reduction as a function of

hydrostatic pressure for the R1400E nNOSred in the presence of CaM ...... 205 Figure 5.8 Observed rate constants for flavin reduction as a function of

hydrostatic pressure for the R1400E nNOSred in the absence of CaM ...... 206 Figure 5.9 Examples of flavin reduction transients observed for FL WT nNOS in the presence and absence of CaM ...... 211 Figure 5.10 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the FL WT nNOS in the presence of CaM 212 Figure 5.11 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the FL WT nNOS in the absence of CaM .. 213 Figure 5.12 Examples of flavin reduction transients observed for FL R1400E nNOS in the presence and absence of CaM ...... 214 Figure 5.13 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the FL R1400E nNOS in the presence of CaM ...... 215 Figure 5.14 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the FL R1400E nNOS in the absence of CaM ...... 216

Figure 5.15 UV-visible absorbance spectra of oxyhaemoglobin (HbO2) and methaemoglobin (MetHb) ...... 218 Figure 5.16 Absorption transients for NO-mediated OxyHb into MetHb conversion (NO synthesis by FL WT nNOS) in the presence (CaM+) or absence (CaM-) of CaM ...... 222 Figure 5.17 Observed rate constants for NO synthesis as a function of hydrostatic pressure measured for FL WT nNOS in the presence of CaM...... 223 Figure 5.18 Absorption transients for NO-mediated OxyHb into MetHb conversion (NO synthesis by FL R1400E nNOS) in the presence (CaM+) or absence (CaM-) of CaM ...... 224 Figure 5.19 Observed rate constants for NO synthesis as a function of hydrostatic pressure measured for FL R1400E nNOS in the presence of CaM .. 225

List of Tables

Table 1.1 Physiological differences of the nitric oxide synthase isoforms, including examples of known functions and locations...... 20 Table 2.1 Oligonucleotides used for the generation of the R1400E mutation in FL rat nNOS ...... 89 Table 2.2 Linear amplification reaction conditions used for the generation of the R1400E mutation in FL rat nNOS ...... 89

Table 3.1 Kinetic parameters obtained for WT and R1400E nNOSred in the presence and absence of CaM ...... 121

Table 3.2 The ge, line widths and intensity data determined from CW EPR

studies on WT and R1400E nNOSred, in the presence and absence of CaM and various ligands ...... 131 Table 3.3 Distances between the FAD and FMN flavin semiquinones in the two- + electron reduced state of WT nNOS, in the presence (WT nNOSred ) - or absence (WT nNOSred ) of CaM, determined from PELDOR spectroscopy ...... 138 Table 4.1 Mass spectroscopy results searched against the generic UniProt (version rat) database ...... 157 Table 4.2 The results of identification, quantification and saturation of flavin observed using HPLC and calculated for FL WT and R1400E nNOS ...... 162 Table 4.3 Kinetic parameters obtained for FL WT and R1400E nNOS in the presence and absence of CaM ...... 166 Table 4.4 NADPH oxidation rates determined for FL WT and R1400E variant nNOS (both CaM+ and CaM- forms) ...... 168 Table 4.5 Mid-point potentials for the four-electron reduction of FL WT nNOS ...... 175 Table 4.6 Distances between the FAD and FMN flavin semiquinone in the two- electron-reduced state of FL WT nNOS, in the presence (FL WT nNOS+) or absence (FL WT nNOS-) of calmodulin, determined by PELDOR spectroscopy ...... 180

Table 5.1 k1 and k2 flavin reduction rate constants and respective amplitudes

observed at different pressures for WT and R1400E nNOSred, in the presence (CaM+) and absence (CaM-) of CaM ...... 200

Table 5.2 k1 and k2 flavin reduction rate constants and respective amplitudes observed at different pressures for FL WT and R1400E nNOS, in the presence (CaM+) and absence (CaM-) of CaM ...... 210

Table 5.3 k1 and k2 rate constants and respective amplitudes of NO-mediated OxyHb into MetHb conversion observed at different pressures (0- 1500 bar) for FL WT and R1400E nNOS, in the presence (CaM+) ...... 221

Abstract

Probing the dynamics and conformational landscape of neuronal nitric oxide synthase Anna Sobolewska-Stawiarz A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences, September 2013. Rat neuronal nitric oxide synthase (nNOS) is a flavo-hemoprotein that catalyses the NADPH and O2-dependent conversion of L-arginine (L-arg) to L- citrulline and nitric oxide (NO) via the intermediate N-hydroxyarginine. nNOS is a homodimer, where the subunits are modular and are comprised of an N-terminal oxygenase domain (nNOSoxy) that binds iron protoporphyrin IX (heme), (6R)- 5,6,7,8-tetrahydro-biopterin (H4B) and L-arg, and a C-terminal flavoprotein or reductase domain (nNOSred) that binds NADPH, FAD and FMN. Regulation of NO biosynthesis by nNOS is primarily through control of interdomain electron transfer processes in NOS catalysis. The interdomain electrons transferred from the FMN to the heme domain are essential in the delivery of electrons required for O2 activation (which occurs in the heme domain) and the subsequent NO synthesis by NOS. Both spectroscopic and kinetic approaches have been used in studying the nature and control of interdomain electron transfer, reaction mechanism and structural changes during catalysis in WT and R1400E nNOS in both full length (FL) and nNOSred. Cytochrome c reduction activity of nNOS was used to determine kinetic parameters for NADPH for FL and nNOSred, WT and R1400E nNOS in the presence and absence of calmodulin (CaM). FL nNOS, where both domains (nNOSred and nNOSoxy) were present, was proven to be more stable and more catalytically efficient than nNOSred by itself. Additionally it was observed that R1400E is still promoting electron transfer despite being thought to lower the affinity of the enzyme to the substrate (NADPH); R1400E also showed lower catalytic efficiency and lower dependence on CaM/Ca2+ compared to the WT. The structure of the functional output state has not yet been determined. In the absence of crystallographic structural data for the NOS holoenzyme, it was important to experimentally determine conformational changes and distances between domains in nNOS. A pulsed EPR spectroscopy (PELDOR) approach has been utilised to gain important and unique information about the conformational energy landscape changes in nNOS. In the presence of CaM, PELDOR results for FL WT nNOS shows a complex energy landscape with multiple conformational states, while in the absence of CaM the interflavin distance distribution matches that exhibited by nNOSred CaM- in the presence of NADP+, suggesting that CaM binding affects some major large-scale conformational changes which are involved in internal electron transfer control in nNOS. A high-pressure stopped-flow technique was also used to perturb an equilibrium distribution of conformational states, to observe the effect of the pressure on the internal electron transfer and to study the kinetics of NADPH oxidation, flavin reduction by NADPH and NO formation. It was shown that high pressure is forcing major changes in the conformational energy landscape of the protein, affecting internal electron transfer. NO formation studies under pressure show that the R1400E mutation in FL nNOS may be affecting protein/NADPH affinity and flavin reduction, but it has no effect on the heme reduction step.

Declaration

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Acknowledgements

This PhD thesis is the result of a challenging journey, to which many people have contributed and given their support.

Foremost, I would like to express my gratitude to my supervisors Dr Stephen Rigby and Professor Nigel Scrutton for their time, help, guidance and encouragement during the course of my PhD. I could not have asked for better supervisors, each inspirational, enthusiastic, supportive and patient.

Special thanks goes to Dr Karl Fisher for proof-reading my thesis (not an easy task!) and providing constructive feedback along with thoughtful and detailed comments and for all the encouraging discussions about the research, future and life in general.

Furthermore, I would like to thank Dr Adrian Dunford for his help and time he spent reading my reports and chapters of this thesis, his endless support in the lab, the patient answers to all my questions and for our interesting discussions about NOS, science and everything beyond.

I would also like to thank Dr Derren Heyes and Dr Sam Hay for all their help and insightful comments, both in my lab work and in this thesis, as well as for their support and inspiration.

All the past and present members of the Scrutton/Munro/Leys groups also deserve my sincerest thanks, their friendship and assistance has meant more to me that I could ever express.

I would also like to thank my parents for all the understanding, faith, unconditional love, care and endless support throughout my whole life. Special thanks to my sister for all the fun, motivation and stimulating discussion about real life and for simply being there for me whenever I needed her.

Last but not least, I would like to thank Andrzej, my best friend, soul-mate and my lovely husband for his endless love, patience, faith and for the unstinting reminder that there is more to life than a PhD thesis. He has been a true and great supporter who always stayed by my side, especially when I was irritable and depressed, and more importantly, I wouldn’t be the person I am today and where I am now if it wasn't for him.

List of Abbreviations

ADP Adenosine diphosphate δ-ALA δ-Aminolevulinic acid 2’(3’)-AMP Adenosine 2′(3′)-monophosphate mixed isomers AMP Adenosine monophosphate Amp Amplitude CaM Calmodulin CaM+ Calmodulin-bound CaM- Calmodulin-free CPR Cytochrome P450 Reductase CW EPR Continuous wave electron paramagnetic resonance DNA Deoxyribonucleic acid dNTP Deoxynucleotide triphosphate DTT Dithiothreitol EDTA Ethylenediaminetetraacetic acid

Em Mid-point potential eNOS Endothelial isoform of nitric oxide synthase EPR Electron paramagnetic resonance FAD Flavin adenine dinucleotide FID Free induction decay FL Full length FL nNOS+ CaM-bound full length neuronal nitric oxide synthase FL nNOS- CaM-free full length neuronal nitric oxide synthase FMN Flavin mononucleotide

H4B (6R)-5,6,7,8-tetrahydrobiopterin Hb Haemoglobin HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High-pressure liquid chromatography iNOS Inducible isoform of nitric oxide synthase IPTG Isopropyl β-D-1-thiogalactopyranoside L-arg L-arginine LB Luria-Bertani

MetHb Methaemoglobin NAD+ β-nicotinamide adenine dinucleotide (oxidised form) NADH β-nicotinamide adenine dinucleotide (reduced form) NADP+ β-nicotinamide adenine dinucleotide phosphate (oxidised form) NADPH β-nicotinamide adenine dinucleotide phosphate (reduced form) NMR Nuclear magnetic resonance NO Nitric oxide NOS Nitric oxide synthase

NOSoxy The oxygenase domain of nitric oxide synthase

NOSred The reductase domain of nitric oxide synthase nNOS Neuronal isoform of nitric oxide synthase + nNOSred CaM-bound reductase domain of nitric oxide synthase - nNOSred CaM-free reductase domain of nitric oxide synthase

OD600 Optical density at 600nm OxyHb Oxyhaemoglobin PAGE Polyacrylamide gel electrophoresis PCR The polymerase chain reaction PELDOR Pulsed electron–electron double resonance ppm parts per million rpm revolutions per minute SDS Sodium dodecyl sulfate SDS-PAGE Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate SOC Super optimal broth with catabolite repression TB Terrific broth Tris Tris(hydroxymethyl)aminomethane UV-vis Absorption spectroscopy in the ultraviolet-visible spectral region v/v volume per volume WT Wild type

List of Amino Acids Abbreviations

Three letter code Single letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid (Aspartate) Asp D Asparagine or Aspartic acid (Aspartate) Asx B Cysteine Cys C Glutamic acid (Glutamate) Glu E Glutamine Gln Q Glutamine or Glutamic acid (Glutamate) Glx Z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Chapter1 – Introduction

Chapter 1 Introduction

Chapter1 – Introduction

Chapter 1 – Introduction

1.1 Nitric oxide synthase

1.1.1 Discovery and isoforms

Nitric oxide synthase (NOS) was first studied in the late 1980s when it was found that nitric oxide (NO) was synthesised directly from the amino acid L-arginine and that it was discovered to act as a signalling mediator in a number of physiological processes including regulation of blood flow/pressure, neurotransmission and immune response. This breakthrough led to The Nobel Prize in Physiology 1998 being awarded jointly to Robert F. Furchgott, Louis J. Ignarro and Ferid Murad "for their discoveries concerning nitric oxide as a signalling molecule in the cardiovascular system".

NOS is a large homodimer enzyme ranging in size from 135 to 160 kDa. Each of its subunits has an N-terminal oxygenase domain (NOSoxy) that binds iron protoporphyrin IX (heme b), (6R)-5,6,7,8-tetrahydrobiopterin (H4B) and L-arginine, and a C-terminal reductase domain (nNOSred) that binds FMN, FAD and NADPH.

There are three mammalian NOS enzymes: neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). All of them are self-sufficient enzymes with two major

1 functional domains fused into a single polypeptide . Heme, FMN, FAD, CaM, H4B are essential as cofactors and NADPH and O2 are essential as co-substrates for all of the NOS isoforms. Neuronal and endothelial isoforms of NOS are typically referred to as constitutive because they are present in tissues under resting conditions. iNOS is not typically expressed in the cellular resting state (exceptions being intestinal and

Chapter1 – Introduction

bronchial epithelial cells, renal tubular epithelial cells) 2. nNOS and iNOS are soluble and predominantly found in the cytosol, while eNOS is membrane associated. The amino acid sequence alignment of all three NOS isoforms has revealed a 50-60% sequence identity and a very high active site conservation 3–6.

This high conservation of active sites makes finding an isoform selective NOS inhibitor a serious challenge. Although the three isoforms share a high level of amino acid sequence identity, they differ in size, intracellular location and functions (Table

1.1).

Table 1.1 Physiological differences of the nitric oxide synthase isoforms, including examples of known functions and locations.

Isoform Mass Primary location Known function nNOS 165 kDa Nervous tissue in both the Neurotransmission. Long-term central and peripheral potentiation. Coordination nervous system. Nerve cells, between neuronal activity and skeletal and heart muscle. blood flow. Pain modulation. eNOS 133 kDa All vascular endothelial Cardiovascular EDRF. cells. Regulation of vascular tone and vasodilation. Inhibition of smooth muscle cell proliferation. Inhibition of platelet aggregation. iNOS 130 kDa Immune and cardiovascular Inflammation and host defence. system as well as many Cytotoxicity against bacteria, other cell types as a viruses and other response of the immune microorganisms. system.

Chapter1 – Introduction

Figure 1.1 Block gene alignment of nitric oxide synthase isoforms and CPR. Blocks indicate binding sites for heme, tetrahydrobiopterin (H4B), calmodulin (CaM), FMN, FAD and NADPH along with positions of PDZ domain (PDZ), auto- inhibitory control element (ACE) and C-terminal extension (CT). Figure adapted from 7.

Figure 1.1 shows the relation between amino-acid sequences of three different isoforms and CPR. iNOS has the shortest sequence, it binds CaM independently to

Ca2+ concentration and is not subject to Ca2+-dependent regulation. The CaM binding sequence in eNOS is modified and its protein sequence contains two additional significant inserts (45 amino acid auto-inhibitory control element (ACE) within the FMN domain and a 42 amino acid extension to the C-terminus (CT) compared to the CPR sequence). nNOS contains modified versions of the above and an additional 220 amino acid extension to the N-terminus (PDZ) 8.

A unique 42-45-amino acid insertion - an auto-inhibitory control element (ACE) - can be found within the FMN-binding subdomain of nNOS and eNOS, but not in

Chapter1 – Introduction

iNOS or CPR. In the absence of CaM, ACE locks the FMN-binding domain to the reductase complex via a network of hydrogen bonds, which block the electron flow from the nNOSred to heme. During binding of CaM, the auto-inhibitory control element is displaced and the enzyme is activated 9. The ACE-deletion mutant of nNOS in the presence of CaM has only 30-50% NO synthesis activity which indicates the disturbance of interdomain interactions between FMN and oxygenase domains in the mutants 10. This shows that the ACE insert may be involved in stabilisation of interdomain interactions in NOS, rather than simply playing the role of an inhibitory element 9.

The N-terminal domain of nNOS is unique to the neuronal NOS isoform and contains a PDZ motif that can be found in other cytoskeletal proteins and enzymes

11. The PDZ domain, also known as the GLGF repeat is a multifunctional protein- interaction motif that has been identified in over 50 different proteins, the majority of which appear to be associated with the cytoskeleton 12. It is usually found in signalling molecules and it often binds to the C-terminus of protein targets 13. The

PDZ-containing domain of nNOS binds to PDZ repeats in postsynaptic density protein 95 (PSD-95) 11 and a novel related protein, PSD-93. PSD-95 is co-expressed with nNOS in several neuronal populations in both developing and mature nervous systems.

Dimer stability also plays an important role in NOS activity. Despite having similar dimeric architecture, the dimers of all three NOS isoforms are not equally stable

(with the rank order of eNOS > nNOS > iNOS) 14,15. It has been shown that during a

H4B- and L-arginine-free purification, all three forms of full length NOSs (expressed in E. coli) were predominantly purified in their dimeric forms. Therefore, H4B presence and binding is not essential for NOS dimerization but it helps to form and

Chapter1 – Introduction

stabilise the dimer. The binding of Zn2+ to NOS, through a pair of cysteines from each subunit, is also not necessary and it has a similar effect on the dimer formation process 16.

The catalytic activities of the NOS isoforms vary significantly; whether activity is measured as cytochrome c reduction or NO formation. Regardless of which parameter is measured, the activities of nNOS and iNOS are higher than that of eNOS. In each case, the nNOSred is able to transfer electrons to cytochrome c at a rate significantly greater than the maximum rate of NO production 17.

Nitric oxide produced by mammalian endothelial nitric oxide synthase (eNOS) or neuronal NOS (nNOS) plays an important role in the cardiovascular and nervous system, respectively 18–20 when NO produced by inducible NOS (iNOS) acts as a cytotoxic agent in the immune system 21.

1.1.2 The reaction

NOS catalyses a two-step reaction that converts L-arginine (L-arg) through

Nω-hydroxy-L-arginine (N-hydroxyarginine, NOHA) to citrulline and nitric oxide.

The reaction occurs within the nNOSoxy of NOS and is NADPH- and O2- dependent.

Chapter1 – Introduction

1 NADP+ 0.5 NADP+ 1 NADPH 0.5 NADPH

L-arginine N -Hydroxy-L-arginine L-citrulline Nitric oxide

Figure 1.2 Reaction catalysed by NOS. Nitric oxide synthase produces nitric oxide and citrulline from arginine, molecular oxygen and NADPH. The chemistry of NOS occurs in two mechanistically distinct steps with Nω-hydroxy-L-arginine as a stable intermediate. Figure was generated with Accelrys Draw 4.1.

The enzyme is active only in its dimeric form as interdomain electron transfer is necessary for NO production. Two flavins (FMN and FAD) in the nNOSred allow electron transfer from NADPH, accepting electrons two at a time, but donating them in a stepwise manner in order to allow a monooxygenation reaction within the nNOSoxy to occur.

Chapter1 – Introduction

Figure 1.3 Domain arrangement and electron flow in the NOS dimer. nNOSred contains NADPH-, FAD- and FMN- binding sites, when the nNOSoxy contains L- arginine-, heme- and H4B- binding sites. nNOS transfers electrons from NADPH via FAD to FMN and the heme of the other subunit where the substrate - L-arginine - is oxidised to L-citrulline and NO. Figure adapted from 22.

Each 1 mol of homodimer NOS contains 2 mols of heme b. The heme iron is in the ferric form (Fe(III)) with cysteine thiolate as the proximal and water as the distal axial ligand 23. The binding of substrate increases the redox potential of NOS but the arginine-bound form stays inactive unless H4B is bound. With bound substrate, oxygen binds to the ferrous heme and the final complex decays 100-fold faster if

H4B is also bound, which explains the role of H4B in NOS catalysis (See section

1.1.3.4) 24–26. Additionally, freeze quench analysis of the products of steps 1-4 (Fig.

1.4) demonstrates that a tetrahydrobiopterin radical is formed 27–31 32.

Chapter1 – Introduction

L-citrulline + NO + H2O

3+ Fe - + L-arg, e , O2 7 1 L-arg 3+ 3 2+ Fe O OH Fe O2 2

+ e- 6 2 + e- 3 1 NOHA2 L-arg 2+ 3+ Fe O 2 Fe OOH

5 3 - - H2O + e , O2 2 L-arg NOHA 1 3+ 4 3+ Fe (FeO)

NOHA -H O 2 4

Figure 1.4 Mechanism of NO synthesis by NOS2 32. Step: (1) Arginine, an electron from the nNOSred-, and O2 are supplied to H4B saturated ferric NOS forming the + e , O2 2+ substrate-bound, transient Fe O2 complex; (2) After supply of the second electron, a transient Fe3+OOH form is generated; (3) Fe3+OOH form undergoes oxygen-oxygen bond scission to lose water and form electrophilic oxyferryl species (FeO)3+; (4) (FeO)3+ then hydroxylates L-arg to form enzyme-bound NOHA and ferric NOS; (5) 2+ when O2 and the third electron from the nNOSred are supplied, transient Fe O2 2+ complex is formed again; (6) Fe O2 accepts a fourth electron to generate a NOHA radical and the heme iron-peroxy Fe3+OOH form; (7) Fe3+OOH undergoes nucleophilic reaction with NOHA radical and forms citrulline, NO and water, with NOS returning to its ferric form. Figure adapted from 32.

Chapter1 – Introduction

The heme reduction rate (Fe(III) → Fe(II)) is determined by the ability of the nNOSred to donate electrons to the nNOSoxy, and it is thought to be a rate limiting step of NOS catalysis 17. The NOS cycle is often compared to the P450 cycle because the product of the reaction, NO, like O2, is a heme diatomic ligand which may cause feedback inhibition 33–35. Self-generated NO controls the NOS catalytic activity by binding to the NOS heme 36–38. NO shows a high affinity to ferrous iron owing to the extremely slow release of NO from the Fe(II)-NO complex. The unproductive side reaction (futile cycle) is the oxidation of Fe(II)-NO to Fe(III) in the presence of dioxygen producing alternative nitrogen oxides rather than NO. This forces the NOS heme reduction rate to remain relatively slow in order to minimise the inherent NO dioxygenase activity which leads to the destruction of the produced

NO in the futile cycle.

1.1.3 Structure, function and dynamics

1.1.3.1 Flavoproteins and flavin cofactors

Flavoproteins are described as proteins containing flavin mononucleotide

(FMN) or/and flavin adenine dinucleotide (FAD). Flavoproteins play an important role in both one- and two-electron transfers in aerobic metabolisms and a number of other biological processes. In some multi-redox-centre enzymes like NADH dehydrogenases, cytochrome P450 systems or nitric oxide synthase, flavins directly participate in the electron transfer forming part of the enzyme, often an active site 39.

Humans’ poor riboflavin diet affects iron handling and was proved to be one of the reasons for anaemia and related diseases 40. Riboflavin was also named as a risk factor for some types of cancer as well as cardiovascular diseases. It was also said that its deficiency reduces the metabolism of other B vitamins 40. Flavin containing

Chapter1 – Introduction

aromatic monooxygenases play a central role in soil detoxification 41 and bioremediation of environmentally harmful compounds like monocyclic phenols or aromatic acids 42. Flavoproteins are also involved in repair of pyrimidine dimers in

UV-damaged DNA 43–45 as well as light-signalling processes in plant phototropism

46–48.

The enzymes of the diflavin reductase family have two tightly bound cofactors, FAD and FMN. Each flavin can exist in three different redox states; the oxidised, semiquinoid, and fully reduced form. In each of these states, the chemical properties of the flavin are very different 49. Flavoproteins form relatively difficult to stabilise semiquinone (one-electron reduced) and hydroquinone (two-electron reduced) forms, which are fundamental for intermediates in the transition between 2-electron and 1- electron donors/acceptors. Although flavin semiquinones can exist in cationic, neutral and anionic forms, only neutral and anionic forms can be found in flavoproteins. Under certain conditions, flavin semiquinones will be formed by almost any flavoprotein (often partial reduction of the protein is necessary) 50.

Neutral and anionic semiquinones are called “blue” and “red” respectively, due to the colour of their concentrated solutions 49.

Chapter1 – Introduction

1 R= 9 8 10 FAD 2

4a 3 7 4 6 5 FMN max= 450 nm)

FAD or FMN (Oxidized)

H+, e- H+, e-

- H+

+ H+ - max= 570 nm max= 490 nm

FADH or FMNH (Semiquinone, blue) FADH- or FMNH- (Anionic semiquinone, red)

H+, e- H+, e-

FADH or FMNH (Reduced, colourless) 2 2

Figure 1.5 Representation of the structures of FMN and FAD in different oxidation states, with the absorption maxima indicated. Figure was adapted from 51 and generated with Accelrys Draw 4.1.

Chapter1 – Introduction

It has also been observed that some of the flavoproteins, for example partially reduced cytochrome P450 reductase (CPR) and inducible NOS, can form two types of FMN semiquinone. One is the catalytically active intermediate form which is produced upon NADPH binding and hydrate ion transfer to the FAD of the flavoprotein, followed by the transfer of one electron to FMN. The other is a slower form of the enzyme, also known as “air-stable” semiquinone due to its remarkable stability in the presence of atmospheric oxygen 52,53. “Air-stable” semiquinone has only been observed after prolonged incubation of partially reduced CPR/inducible

NOS under aerobic conditions; it does not seem to be a part of the catalytic cycle as it does not reduce cytochrome c 54. It is likely that the “air-stable” semiquinone forms of other flavoproteins could also be different from the active catalytic intermediate.

Flavoproteins are one of the most-studied enzyme families partly because of their spectroscopic properties. The very interesting part of the flavin cofactor is its tricyclic isoalloxazine moiety presented in Figure1.5. Both, FMN and FAD, can exist in various oxidation states. Each of the states (oxidised, semiquinone or reduced) has specific electronic, spectral and chemical properties. The maximum absorptions significantly differ between different flavin oxidation states making them easy to recognize, distinguish and follow using UV spectroscopy. Because of that, flavin cofactor is a great reporter of changes occurring within the active site.

1.1.3.2 Reductase domain

The reductase domain of NOS (NOSred) belongs to a large protein family that includes NADPH-dependent CPR, sulphite reductase flavoprotein and novel protein-

1. These reductases share a conserved organisation of FMN, FAD and NADPH-

Chapter1 – Introduction

binding domains 55–57. The FMN-binding domain is homologous to small electron- carrier flavodoxins 58, whilst FAD and NADPH-binding domains are related to

+ 59 ferredoxin-NADP reductase . NOSred can be expressed and purified independently

60 of its nNOSoxy and it can transfer electrons from NADPH to FAD to FMN and artificial acceptors such as cytochrome c, but it is unable to carry out the full catalytic cycle of NOS and it cannot convert L-arginine to L-citrulline and produce

NO. The internal electron transfer in nitric oxide synthesis begins with the transfer of hydride ion from NADPH directly to the N5 of the FAD in the nNOSred. For this step, the presence of an aromatic residue (for example F1395 in rat nNOS or F1400 in human nNOS) is necessary.

The aromatic residue lies penultimate to the start of the C-terminal control element and its side chain shields the isoalloxazine ring of the FAD (presented in Figure 1.6) and then swings towards the FMN domain allowing the nicotinamide group to access the FAD. This aromatic residue is also known to regulate NADPH binding affinity in related flavoproteins and it is thought that its phenyl side chain is part of a conformational trigger mechanism that negatively and positively controls NOS electron transfer depending on the presence of calmodulin 61.

In the next step, an electron must then be transferred from newly formed FAD hydroquinone to the FMN, which passes one electron at a time to the heme and acts as a shuttle 10,62,63. During the electron transfer, FMN oscillates between its hydroquinone and semiquinone forms, whilst the FAD passes through all three oxidation states 64,65.

Chapter1 – Introduction

Figure 1.6 Representation of the position of an aromatic residue F1395 within rat nNOSred. Figure shows the representation of the position of an aromatic residue F1395 in rat nNOSred. The aromatic residue F1395 shields the isoalloxazine ring of the FAD and then swings towards the FMN domain allowing the nicotinamide group to access the FAD. FMN is shown in pink, FAD is shown in dark green, F1395 aromatic residue is shown in blue/navy and NADPH is shown in yellow. The figure was generated with PyMOL (pdb 1TLL).

Binding calmodulin to the NOSred activates substrate-independent NADPH oxidation and is thought to promote internal electron transfer from the flavins in nNOSred to the

62 heme (in nNOSoxy) which is rate-limiting for the NO production catalysed by NOS

66 67 . NOSred is uniquely tuned to control nitric oxide production . Both, nNOS and eNOS contain a 21-42 residues long C-terminal tail which represses the transfer of the electrons. Additionally, nNOS and eNOS contain a short auto-inhibitory control element in the FMN-binding domain that interferes with calmodulin binding and

Chapter1 – Introduction

inhibits the transfer of electrons. Both these regulatory elements, when phosphorylated, can further modulate the activity of nNOS and eNOS. Finally, the

CD2A regulatory element is thought to be responsible for CaM/Ca2+ dependence by its interaction with the calmodulin binding peptide 68. CD2A has been found in the connecting domain between the FMN and FAD binding subdomains 68. Based on the structure of nNOS FAD/NADPH domain 69 CD2A has a surface hairpin loop which is very likely to be within interaction distance of the NOS CaM-binding site. In iNOS, CD2A is 5 residues shorter than in nNOS and eNOS, which may indicate that the longer CD2A loop contributes to Ca2+ dependence, although its effect is masked by the dominant ACE in the FMN subdomain.

The crystallographic structure of the dimeric nNOSred was solved at 2.3 Å resolution

70 and it revealed 4 major domains: i) FMN binding domain (residues 750-942), ii) connecting domain (943-989 and 1039-1170), iii) FAD binding domain (990-1038 and 1171-1231) and iv) NADPH binding domain (1232-1396). In the NADPH- binding domain, co-substrate NADP(H) binds at one end of the five-stranded parallel

β-sheet, as in related reductases 55,56,59. The negatively charged phosphate groups of

NADP(H) are held by a network of ionic interactions, including an Arg1400 from the regulatory C-terminal tail (Arg1400 is conserved in nNOS and eNOS, but not in iNOS). The guanidinium group of nNOS Arg1400 forms a salt bridge with the 2’- phosphate group of NADP(H), and this phosphate differentiates NADPH from

NADH. The R1400E variant shows about a 5-fold faster rate of electron transfer from flavins to cytochrome c in the absence of CaM/Ca2+ and it has a lower dependence on CaM/Ca2+ compared to the WT 70. Results for variant R1400E show that introduction of Glu1400 instead of Arg1400 repels rather than attracts NADPH, destabilises and disorientates the C-terminal tail, relieving repression and supporting

Chapter1 – Introduction

internal electron transfer despite its decreased affinity for NADPH. CaM- activity of this variant is only about 30% lower than the activity of CaM+ wild-type NOS. It is thought that cNOS-specific Arg1400 helps maintain the repressed or locked electron- accepting position of the FMN domain upon NADPH binding to the CaM- NOS 70.

Available biochemical data suggest a possible model for the NOS mechanism in which the FMN domain undergoes large scale movements to transfer the electrons between reductase and oxygenase domains (domain shuffle hypothesis) 70, although, the type of motion occurring during the catalytic cycle is not certain as no structures of NOSred in the open conformation have been reported.

Crystallographic data have observed that the distance between FAD and FMN isoalloxazines is about 5 Å and the FMN to heme distance is about 15 Å in the electron-accepting and electron-donating positions, respectively 55,70. Electron transfer from FAD to FMN, being only 5 Å apart, is extremely rapid, but that also means that with the heme 15 Å away, FMN will be unable to interact with the nNOSoxy effectively. When FMN and FAD are in close contact, FMN is inaccessible to exogenous redox partners. It is difficult to imagine a conformation in which FMN is in close contact with both FAD and heme. The crystallographic structure indicates that catalytically competent electron transfer from FMN to the nNOSoxy cannot occur

70 in the nNOSred conformation observed in the crystal structure . Thus for the FMN to be positioned for reduction by FAD, it will be necessary for the FMN to move substantially in order to deliver an electron to the heme. Rotational flexibility of the

FMN domains has also been observed 70.

Chapter1 – Introduction

Figure 1.7 Structure of rat nNOSred. Figure A shows the structure of rat nNOSred dimer (one monomer is shown in green/blue, the other – in orange/red; FMN, FAD and NADP+ for each monomer are also visible). Figure B shows the monomer structure of rat nNOS. nNOS can be divided into four subdomains; the FMN- binding domain (blue) with the FMN shown in red, the connecting domain (with flexible hinge and β–finger) shown in purple, FAD-binding domain (green) with the FAD shown in pink and NADPH-binding domain (grey) with NADP+ shown in yellow. The figure was generated with PyMOL (pdb 1TLL).

Chapter1 – Introduction

1.1.3.3 Oxygenase domain

In almost all forms of life, heme is an integral part of proteins involved in fundamental biological processes such as electron transfer during respiration, photosynthesis, enzyme catalysis, metabolism and oxygen transport. There are a few different heme groups existing in enzymes, nitric oxide synthase (similarly to cytochrome P450) contains a heme b with iron coordination to a proximal cysteine and water as the distal ligand. Binding of substrate to cytochrome P450 or NOS changes the conformation of the active site, usually displacing water from the distal axial position of the heme iron, enabling dioxygen binding and sometimes changing the state of the heme iron from low-spin to high-spin.

2  3

a b 1 4

 

d c 8 5 7  6

heme b H2O/Cys

Figure 1.8 Chemical structures of heme. Figure A shows a general structure of heme b. Figure B shows a structure of heme b in NOS (with its coordination bond to proximal cysteine and water as the distal ligand). Figure was generated with Accelrys Draw 4.1.

Chapter1 – Introduction

Figure 1.9 Structure of rat nNOSoxy. Figure shows the structure of rat nNOSoxy dimer 71. The two subunits are coloured in blue and green, respectively. Each subunit contains a heme-binding site with heme in red, L-arginine-binding site with L- arginine in orange, a binding site for tetrahydrobiopterin with H4B in yellow. At the dimer NOS interface, a Zn2+ zinc ion was also found being tetrahedrally ligated by 2 pairs of cysteine residues, one from each subunit and it is thought to be important for structural integrity of the dimer (zinc ion shown in magenta). The figure was generated with PyMOL (pdb 1ZVL).

Nitric oxide synthase has a dimeric structure in which an N-terminal nNOSoxy

72–74 contains the binding sites for heme, H4B, L-arginine . The rate limiting step for

36,38,66 NO synthesis is a heme reduction step (Fe(III) → Fe(II)) , which enables O2

1,27,75 binding and substrate oxidation to occur within the nNOSoxy . To catalyse

Chapter1 – Introduction

oxygen activation NOS uses a similar mechanism to cytochrome P450 enzymes.

Two-step heme reduction is involved in the oxygen activation where donated protons help to break the O-O bond and where the reactive heme-oxyferryl version of NOS is formed. The flavoprotein domain of nitric oxide synthase directly donates the first

27 electron whilst the second electron is provided via the bound H4B cofactor , which is a novel feature of NOS because the other cytochrome P450 enzymes are supplied with all necessary electrons by their reductase domains.

As a part of pharmaceutical development, a number of crystal structures have been solved including those for truncated oxygenase domains of murine iNOS 3,76, human iNOS 4,77, bovine eNOS and human eNOS 6. The X-ray crystal structure of rat nNOSoxy (without PDZ-binding region) was also determined to 2.0-2.5 Å resolution

71 in a form with H4B bound and in the presence/absence of L-arginine .

Crystallographic structures reveal key sites which can be used for designing selective

NOS inhibitors. Highly conserved active sites among nitric oxide synthase isoforms make the design of selective inhibitors very difficult and challenging. There is no selective NOS inhibitor for any of the NOS isoforms available for clinical use 78.

1.1.3.4 Function of tetrahydrobiopterin

Tetrahydrobiopterin was first described by Hopkins in 1895 then characterised by Watt in the 1960s. It was initially identified as a cofactor for phenylalanine hydroxylase and other aromatic amino acid hydroxylases but in 1989, soon after nitric oxide synthase was isolated, (6R)-5,6,7,8-tetrahydrobiopterin was identified as a required NOS cofactor by Stuehr et al. 79 and Tayeh and Marietta 80.

H4B binds within the dimer interface and helps to stabilise the quaternary structure of the active dimeric enzyme form 3,81. During the catalytic cycle,

Chapter1 – Introduction

tetrahydrobiopterin remains permanently bound to nitric oxide synthase, cycling between the fully reduced and the one-electron oxidised forms 82. It is required to act as an electron donor during oxygen activation and to recapture an electron from the ferrous nitrosyl complex in order to trigger NO release 37 which is a very important function because the ferrous NO complex is effectively a dead-end complex, with sub-nanomolar affinity for NO and a high reduction potential 26. The chemistry of oxygen activation requires two-electron reduction which is inconvenient for free- radical formation which is why a high-potential one-electron donor-acceptor is required. Firstly, it is thought that a tetrahydrobiopterin donates an electron to the ferrous-dioxygen complex in the nNOSoxy to activate it in order that it is able to release NO later in the cycle by recapturing the electron. This mechanism is unusual in enzyme chemistry and it may explain why NO synthesis is relatively slow compared to similar chemical reactions 37.

When H4B is limited, electron transfer from NOS becomes uncoupled from L- arginine oxidation, the ferrous-dioxygen complex dissociates and superoxide is

83–85 produced from the nNOSoxy .

Chapter1 – Introduction

A B

Figure 1.10 Role of tetrahydrobiopterin in eNOS activity regulation in vascular disease. In healthy vascular endothelium (A), where availability of H4B is not limited, calmodulin (CaM) is activated by Ca2+ and electrons donated by NADPH flow from FAD to FMN (in the nNOSred of one monomer) through H4B to the ferrous-dioxygen complex (Fe, in the nNOSoxy of the other monomer). Reduction of molecular oxygen is coupled to L-arginine oxidation and L-citrulline generation and concurrent NO production. When availability of H4B is limited (B), electron transfer becomes uncoupled from L-arginine oxidation, then the ferrous-dioxygen complex - dissociates and superoxide (O2∙ ) is produced from the nNOSoxy. Figure adapted from 86.

Superoxide generation by NOS has been observed in a number of experimental and clinical vascular disease states, diabetes, hypertension and atherosclerosis. NOS uncoupling in vascular disease may have an important effect on NO bioavailability because NO production is reduced and superoxide production is increased, leading to further reductions in NO bioactivity 86.

Chapter1 – Introduction

Figure 1.11 Structural arrangements of the heme and tetrahydrobiopterin in nNOSoxy dimer. The two subunits are coloured in blue (chain A) and pink (chain B). Each subunit contains a heme-binding site with heme in red, L-arginine-binding site with L-arginine in green (spherical representation) and a binding site for tetrahydrobiopterin with H4B in yellow. A tetrahydrobiopterin ring in each subunit is held in place by two aromatic residues. In chain A (blue) tetrahydrobiopterin ring is hold by Trp678 (in light blue, chain A) and Phe691 (in dark pink, chain B) when in 678 691 chain B (pink) H4B ring is held in place by Trp (in purple, chain B) and Phe (in navy blue, chain A). The figure was generated with PyMOL (pdb 1ZVL).

Chapter1 – Introduction

Two aromatic residues, a Trp from one NOS subunit and a Phe from the other, hold the tetrahydrobiopterin ring whilst a few –OH groups from the 6-position dihydroxypropyl side chain of H4B form hydrogen bonds to the protein backbone.

Additionally, most of the heteroatoms in the tetrahydrobiopterin ring make hydrogen bonds with the neighbouring protein groups, with the most important being a hydrogen bond from N3 to the heme propionate which is thought to be the electrons’

87 access way between pterin and heme . The disruption in the H4B position in NOS leads to the loss of tetrahydrobiopterin from the active site, enzymatic uncoupling and destabilisation of NOS dimers 86. In room temperature solutions, tetrahydrobiopterin is very unstable and it undergoes 2-electron oxidation, losing

100% of its activity in less than 4 hours. When bound to NOS H4B appears to be stable for at least a few hours 88. There is no certain explanation of how NOS stabilises H4B but it is thought to be related to the architecture of the NOS pterin- binding site where, when bound, H4B may be protected by protein from oxidation and hydrolysis. Comparison of the stability and geometry around C6 atoms in tetrahydrobiopterin and 7,8-dihydrobiopterin has shown that the NOS H4B-binding site is compatible only with tetrahedral geometry around C6 in H4B and this prevents its oxidation. The trigonal planar geometry around C6 in 7,8-H2B is incompatible with the binding environment and its stability is not increased by binding to NOS 88.

Chapter1 – Introduction

10 2' 4 5 1' 3 6

7 8 2 9 1

5,6,7,85,6,7,8-Tetrahydrobiopterin-Tetrahydrobiopterin 7,87,8-Dihydrobiopterin-Dihydrobiopterin

Figure 1.12 Chemical structures of 5,6,7,8-tetrahydrobiopterin and 7,8- dihydrobipterin. The atoms are labelled on tetrahydrobiopterin; the same labelling applies to dihydrobiopterin. Figure was generated with Accelrys Draw 4.1.

Overall H4B has several independent but complementary roles in the physical stabilisation of NOS in its active dimeric form and in its participation in the NOS catalytic reaction.

1.1.3.5 Role of calmodulin in control of electron transfer

Calmodulin (CaM) is a member of the EF-hand protein family. Proteins from this family bind calcium ions through very similar structural domains (EF-hand motif).

CaM is a small (17 kDa) protein which activates NOS and a variety of other target proteins 89,90 and plays the role of intracellular receptor for Ca2+ ions. CaM single polypeptide chain (148 amino acids) contains trimethyllysine, a unique methylated amino acid. The crystal structure of free calmodulin shows that it has a lobe-helix- lobe conformation and four binding sites for Ca2+ (two on each loop) 89. When calmodulin binds Ca2+, conformational changes occur which result in making the

Chapter1 – Introduction

helix linker looser, thus allowing the Ca2+-bound helical lobe arms to wrap around the calmodulin-binding site of target proteins 91,92.

When binding to NOS, the helix linker of the CaM caves in the middle of the CaM- binding site so that its C-terminal lobe interacts with NOS N-terminus and vice versa

(CaM is bound to NOS in an antiparallel manner) 90. Studies on CaM-binding peptides from different isoforms of NOS have shown that some of the forms have more hydrophobic residues contributing to CaM binding than others. This may explain why CaM has such a high affinity to iNOS that the presence of Ca2+ ions is not necessary for binding to occur 93,94.

Calmodulin promotes an interaction between FMN and the nNOSoxy in NOS, because Ca2+/CaM binding is necessary for both heme reduction and NO synthesis

22. iNOS binds CaM irreversibly as a subunit and independently of Ca2+ presence, whereas both nNOS and eNOS are strictly dependent on Ca2+ concentration 95.

Chapter1 – Introduction

A B

Figure 1.13 Ca2+-calmodulin complex formation. Figures A and B show the calmodulin structure (green) wrapped around the calmodulin-binding site of nNOS (red). Figure C shows that calcium ions are essential for Ca2+-calmodulin complex formation, calmodulin binding and NOS activity. When calcium ions are present, the two bind each globular end of calmodulin, the helical arm region changes conformation (the active complex) and then wraps around the calmodulin-binding site of NOS. Figures A and B were generated with PyMOL (pdb 2O60), Figure C was adapted from 96.

Chapter1 – Introduction

In laser flash photolysis experiments for nNOS and eNOS, internal electron transfer was not observed in the absence of CaM showing that CaM is the required factor in

97,98 controlling the transfer of electrons between the nNOSred and heme . Two primary steps are involved in the formation of the NOS output state. Firstly (I),

CaM-binding triggers the dissociation of the FMN domain from its reductase binding

99 site. Secondly (II), subsequent re-association of FMN with the nNOSoxy occurs .

CaM-binding to nNOS or eNOS unlocks the input state where the FMN domain is able to move between the FAD and heme domains 97,99. The state of NOS after step

(I) is competent to reduce cytochrome c, which requires accessibility of FMN to cytochrome c, but is not sufficient for NO production. Production of NO requires an

100 additional CaM-dependent interaction between FMN and the nNOSoxy .

Previously, CaM control of electron transfer in NOS was believed to be located

101 within the nNOSred of the enzyme only , but recently it has been demonstrated that

CaM is able to control NOS on multiple levels, not only within the FMN domain but also outside the reductase complex 99.

Chapter1 – Introduction

1.2 Enzymes related to NOS

The structures and mechanisms exhibited by NOS are also evident in several related enzymes, previous studies of which have informed research on NOS carried out by myself and others.

1.2.1 Cytochrome P450 reductase

Cytochrome P450 reductase (CPR) is present in the endoplasmic reticulum. It is monomeric with a molecular weight of about 75-80 kDa (depending on source) and it supplies the reducing equivalents required for catalysis by a number of different cytochrome P450 enzymes 102–104. The molar ratio of total P450 content to

CPR in microsomes is in the order of 5-10:1 105–107. CPR supports the catalysis of a number of different P450 enzymes having only one single gene in animals 108, in contrast to higher plants where several different isoforms of CPR exist. The precise functions of these isoforms are not known. In CPR, similarly to NOS and other diflavin reductases, FAD accepts two reducing equivalents from NADPH and FMN acts as a one electron carrier for the transfer from NADPH to the heme protein, in

109 which the FMNH∙/FMNH2 couple donates electrons to cytochrome P450 . CPR, like the flavin-containing domain of sulphite reductase, has an additional domain that is unique to diflavin reductases, which connects the FMN and FAD domains.

Additionally, microsomal CPR also has a 20-30 amino acid N-terminal hydrophobic fragment which serves as a membrane anchor and is necessary for binding to the membrane and for interaction with cytochrome P450 50.

Chapter1 – Introduction

Figure 1.14 Structure of rat CPR. Figure A shows the structure of rat CPR dimer 55(one monomer is shown in blue, the other – in green; FMN, FAD and NADP+ for each monomer are also visible). Figure B shows the monomeric structure of CPR. CPR can be divided into four subdomains; the FMN- binding domain (blue) with the FMN shown in red, the connecting domain shown in purple, FAD-binding domain (green) with the FAD shown in pink and NADPH-binding domain (grey) with NADP+ shown in yellow. Both figures were generated with PyMOL (pdb 1AMO).

Chapter1 – Introduction

The crystal structure of recombinant rat CPR was obtained for the protein after removal of the membrane anchor to allow for successful crystallisation 55. The truncated protein no longer reduces cytochromes P450 110,111 but keeps its full catalytic properties in terms of reducing artificial electron acceptors. A number of electron acceptors such as cytochrome P450, cytochrome c, dichlorophenol indophenol (DCPIP) and ferricyanide can be reduced by CPR in vitro. These reactions are often used as convenient assays of enzyme activity. Cytochrome c reduction is the most popular in enzymological studies representing electron transfer from NADPH to the acceptor through both flavin cofactors 50. Many important proteins also accept electrons from CPR in their physiological functions, for example

112 113 114 cytochrome b5 , heme oxygenase or squalene epoxidase .

1.2.2 Cytochrome P450

The cytochrome P450 (CYP) superfamily is one of the largest, oldest and most diverse known superfamilies of enzymes. Enzymes from this group catalyse the oxidation of a number of organic substances. Cytochromes P450 take part in the bioconversion of xenobiotics (antibiotics) and the biosynthesis of compounds such as steroidal hormones, fatty acids, eicosanoids, fat soluble vitamins and bile acids.

CYPs are also the major enzymes involved in drug metabolism and bioactivation, conversion of alkanes, terpenes, aromatic compounds as well as degradation of herbicides and insecticides 115,116. The terminology P450 is uncommon for enzymes because it is not based on function, but describes the spectral properties of this b- type heme containing red pigments, which display an absorption band at 450 nm of their reduced carbon-monoxide bound form 116. This spectral feature is unusual for cytochromes and is induced by a cysteine thiolate group forming the fifth ligand of

Chapter1 – Introduction

the heme iron which classifies cytochrome P450 enzymes as hemethiolate proteins

116.

Cytochrome P450s in humans are mostly membrane-associated proteins 117 located either in the inner membrane of mitochondria or in the endoplasmic reticulum of cells 116. P450s metabolise a number of endogenous and exogenous chemicals, potentially toxic compounds and products of endogenous metabolism. P450s play a role in hormone synthesis and breakdown (for example estrogen and testosterone synthesis and metabolism) as well as cholesterol synthesis 116.

The simplified catalytic cycle for P450 reactions is shown in Figure 1.15. The system is thought to be dynamic, steps don’t always proceed in a linear order around the cycle and substrate can be bound and released at other steps along the cycle. Rate limiting steps in the cytochrome P450 reaction vary considerably, depending on the reaction and the in vitro setting. Heme iron reduction (reaction where first electron is donated), C-H bond breaking, step following product formation (it is thought that rate of this step is related to conformational change) and rate of transfer of the second electron have all been proposed as possible rate limiting steps (depending on the conditions of the reaction) 115.

Chapter1 – Introduction

- ROH Fe 3+ + RH 9 1 Fe3+ ROH Fe3+ RH NADPH-P450 reductaseoxy

e- 8 2 NADPH-P450 reductasered

X (FeOH)3+ R∙ Fe3+CORH Fe2+ RH

3 7 + O2 + RH

3+ 2+ (FeO) RH Fe -O 2 RH

6 4 NADPH-P450 reductaseoxy - - H2O e 2+ 5 2+ - Fe -OOH RH Fe -O2 RH red NADPH-P450 reductase + H+

Figure 1.15 Catalytic cycle for cytochrome P450. Reaction: (1) Substrate RH is bound to the enzyme inducing a change in the conformation of the active site (often displacing a water molecule from the distal axial coordination position of the heme iron); (2) Electron is transferred from NADPH cytochrome P450 reductase, ferric heme iron is reduced to its ferrous form; (3) O2 binds to the distal axial coordination position of the heme iron; (4) Second electron is transferred and dioxygen is reduced to a negatively charged peroxy group; (5) The peroxy group is rapidly protonated by local transfer from water or from surrounding amino-acid side-chains; (6) One molecule of water is released and a highly reactive iron(III)-oxo species is formed; (7-8) RH in the active site reacts with the highly reactive iron(III)-oxo species; (9) The oxidised substrate is released, enzyme returns to its initial state with a water molecule returning to occupy the distal coordination position of the heme iron. Reaction (X): if carbon monoxide (CO) binds to reduced cytochrome P450, the catalytic cycle is interrupted. This reaction yields the classic CO difference spectrum (inset) with a maximum at 450 nm. Figure was adapted from 115.

Chapter1 – Introduction

1.2.3 Flavocytochrome P450 BM3

Flavocytochrome P450 BM3, a fatty acid hydroxylase from the soil bacterium Bacillus megaterium, belongs to the P450 cytochrome superfamily of heme b-dependent monooxygenases 118 and it was the first enzyme class to be characterised in which a heme domain with cysteinate coordinated iron is linked to a diflavin reductase 119. Due to cytochrome P450 genes being designated CYP, P450

BM3 is also called CYP102A1 according to Nelson’s classification system. Using a number of different engineering strategies, P450 BM3 was redesigned to catalyse the oxidation of non-natural substrates as diverse as pharmaceuticals, terpenes and gaseous alkanes along with the NADPH-dependent hydroxylation of several long- chain fatty acids 118–121. Studies of the purified P450 BM3 (119 500 Da) revealed that the 55 kDa P450 heme domain was fused to a 65 kDa reductase domain containing two flavin groups, FAD and FMN, in an equimolar ratio 122–124 which makes P450

BM3 highly similar to nitric oxide synthase. The highest determined catalytic activity for a P450 monooxygenase (about 17000 min-1 with arachidonate 65) has been associated with the high efficiency of the electron transfer from NADPH cofactor, through the reductase domain and onto the P450 heme in BM3 125. P450

BM3 has been extensively studied as it has potential uses in a variety of biotechnological processes including drug metabolite production, the development of process-scale techniques, studying the general mechanistic aspects of P450 chemistry and for improving selectivity control to allow the synthesis of fine chemicals 118.

1.2.4 Flavocytochrome b2

Chapter1 – Introduction

Flavocytochromes b2 are located in yeast mitochondria and they belong to the

2-hydroxyacid dehydrogenase protein family. Flavocytochromes b2 are a well-known and studied protein group which include L-lactate dehydrogenases from

Saccharomyces cerevisiae, one of the most studied flavocytochromes b2.

Flavocytochrome b2 is a homotetramer. Each subunit has a molecular mass of about

57 kDa and two domains linked by a short “hinge” peptide; N-terminal cytochrome domain binding b-type heme group and the larger C-terminal flavodehydrogenase domain binding FMN. Flavocytochrome b2 catalyses the reaction where L-lactate is oxidised and pyruvate is produced and used in the Krebs cycle, with one ATP molecule being produced for every L-lactate used. The FMN reduction is the first

126–129 electron transfer step in the flavocytochrome b2 catalytic cycle , with a rate of electron transfer in the range 1500-2000 s-1, which is several times faster than the rate of catalytic turnover. The electron transfer from FMN semiquinone to heme, is the rate-limiting step in the flavocytochrome b2 catalytic cycle, being much slower than the FMN reduction step 125,130.

1.2.5 Cellobiose dehydrogenase

Cellobiose dehydrogenase (CDH) is an extracellular flavocytochrome formed by a number of wood-degrading fungi and also by various phytopathogenic fungi 131.

The complexity of woody tissues makes them resistant to microbial degradation, but also very energetically rewarding. That is why certain fungi use secreted enzymes, for example CDH, to degrade cellulose and hemicellulose to soluble carbohydrate components. CDH is a monomer with a molecular mass of about 90 kDa and two domains, one containing FAD and the other containing b-type heme as a cofactor.

During catalysis, FAD is reduced to FADH2 in a two-electron process just to be

Chapter1 – Introduction

reoxidised by the heme group in two single-electron steps 132. Once again, the FAD- heme electron transfer step is rate-limiting.

1.2.6 Flavohemoglobin

Flavohemoglobin is the most studied of the nitric oxide dioxygenases and it

- catalyses the conversion of nitric oxide (NO) to nitrate (NO3 ) It contains two domains: oxydoreductase FAD- and NAD(P)H-binding domain and N-terminal b- type heme-binding domain 133,134. The mechanism of this reaction involves the initial two-electron reduction of FAD by hydride transfer from NAD(P)H. Then a single

2+ electron is passed from the reduced FAD to the heme generating a FADsq-Fe state

3+ - of the enzyme which is now able to bind O2, leading to a ferric-superoxo (Fe -O2 )

134 complex formation . Next, the O2-bound enzyme reacts rapidly with NO forming a

- ferric-peroxynitrite intermediate which goes on to rearrange, releasing NO3 and

3+ 3+ generating the FADsq-Fe (FAD semiquionone-Fe ) complex. Later, a second electron is transferred from the FAD to the ferrous-heme which then goes through the oxygen-binding and peroxynitrite formation steps once more to complete the cycle. Rapid conversion of peroxynitrite to nitrate has been proposed as the last step of the flavohemoglobin catalytic cycle 135. Due to the potential danger of the overproduction of NO, it has been proposed that the main role of flavohemoglobins is to prevent NO toxicity (nitrosative stress), protect various bacteria, yeast and fungi against growth inhibition and damage mediated via NO 134,136,137 and to regulate NO signalling 138.

1.2.7 Methionine synthase reductase

Methionine synthase reductase (MSR), reactivates the catalytically inert cobalamin (II) form of methionine synthase which is necessary for folate and

Chapter1 – Introduction

methionine metabolism 139. Methionine synthase reductase is a soluble monomeric protein with molecular mass of 78 kDa and equimolar concentration of FAD and

FMN 140. Its reduction by NADPH results in the formation of an air stable semiquinone similar to that observed in CPR. The amino acid sequence of MSR is homologous at the N-terminus to the flavodoxin-like FMN-containing domain and linked by a hinge region to an NADPH-flavodoxin oxidoreductase-like FAD and a

NADPH-binding domain found in CPR 140.

Methionine is an essential amino acid coded by the single codon - AUG, also known as the initiation codon because it indicates the mRNA’s coding region where translation into protein begins. Methionine, in its activated form, S- adenosylmethionine, is the universal methyl donor in several cellular transmethylation reactions 139–141. Demethylation of methionine leads to the production of homocysteine which can be remethylated in a reaction catalysed by methionine synthase, a cobalamin-dependent enzyme. Cobalamin cofactor bound to methionine synthase acts as an intermediate methyl group carrier between methyltetrahydrofolate and homocysteine and plays an important role in the methyl transfer reaction 139. Whilst cycling between methylcobalamin(III) and cobalamin(I)

(highly reactive oxidation state), the cofactor may be oxidised to the cobalamin(II) inactivating enzyme at the same time. The reactivation of the enzyme activity is dependent on the reductive methylation of cobalamin(II) in a reaction in which S- adenosylmethionine provides the methyl group 142.

Megaloblastic anaemia, developmental delay, hyperhomocysteinemia and hypomethioninemia are all caused by a deficiency of methionine synthase activity

143,144. Additionally, elevated levels of homocysteine correlate with the incidence of cardiovascular diseases, neural tube defects and Alzheimer’s disease 66,145–147.

Chapter1 – Introduction

Figure 1.16 Role of methionine synthase reductase (MSR). Methyl groups of methyltetrahydrofolate (CH3-THF) are transferred to homocysteine via methionine synthase-methylcobalamin(III) as an intermediate methyl carrier. The reductive methylation of methionine synthase-cobalamin(II) to methionine synthase- methylcobalamin(III) is the mechanism by which S-adenosylmethionine, together with an electron from MSR, reactivates the enzyme.

Chapter1 – Introduction

1.2.8 Sulphite reductase

Sulphite reductase (SiR) is a soluble oligomeric protein consisting of two

148–150 subunits in a stoichiometry α8β4 . Subunit α (SiR-FP) is a 66 kDa FAD- and

FMN- containing flavoprotein similar to microsomal CPR 148,151 while subunit β

150,152–154 (SiR-HP) contains siroheme and an Fe4S4 cluster . Each subunit α has one

FAD and one FMN bound 152,153. SiR is an important enzyme in the metabolism of sulphur where it catalyses the reduction of sulphite to hydrogen sulphide and water

2- 155,156 (SO3 + electron donor → H2S + oxidised donor + 3H2O) . Reduction of sulphite to sulphide occurs at the heme catalytic site when the flavoprotein subunit of

SiR delivers 6 electrons from 3 mol of NADPH 157,158. Electrons are transferred from

NADPH to FAD, and on to FMN, from which they are transferred to the metal centre where they reduce the siroheme-bound sulphite.

1.2.9 Novel reductase 1

Novel reductase 1 (NR-1) is a diflavin reductase with molecular mass of 67 kDa and strong sequence similarities to CPR and NOS. The purified recombinant protein binds FMN, FAD and NADPH and exhibits a UV-spectrum with maximum absorbance at 380, 460 and 580 nm 57. Like CPR, the addition of NADPH under aerobic conditions will cause a decrease in the absorbance at 380 and 460 nm, an increase in the absorbance at 580 nm and the production of an air-stable semiquinone form 52,159,160. Purified NR-1 reduces cytochrome c and metabolises the one-electron acceptors doxorubicin and potassium ferricyanide 57. The amino acid sequence of

NR-1 is 41% similar to sequences of iNOS and 44% similar to the sequence of CPR

57. Novel reductase 1 has a domain organisation similar to other diflavin reductases with particularly strong sequence conservation in the regions known to be involved

Chapter1 – Introduction

in FMN, FAD and NADPH binding. The lack of amino-terminal region in NR-1, the hydrophobic 60-amino acid terminal anchor domain present in CPR which is involved in tethering the molecule to the endoplasmic reticulum, suggests a different cellular location for the enzyme 37,55,161,162. The exact biological role of NR-1 is unknown, but several immunochemical experiments have proved that recombinant

NR-1 is cytoplasmic and highly expressed in a panel of human cancer cell lines.

This indicates that this novel reductase may play an important role in the metabolic activation or deactivation of bioreductive anticancer drugs and other chemicals activated by one-electron reduction 163.

1.3 Nitric oxide and the importance of NOS

Nitric oxide (NO) was among the first gases to be discovered. It was discovered by Joseph Priestley in 1772, two years after his discovery of oxygen (O2).

For more than 200 years, this colourless and odourless gas was considered highly toxic. Several chemists died of toxic-shock syndrome after inhaling large amounts of

NO, either intentionally or accidentally. No one thought that small quantities of this lethal agent would play a crucial function in humans 164, however, over the last 30 years, a number of important discoveries have been made which have revealed the significant biological role played by nitric oxide. In 1980 Furchgott and Zawadzki reported that endothelial cells release a substance they called EDRF – endothelium- derived relaxing factor, which was responsible for the relaxation of the vascular smooth muscle. Seven years later Palmer, Ferrige and Moncada 165 suggested that

EDRF is in fact NO and that it is produced by the oxidation of L-arginine. This theory was then proven in 1992 by Malinski (NO was then named “the molecule of the year”) 166,167. The role of NO was firstly defined in the cardiovascular system, but as the research expanded, its ultimate function and mechanism had been identified in

Chapter1 – Introduction

all systems of the human body. It is thought that NO has a dual action in biological systems; in low concentrations, it regulates the physiological functions but at high concentrations, it may contribute to the pathogenic processes 168. Knowledge about the physiology of NO signalling cascades could have clinical applications, mainly considering therapeutic potential of NO donors, or antagonists of NO-synthases

164,169.

1.3.1 Nitric oxide – cardiovascular physiology concepts

In cardiovascular systems, vascular endothelial cells contain a Ca2+ dependent constitutive NOS - eNOS. eNOS synthesises NO in bursts lasting a few minutes to maintain the constant level of NO necessary to maintain normal stable blood pressure. NO synthesised by endothelial cells diffuses out in all directions.

About 70-90 % of released NO is washed away by blood where it is used to prevent platelet aggregation and the formation of blood clots. The remaining NO diffuses to the walls of arteries and veins (smooth muscle) 170. Then, NO is attached to the Fe2+ of the heme part of soluble guanylyl cyclase leading to the activation of the enzyme.

Guanylyl cyclase turns guanosine triphosphate (GTP) to cGMP thus activating cGMP-dependent protein kinases. cGMP-dependent protein kinase catalyses the phosphorylation of different proteins, activating or inhibiting some ion channels and regulating the activity of phosphodiesterases 171. The consequences of these processes are biological actions such as the relaxation of smooth muscle cells, cardial protection, neuronal plasticity and many more. Relaxation of the surrounding smooth muscles allows the blood vessel to dilate resulting in lowered blood pressure.

The cardiovascular system maintains a constant level of NO at a given blood flow.

When blood flow increases, the endothelium releases more NO to maintain its constant concentration in blood. If the necessary amount of NO is not produced, the

Chapter1 – Introduction

vascular muscles do not relax to the appropriate degree, blood pressure is increased and hypertension occurs 170,172,173. Nitroglycerin has been used for cardiac treatment for more than 100 years, but the background of its activity was not explained until the discovery of the biological role of NO. It was proven that nitroglycerin undergoes metabolic degradation in biological tissue, and one of the degradation products is NO. Thrombocytes in the blood can also release NO preventing blood coagulation, formation of thrombi and subsequent blockage of arteries 170. The pathology of this process leads to coronary thrombosis and is a major cause of stroke

169. Under heavy stress, exercise and other conditions when the heart has to pump blood quickly, NO is also produced by myocytes as well as by the endothelial cells.

Myocyte NO production is stimulated by release of adrenaline which is triggered by the nervous system. It is an important function of myocytes because the heart is much more sensitive to a low supply of NO than to a low supply of oxygen 174.

Surprisingly, cutting-off NO production/supply in the heart will terminate human life within 10-15 seconds, whilst it can last 5-7 minutes without an oxygen supply 170.

1.3.2 Nitric oxide and the nervous system

NO produced in certain neurons in the peripheral nervous system acts as a neurotransmitter and helps to control the cardiovascular, respiratory and digestive systems. In the central nervous system, NO acts as a neurotransmitter in the cerebellum, and is responsible for the interactions between endothelial cells and smooth-muscle cells in blood vessels. NO can also affect functions of the hippocampus, a part of the brain which is involved in learning and the formation of memory 175,176. NO is essential for establishing long-term potentiation memory, but there is no evidence that it affects short-term potentiation memory. Long-term potentiation is a persistent increase in synaptic strength following high-frequency

Chapter1 – Introduction

stimulation of a chemical synapse. As memories are thought to be encoded by modification of synaptic strength, long-term potentiation is considered one of the major cellular mechanisms related to learning and memory. Long-term potentiation memory is usually frequently used; in consequence the strength of the synaptic contact increases and NO acts as a retrograde messenger 177. The inhibition of NO released in the hippocampus prevents long-term potentiation and decreases learning capabilities 170,178,179. Under pathological conditions the vital role of nitric oxide in the brain can change diametrically. For example in 4-6 minutes after stroke, when the supply of blood to the brain is limited, a massive NO release is observed. Nitric oxide concentration in the brain can reach a level about 100 times higher than physiological NO concentration. This highly non-physiological concentration of NO usually leads to quasi-reversible or irreversible brain damage 170,180.

1.3.3 Role of NO in the immune system

As a part of the immune system, NO acts differently than in the cardiovascular or neuronal systems. Any kind of infection, including bacteria, viruses or cancer, will lead to the production of cytokines. Most cells in the human body can express NOS. Certain cytokines, produced by infected cells, will carry the message of the infectious state to the neighbouring cells and will give a signal to translate the DNA sequence and start NOS production. After translation is complete and all the prosthetic groups are present and in place, NOS will continuously produce extensive amounts of NO for several hours. The total amount of produced

NO will be therefore much higher than that produced by endothelial cells (few minutes) or neurons (few seconds). In rapidly dividing tumour cells or pathogens, the large amount of NO produced causes local DNA synthesis inhibition and a cytostatic effect on the proliferation of those cells. Very high NO concentration can

Chapter1 – Introduction

also inhibit ribonucleotide reductase activity slowing the proliferation of smooth muscles around major arteries and cardiac myocytes. It was proven than NO is not toxic even in higher concentrations as long as it fits in normal physiological limits.

This is a very important feature of NO because DNA synthesis is a fundamental step not only in the proliferation of tumour cells but also in the proliferation of normal cells. As a result of this limitation, nitric oxide is more likely to just limit and inhibit the proliferation of tumour cells rather than actually kill them 166.

1.3.4 Side effects of uncontrolled nitric oxide production

NO is essential to the everyday activities of many cells and tissues in the body, because of that any deviation in NO production in the body may be toxic and can cause many different diseases. In most life-threatening diseases like hypertension, atherosclerosis and diabetes, the net concentration of NO is lower than in a healthy system, which does not necessarily mean that the expression of NOS is lower 170.

Chapter1 – Introduction

Figure 1.17 Side effects of nitric oxide release. Figure shows the side effects of uncontrolled NO production, when NO concentration is too high or too low.

In cases of hypertension, enzyme expression is higher than in a healthy system but the net concentration of NO is lower due to the endothelial L-arginine concentration being too low. It has been shown that if there is a deficiency of endothelial L- arginine in the cell, NOS can donate an electron not only to L-arginine but also to its

-• other substrate, oxygen (O2), to produce superoxide (O2 ). Simultaneously produced

-• - NO and O2 will react very quickly to generate peroxynitrite (OONO ). In the presence of certain reactive centres, HOONO may undergo homolytic cleavage, generating the hydroxyl free radical (∙OH) and nitrogen dioxide free radical (∙NO2).

+ It may also undergo heterolytic cleavage to nitronium cation (NO2 ) and hydroxide

- + anion (OH ). Most of these cleavage products (∙OH, ∙NO2 radicals and NO2 ) belong to a group of the most reactive and the most damaging species in biological systems

Chapter1 – Introduction

and they may greatly contribute to heart and brain damage. The low concentration of

NO produced in the heart in the atherosclerotic cardiovascular system is thought to be the main reason for heart attacks 180.

Another serious disease associated with NO is septic shock. It is usually initially caused by a bacterial toxin entering the blood circulation through infections, wounds or surgical procedures. Every year about 50-70 million people suffer septic shock and over a half of them do not survive. During septic shock, the immune system tries to fight the infection by releasing non-physiologically high amounts of NO. These high NO concentrations dramatically decrease blood pressure, which is followed by failure of the vital organ system, especially the liver, kidney and heart. A massive release of NO is also observed during heart attack, brain stroke, and any other conditions which limit the supply of blood and oxygen (ischemia) to organs. If blood flow is not restored within several minutes, NOS starts to produce superoxide in

-• addition to NO. The concurrent release of both NO and O2 may lead to serious quasi-reversible or even irreversible damage to the brain or heart 170.

Nitric oxide also plays an important role in the regulation of both female and male reproductive systems. Similarly to other biological systems, NO is involved in physiological and pathological processes and knowledge about its activity may be clinically important. NO regulates follicle maturation and ovulation in ovaries, controls oestrogen levels in the body and is a very important regulator during pregnancy and childbirth 181. It is also thought that NO stimulates the movement of sperm and plays an important role in promoting fertilisation 182. Regulating the erection by non-noradrenergic, non-cholinergic (NANC) neurotransmission, nitric oxide also affects the male reproductive system. NO not only plays the role of mediator of the erection but it is also important for the spermatogenesis process

Chapter1 – Introduction

where it modulates blood flow, cell permeability and myofibroblast contractility in testis 183.

Knowledge about NOS activity and NO targets may have clinical applications related to the therapeutic potential of new NO donors or antagonists of NOS. It is also necessary to specifically understand the action of NO within the different systems in order to minimise the adverse effects and to maximise desirable effects of

NO in any pharmacological applications 169.

1.4 Overview of EPR spectroscopy

Electron Paramagnetic Resonance (EPR) spectroscopy, sometimes called

Electron Spin Resonance (ESR) spectroscopy, is a branch of magnetic resonance spectroscopy that uses microwave radiation to probe species with unpaired electrons, such as radicals, radical cations and anions and certain transition metals in the presence of a static magnetic field.

In many ways, the physical basis of EPR theory and methods are very similar to the related and better known technique of Nuclear Magnetic Resonance (NMR) spectroscopy. The main difference is that EPR focuses on the interactions between an external magnetic field and unpaired electrons of the system whereas NMR is focused on the nuclei of individual atoms. The electromagnetic radiation used in

NMR is typically confined to the radio frequency range between 300 and 1000 MHz, while EPR is usually performed using microwaves in the 3-400 GHz range. In EPR, the frequency is typically held constant, while the magnetic field intensity is varied, by contrast in continuous wave NMR, the magnetic field strength is constant and the radio frequency is changed. This reflects differences in the technology available as scanning microwave sources are very difficult to make. Furthermore, because of the

Chapter1 – Introduction

short relaxation times of electron spins in comparison to nuclei, EPR experiments must often be performed at very low temperatures (even 2-10 K), which requires liquid helium as a coolant. EPR is roughly 1000 times more sensitive than NMR spectroscopy due the higher frequency of electromagnetic radiation used in EPR in comparison to NMR. Despite higher sensitivity, EPR spectroscopy is less widely used than NMR because there are not that many existing stable molecules with unpaired electrons. Although EPR is limited to investigation of compounds and materials with unpaired electrons, it is the most direct and useful spectroscopic method for probing the properties of these specific systems. Another advantage is that sample preparation is simple and EPR does not cause destruction or activation in the sample. By probing the fundamental splitting of energy levels of spins with regard to their orientation in the external magnetic field, interactions between paramagnetic spin systems and their local environments can be detected and labelled species can be observed in situ either biologically or in chemical reactions 184.

1.4.1 Physical origin of the EPR signal

An electron is a negatively charged particle with certain mass (me) and which has two main kinds of movement. The first one is spinning around the nucleus, which brings orbital magnetic moment. The other one is spinning around its own axis, which creates spin magnetic moment. The magnetic moment of a molecule is mainly contributed by the unpaired electron’s spin magnetic moment:

√

where MS is the total spin angular moment; S is the spin quantum number and h is

Planck’s constant (6.62606957∙10-34 Js). Generally the secondary spin quantum

Chapter1 – Introduction

number (mS), a component of the total spin angular moment, can have 2S+1 different values ranging from –S to +S. In the z direction, for the single unpaired electron mS can only have two possible values: +1/2 and -1/2.

The spin magnetic moment of the electron (µS) is directly proportional to mS

(Equation 1.2). The negative sign in this equation is due to the fact that the spin magnetic moment of the electron is collinear but antiparallel to the spin angular

184 momentum of this electron. The quantity gSµB is called the magnetogyric ratio .

where gS is the free electron g-factor (the theoretical g-factor value of the free unpaired electron in vacuum is 2.00232); mS is the secondary spin quantum number

(±1/2 for a single unpaired electron) and µB is the Bohr magneton (the magnetic moment for one unit of quantum mechanical angular moment), which can be described with the Equation 1.3 below:

where e is the electron charge, h is Planck’s constant and me is the electron mass.

The Bohr magneton has the value of 9.274 009 68(20)∙10-24 JT-1.

The magnetic moment (µS) interacts with the applied magnetic field (B), and this interaction can be described by the energy below:

For single unpaired electron, there are only two possible energy states: +1/2 (excited state) and -1/2 (ground state):

Chapter1 – Introduction

The EPR spectroscopy requires a source of radiation, a sample, detector and an external magnetic field. The source of the microwaves generates radiation with energy:

where h is Planck’s constant and is the frequency of the radiation (in units of Hz or cycles per second) with its corresponding wavelength λ (in meters) according to the relation below:

where c is the speed of light, 2.99792∙108 ms-1.

As mentioned before, in EPR the frequency υ is constant and the magnetic field intensity (B) is variable. The energy values of the states are a function of the external magnetic field according to the Equations 1.4 and 1.5 and so is the energy difference

(ΔE):

( ) ⁄ ⁄

where the E+1/2 is the energy of the excited state;E-1/2 is the energy of the ground state; gS is the free electron g-factor; µB is the Bohr magneton and B is the intensity of the magnetic field. In the absence of the external magnetic field, E+1/2=E-1/2=0. In the presence of the external magnetic field, the Zeeman splitting effect occurs

(Figure 1.18) 184,185.

Chapter1 – Introduction

Figure 1.18 Zeeman splitting effect. Splitting of the energy levels of a free electron in an external magnetic field is called a Zeeman splitting effect and the energy levels are often referred to as Zeeman levels 185.

The concept of magnetic resonance is that the energy difference between the two states increases with increasing intensity of the magnetic field and is tuned to become equal to the energy of an electromagnetic wave with fixed frequency

(radiation) produced by the source. The resonance condition for a two-level system in EPR is when the energy produced by source (Equation 1.6) is equal to the energy difference between two states produced by the external magnet (Equation 1.8):

Chapter1 – Introduction

where gS is the free electron g-factor; µB is the Bohr magneton, B is the intensity of the magnetic field, h is Planck’s constant and υ is the frequency of the radiation.

From the relation shown in the Equation 1.9, it can be seen that there are infinite pairs of υ and B that fit this relationship. The magnetic field for resonance is not a unique fingerprint for the identification of a compound because spectra can be acquired at different microwave frequencies. The fingerprint of the molecule is the g-factor (g) which contains the chemical information that lies in the interaction between an electron and the electronic structure of the molecule. That is why the aim of the EPR experiment is determination and chemical interpretation of the value of g-factor in the external magnetic field. After the Equation 1.9 is rearranged, the following relation can be established:

[ ]

where, as before, g is the electron g-factor; µB is the Bohr magneton, B is the intensity of the magnetic field, h is Planck’s constant and υ is the frequency of the radiation. The g-factor is dimensionless and it is a constant of proportionality, whose value is the property of the electron in a certain environment. After the numeric values of h and µB have been inserted into the Equation 1.10, the g value can be given by Equation 1.11:

The free unpaired electron in a vacuum has a g-value ge=2.00232, however, when

184 the electron is in a certain environment, its g-factor may be different from ge .

Chapter1 – Introduction

Equation 1.9 is the fundamental equation of the EPR spectroscopy; it permits a large combination of frequency and magnetic field values, but the great majority of EPR measurements are made with the microwaves in the X-band region (~ 9 GHz).

During the experiment, the collection of the paramagnetic centres is exposed to microwaves at a fixed frequency. By increasing an external magnetic field, the gap between the mS=+1/2 and mS=-1/2 energy states is widened until it matches the energy of the microwaves (as represented by the Equation 1.9 and the double arrow in the Figure 1.18). At this point the unpaired electrons can move between their two energy spin states. Since there are typically more electrons in the lower energy state, there is a net absorption of energy, and it is this absorption that is monitored and converted into a spectrum. A simple absorption spectrum will appear similar to the spectrum A in Figure 1.19, however, a phase-sensitive detector is used in EPR spectrophotometers to convert the normal absorption signal into its first derivative.

Then the absorption spectrum is presented as its first derivative in the spectrum, which is similar to the spectrum B in Figure 1.19. Presenting first derivative is the most common way to record and present CW-EPR spectra. There are three main purposes of using derivative methods in EPR spectroscopy: a) spectral discrimination, as a qualitative fingerprinting technique to highlight small structural differences between nearly identical spectra; b) spectral resolution enhancement, as a technique for increasing the apparent resolution of overlapping spectral bands in order to more easily determine the number of bands and their wavelengths; c) quantitative analysis, as a technique for the correction for irrelevant background absorption and as a way to facilitate multicomponent analysis 186.

Chapter1 – Introduction

Signal

A A Absorbance

B First Derivative

Magnetic Field Strength [G]

Figure 1.19 Idealised EPR spectrum of a free electron. The spectrum shows the absorption signal and the collected first derivative 185.

All spectra collected for the purpose of this thesis were collected using spectrophotometer operating at the X-band (with the frequency in range 8-10 GHz).

The microwaves with frequencies in the X-band range are the most common frequencies used in EPR spectroscopy, because they provide the optimum between the sensitivity and the frequency of the resonance. At this point, most of the EPR studies start at X-band frequency and then they may be repeated at the frequencies that are a few times lower than or greater than X-band. Deviation from the X-band almost always means a loss of sensitivity and adds extra difficulties to the experiment. The added difficulty of the experiment outside the X-band is only

Chapter1 – Introduction

acceptable if extra information is obtainable in addition to that which X-band spectroscopy provides 187.

In the X-band, the resonance field for a free electron (ge=2.00232) is around 3400

Gauss. Higher fields are required for g lower than 2.00. Most of the biological studies will not require detection of g-values lower than approx. 1.2 corresponding to a field of approx. 5700 Gauss. The majority of X-band spectrophotometers have magnets that can be scanned from 0 to 6000 Gauss (0-0.6 Tesla) 184.

1.4.2 PELDOR spectroscopy

The pulsed electron electron double resonance method (PELDOR) also known as double electron-electron resonance (DEER) is an EPR spectroscopy technique which allows for the measurement of long-range distances, on the nanometer scale, between unpaired electrons. PELDOR spectroscopy uses two separate microwave frequencies to examine the dipolar coupling between two electron spins in order to make a distance measurement.

Opposite to the CW EPR technique, where the frequency of the radiation is kept constant when magnetic field is scanned to detect any resonance in the sample, in the pulsed EPR technique short and intense microwave pulses, consisting of a finite bandwidth of frequencies, are applied, the signals coming from the sample are digitised, and Fourier Transformation is performed to obtain the EPR spectrum in the frequency domain 188.

All electrons have spin (see above), which gives rise to a magnetic moment. When an electron spin is placed in a magnetic field, the force is being used on the spin, causing its magnetic moment to precess around the strong z axis. The angular

Chapter1 – Introduction

frequency of this precession is called the Larmor frequency (ωL) and is related to the magnetic field (B) and gyromagnetic ratio (ϒ) by (Equation 1.12):

Each of the electron spins precessing around the z axis will assume one of the quantum states and align either parallel (spin quantum number ms=-1/2) or antiparallel (ms=+1/2) to the magnetic field (Figure 1.20 A).

The parallel state has lower energy, and the antiparallel state has higher energy, and according to the Maxwell-Boltzmann distribution there are more electrons in the lower energy state than in the higher. Because of the magnetisation being a vector sum of all the individual magnetic moments in the sample, for a large enough number of spins the resultant net magnetisation will be along +z axis and exactly

188,189 parallel to the external magnetic field B0 (Figure 1.20 B) .

Chapter1 – Introduction

A B A A

Figure 1.20 A collection of spins in an external magnetic field, B0. Figure A presents a collection of spins aligned either parallel or antiparallel to the external magnetic field, B0. Figure B shows a small net magnetisation along +z axis, M, 190 positioned parallel to the external magnetic field, B0 .

As the B0 static magnetic field is used to establish the equilibrium condition, an additional external magnetic field B1 is needed to perform a pulsed EPR experiment.

Additionally the detection coils must lie in the x-y plane so that the magnetic field from the sample after perturbation from equilibrium is not masked by the larger external B0 field. Only transverse magnetisation, or magnetisation having an x-y component, will give a signal. Usually B1 is much weaker than B0 and the secret of successful pulsed EPR experiments lies in the manipulation of the magnetisation by

B1 pulses that have specific tip angles and then the detection of the magnetic behaviour during its return to equilibrium. Pulses are usually named by their tip

Chapter1 – Introduction

angles, and the most common tip angles are π/2 (90 degrees) and π (180 degrees). A

π/2 pulse is called a saturating pulse because it will tip the magnetisation into the x-y plane zeroing the magnetisation along the z axis. A π pulse is called an inversion pulse because it tips the magnetisation 180 degrees (it flips it over), exchanging the populations of the quantum states (Figure 1.21) 188,190.

Figure 1.21 Common pulses and the populations of quantum states in comparison to the thermal equilibrium.

Because the electron spins interact with their surroundings, after tipping the magnetisation into the x-y plane with a π/2 pulse the spins will establish phase coherence and the transverse magnetisation will decay away and return to equilibrium through the relaxation process after the pulse ends. During relaxation, the magnetisation rotates in the x-y plane at the Larmor frequency (Equation 1.12)

Chapter1 – Introduction

until it reaches equilibrium. This precessing magnetisation generates currents and voltages in the detection coil (resonator) that are detected as an EPR signal. This is called a Free Induction Decay (FID) and it is then Fourier transformed in order to obtain a frequency-domain spectrum 188.

After an intense microwave pulse, the magnetisation will interact with its surroundings and return to equilibrium (relaxation), described by two time constants,

T1 and T2. The spin-lattice relaxation time, T1, describes how quickly the magnetisation recovers its longitudinal component along the z axis (it is the amount of time in which the energy absorbed from the pulse is dissipated to the lattice as the system returns to equilibrium). The T2 is the transverse relaxation time, and it describes how quickly the net magnetisation dissipates in the x-y plane. Because the detection coil lies in the x-y plane, the transverse relaxation usually determines the length of time that is needed to observe a signal. A single π/2 pulse produces a FID, and the Fourier transform of the FID will give the frequency-domain EPR spectrum.

Immediately after the pulsed is turned off, relaxation begins and the signal starts to disappear. It would be ideal to start collecting the signal immediately after the pulse is turned off; however the spectrometer cannot read the signal directly after the microwave pulse because the receiver would be destroyed by the high power. Also the signal would be masked as it is in the nW range while the applied pulses are about 1 kW. Because of that, there is a lag (dead time) between the end of the pulse and when the signal can be measured. The dead time is typically around 70-80 ns and it depends on the quality factor of the resonator. Because an EPR spectrum is usually inhomogeneously broadened, the disappeared signal can be restored with an additional pulse to produce echo. If the second pulse is applied at a time t after dephasing begins, the echo maximum will occur at time t after the second pulse. This

Chapter1 – Introduction

second pulse produces a signal after the dead time, and the echo shape resembles two back-to-back FIDs 188,189.

Figure 1.22 The magnetic behaviour during a Hahn echo as a function of pulse position 191. From left to right, a spin (represented by arrow) is tipped into and rotated to the x-y plane with π/2 pulse producing a Free Induction Decay (FID). External magnetic field then cause the spin to precess around the z-axis (‘free precession’). By inverting the spin 180o around the y-axis in the middle of the free precession period with a π pulse, the original spin orientation is recovered, allowing another spin manipulation to occur. An accurate echo (Hahn echo), caused by π pulse, is a reversed FID followed by a normal FID, which can be Fourier transformed to obtain an EPR spectrum. The longer the time between the pulses becomes, the smaller echo will be due to spin relaxation.

In the four-pulse PELDOR, used in this thesis, the EPR signal is a modulation of echo intensity of one spin population as the pump pulse timing of a second spin population is changed. The timing of the three observed frequency pulses remains constant, however the pump frequency pulse starts at a time slightly before the first

Hahn echo, and the timing of its application is varied over several repetitions of the

PELDOR pulse sequence. The resulting oscillations in the integrated echo intensity occur at the dipolar interaction frequency. Because the dipolar frequency is inversely proportional to the cube of the distances between spins, longer distances are related

Chapter1 – Introduction

to lower frequency oscillations. Because of that, the range of obtainable distances with the PELDOR spectroscopy is approx. 2-8 nm 188.

Figure 1.23 The four-pulse PELDOR sequence. The mw detection subsequence at ωA is applied to the detection spins (spins A), when the pump pulse at frequency ωB is applied to the pump spins (spins B). The refocused echo from the detection spins at time 2(τ1+ τ2) is observed as a function of time (t). t0 is a spectrophotometer’s dead time 192.

During a four-pulse PELDOR experiment (Figure 1.23), initial π/2 pulse tips the magnetisation into the x-y plane. The A spins precess at the frequency ωA (but appear stationary in the rotating frame). After the pulse ends, the field inhomogeneities and different resonance fields dephase the spins. Because of the dipolar interactions, the angular frequency is changed to the value of ±1/2ωAB, depending on the quantum state of the coupled B spin. The π pulse reverses the

Chapter1 – Introduction

dephasing spins and refocuses the A spins to ωA resulting in an undetected Hahn echo. When the spins go through the relaxation process again, the dipolar frequency is extracted by the application of an inversion “pump” pulse to the B spins, which changes the dipolar contribution experienced by the A spins from +1/2ωAB to -

1/2ωAB, and vice versa. The change of the dipolar contribution causes these spins to acquire a lag phase, which affects their ability to be refocused by the final π pulse applied at the observed frequency, because of that the refocused echo intensity is also affected. The modulation of the four-pulse PELDOR echo can be obtained from the relation (Equation 1.13):

( )

where υDD is the dipolar coupling, t is the timing of the ‘fourth’ pulse, and τ1 is the time between the π/2 pulse and first π pulse.

When the modulation pattern of the refocused echo is obtained it is then Fourier transformed in order to obtain a frequency-domain spectrum. The Fourier transform is used to transform the function of time into the function of frequency so that the dipolar coupling values can be obtained and the distances between two spins can be determined. PELDOR data analysis provides information about one or more probability distributions corresponding to the interspin distances.

PELDOR spectroscopy can observe and resolve a dipolar interaction in order to measure distances between two electron spins that are far enough apart. Two spins in a molecule do not precess independently. If one spin can have different energy states depending on which state a nearby spin is in, these two spins are coupled. Weak couplings between two electron spins can be used to determine distances between paramagnetic centres in the nanometer range. This is particularly important for

Chapter1 – Introduction

disordered systems in the solid state, where the measurement of dipolar couplings is one of the very few methods which access the distance range from 2-8 nm.

Dipolar coupling forces each spin to generate a magnetic field that is orientated parallel to the nuclear spin vector. The strength of the dipolar coupling depends strongly on the spin-spin distance r-3 as well as the angle θ between the axis connecting the two spins and the external magnetic field (Figure 1.24) (Equation

1.14):

where g1 and g2 are the g-factor values for two electrons, µB is the Bohr magneton,

µ0 is the magnetic moment.

Figure 1.24 The dipolar coupling dependence on the interspin distance (r) and the angle (θ) to the external magnetic field.

Chapter1 – Introduction

Rearranging Equation 1.14 and inserting values for the numerical constants yields the following (Equation 1.15):

√

where νDD is in MHz and r is in nm. Therefore, using Equation 1.15, the distance between two unpaired electrons can be determined from the pulsed EPR spectra.

1.5 Aims of the project

In this thesis rat neuronal full length nitric oxide synthase (FL nNOS) and its cleavable reductase domain (nNOSred) are studied. The overall aim of the work is to obtain a more detailed understanding of the thermodynamic and kinetic properties of nNOS along with the conformational energy landscape changes possibly occurring in nNOS during catalysis.

The cytochrome c activity of nNOSred and FL nNOS (both WT and R1400E) is used to obtain kinetic parameters for NADPH, in the presence and absence of calmodulin

(CaM), as well as to study the internal electron transfer from NADPH through FAD,

FMN to cytochrome c. These studies enable the observation of how the presence of heme and nNOSoxy in FL nNOS is affecting the catalytic efficiency of nNOSred and

FL nNOS, as well as the electron transfer from NADPH through the nNOSred to the artificial acceptor (cytochrome c).

During this project, electron paramagnetic resonance (EPR) spectroscopy is used to identify a radical signal in the enzyme, such as a flavin in its semiquinone state. The flavin cofactors found in nNOS can be titrated to their unpaired-electron

(semiquinone) forms and thus used to generate a paramagnetic signal that can be

Chapter1 – Introduction

followed by EPR spectroscopy. In addition, EPR is also used to gain an understanding of how CaM and ligands (NADP+, ADP) can affect the redox potentials of flavin cofactors and/or the semiquinone formation. This work is also undertaken in this thesis to develop a better understanding of the role of NADPH and

CaM in the control of electron transfer and reactivity in NOS. In the absence of crystallographic structural data, it is also important to experimentally determine possible conformational changes and distances between flavins in nNOS. To do this, pulsed electron-electron double resonance (PELDOR) is used to provide the interflavin distance distributions measured for either nNOSred or FL nNOS.

Determined distances between flavins are used for better characterisation of internal electron transfer and its regulation, and to address conformational equilibrium in nNOS.

With chemical reactions often being highly pressure dependent, a perturbation of the elementary steps of electron transfer within nNOS by pressure therefore offers the possibility for a detailed characterisation of the enzyme mechanism. High hydrostatic pressure has not been extensively used in nitric oxide synthase in the past partly because of the complexity of the reaction catalysed by nNOS. Another goal of the project is to evaluate the effect of high hydrostatic pressure on the internal electron transfer, flavin reduction, NADPH oxidation and NO formation rates using a high pressure stopped flow approach. As NOS is thought to be very flexible, this work is undertaken to develop a better understanding of the possible domain movement and flexibility perturbations which may occur after applying pressure to the protein. The effects of high pressure are studied on WT nNOS along with the

R1400E key variant (an Arg1400 mutation in the NADPH-binding site mentioned in

Chapter 1, Section 1.1.3.2) which is thought to repel NADPH binding, destabilise

Chapter1 – Introduction

and disorientate the C-terminal tail but still promote the internal electron transfer.

The aim is also to observe which part of nNOS (nNOSred or nNOSoxy) is affected most by the introduction of the R1400E mutation, as well as to see if the rate limiting step of nNOS catalysis changes when Arg1400 is replaced with Glu.

Chapter 2 – Materials and methods

Chapter 2 Materials and methods

Chapter 2 – Materials and methods

Chapter 2 – Materials and Methods

2.1 Materials

All chemicals used were of laboratory reagent grade or of equivalent purity, with the exception of the methanol and water used in FAD and FMN identification and quantification using HPLC, which were of HPLC grade (Sigma Aldrich).

BL21(DE3) Singles, NovaBlue Singles E. coli competent cells and SOC Broth were from Novagen.

Tetrahydrobiopterin (H4B) was from Enzo Life Sciences. Calcium chloride, diaminoethane-tetraacetic acid (EDTA), dipotassium hydrogen orthophosphate anhydrous, ethanol, glycerol, hydrochloric acid, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), magnesium chloride, potassium chloride, potassium dihydrogen orthophosphate, sodium chloride, sodium dodecyl sulphate

(SDS), sodium dithionite and sodium hydroxide were from Fisher Scientific. 5- aminolevulinic acid hydrochloride (δ-ALA), ampicillin sodium, Auto Induction TB base including trace elements medium, carbenicillin disodium, dithiothreitol (DTT), isopropyl β-D-1-thiogalactopyranoside; dioxan free (IPTG), LB-Agar broth, yeast extract, 2-amino-2-hydroxymethylpropane-1,3-diol (Tris) and tryptone were from

Formedium Ltd. Nicotinamide adenine dinucleotide phosphate-oxidised form

(NADP+) and nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) were from Melford Laboratories Ltd. Eco RI-HF, Hin dIII, Nde I, restriction enzyme buffers and Sac I-HF were from New England BioLabsInc. Adenosine 5'- diphosphate monosodium salt trihydrate (5'-ADP), adenosine 2′(3′)-monophosphate mixed isomers (2′(3′)-AMP), benzyl viologen dichloride (BV), catalase, calmodulin

Chapter 2 – Materials and methods bovine, flavin adenine dinucleotide disodium salt hydrate (FAD), riboflavin 5′- monophosphate sodium salt hydrate (FMN), 2-Hydroxy-1,4-naphthoquinone (HNQ), human haemoglobin, imidazole, L-ascorbic acid, L-arginine monohydrochloride,, methyl viologen dichloride hydrate (MV), cytochrome c from equine heart and phenazine methosulfate (PMS) were from Sigma-Aldrich.

Q-Sepharose resin was from Amersham PharmaciaBiotech. Prestained Protein

Molecular Weight Marker and GeneRuler 1 kb DNA ladder were from Fermentas.

2’5’-adenosine diphosphate (ADP)-Sepharose affinity resin was from GE Healthcare

Bio-Sciences AB. Ni-IDA Metal Chelate resin was from Generon. InstantBlue

PAGE gel stain was from Novexin Ltd. Complete EDTA-free, protease inhibitor cocktail tablets were from Roche Diagnostics GmbH. QIAprep Spin

Miniprep/Midiprep/Maxiprep Kits were from QIAGEN.

EPR tubes were from Wilmad-LabGlass. Dialysis membranes were from Medicell

International Ltd. Membrane discs for Amicon pressure concentrators were from

Generon Ltd. Vivaspin columns (30000 kDa MW cut off) were from Santorius

Stedim Biotech. Carbon monoxide, helium and oxygen were from BOC. Glass distilled, deionised water, distilled water, Eppendorfs, tips, and Falcon tubes were used throughout.

Terrific Broth (TB) medium consisted of (per litre): 12 g tryptone, 24 g yeast extract,

4 mL glycerol, made up to 900 mL with deionised water. Upon cooling of the autoclaved medium (121 °C for 20 min), 100 mL of sterile solution of 0.17 M potassium dihydrogen orthophosphate and 0.72 M dipotassium hydrogen orthophosphate was added. Luria-Bertani (LB) medium consisted of (per litre): 10 g tryptone, 5 g yeast extract and 10 g sodium chloride. LB-Agar medium consisted of

Chapter 2 – Materials and methods

(per litre): 10 g tryptone, 5 g yeast extract, 10 g sodium chloride and 15 g agar. For optimal cell growth and yield all media were prepared using complete mixture from

Formedium and autoclaved before use.

The plasmid pCRNNR encoding both WT and R1400E variant rat nNOSred co- expressing CaM (WT nNOSred, R1400E nNOSred) was a gift from Dr Anne Marie

Quinn (The University of Manchester). The plasmid pCWORI encoding full length

WT rat nNOS (FL WT nNOS-, calmodulin-free form) was a gift from Dr Dennis J.

Stuehr (Cleveland Clinic, Cleveland, Ohio, US). FL R1400E rat nNOS was prepared using site-directed mutagenesis, which is detailed in Chapter 2, Section 2.2.1.1.

2.2 Methods

2.2.1 Recombinant DNA techniques

2.2.1.1 Site-directed mutagenesis of FL R1400E nNOS

The R1400E variant was constructed by site-directed mutagenesis technique using pCWORI FL WT nNOS as a template and the primers were designed in forward and reverse direction (Table 2.1). Linear amplification reaction, for the generation of the R1400E variant, was carried out in a 50 µL reaction volume with 3

µL of QuickSolution, 125 ng of the required oligonucleotide-primers, 200 µM dNTPs, 1 x reaction buffer and 10 ng backbone dsDNA template and 1 µL of

PfuTurbo DNA polymerase (2.5 U/µL). The conditions used for the linear amplification reaction are shown in Table 2.2.

Chapter 2 – Materials and methods

Table 2.1 Oligonucleotides used for the generation of the R1400E mutation in FL rat nNOS.

Direction Primer sequence (5’ → 3’) Forward T GGA GTC ACC CTC GAA ACG TAT GAA GTG ACC AAC CGC C

Reverse G GCG GTT GGT CAC TTC ATA CGT TTC GAG GGT GAC TCC A

Table 2.2 Linear amplification reaction conditions used for the generation of the R1400E mutation in FL rat nNOS.

Segment Cycles Temperature Time 1 1 95 °C 1 min 2 18 95 °C 50 sec 60 °C 50 sec 68 °C 8 min 10 sec 3 1 68 °C 7 min

Parental supercoiled dsDNA was removed by the addition of 1 µL of Dpn I (10

U/µL), gentle mixing and incubating the sample for 1 hour at 37 °C. 2 µL of the Dpn

I-treated DNA of R1400E was transformed into NovaBlue E. coli competent cells

(See section 2.2.1.2.1), isolated by mini-prep (Section 2.2.1.3) and verified by automated DNA sequencing (MWG Eurofins).

Chapter 2 – Materials and methods

2.2.1.2 DNA transformation of cells

2.2.1.2.1 NovaBlue E. coli competent cells

50 µL of NovaBlue E. coli competent cells were thawed on ice and 50-100 ng of plasmid DNA (about 1-2 µL) added, cells were incubated on ice for a further 5 minutes. The cells were heat shocked in a 42 °C water bath for exactly 30 seconds and then gently returned to ice for 2 minutes. Ten reaction volumes (10 x 50 µL =

500 µL) of SOC medium were added and cells were plated out onto LB-Agar plates containing 100 µg/mL ampicillin or 100 µg/mL carbenicillin, inverted and incubated at 37 °C overnight.

2.2.1.2.2 BL21(DE3) E. coli competent cells

50 µL of BL21(DE3) E. coli competent cells were thawed on ice prior to the addition and gentle mixing of 50-100 ng of plasmid DNA (about 1-2 µL). After that cells were left on ice for a further 30 minutes. Then cells were heat shocked in a 42

°C water bath for exactly 30 seconds and returned to ice for 2 minutes. Ten cell volumes (10 x 50 µL = 500 µL) of SOC medium were added and cells were incubated with shaking at 37 °C for one hour. After that 100 µL of those cells were plated onto LB-Agar plates, containing 100 µg/mL ampicillin or 100 µg/mL carbenicillin, inverted and incubated at 37 °C overnight.

2.2.1.3 Preparation of DNA

For plasmid DNA isolation, a single transformed colony of NovaBlue XL1

SinglesTM Competent Cells E. coli (Section 2.2.1.2.1) was picked from a LB-agar plate and used to inoculate 5 mL LB broth containing 100 µg/mL ampicillin.

Cultures were grown overnight at 37 °C with vigorous shaking. Preparation of the

Chapter 2 – Materials and methods plasmid DNA from these cells was carried out using the QIAprep Spin MiniPrep Kit using the supplied reagents and following the manufacturer’s instructions throughout.

QIAprep Spin MiniPrep Kit was designed for isolation of up to 20 µg high-purity plasmid. In cases where larger amounts of DNA were necessary, QIAprep Spin

MidiPrep Kit and QIAprep Spin MaxiPrep Kit, using the supplied reagents and following the manufacturer’s instructions throughout, were used.

Restriction digest of the plasmid DNA and agarose gel electrophoresis were carried out to verify the presence and correct identity of the resultant plasmid DNA.

2.2.1.4 Enzymic DNA modifications

2.2.1.4.1 Restriction digestion of pCRNNR

Restriction digests of the plasmid DNA (pCRNNR) were carried out in a total volume of 10 µL, containing 2.5 µL of 6x restriction buffer, 1 µg DNA and 10 units of enzyme/s (Eco RI, Sac I or a mix of Eco RI + Sac I). The volume was made up to 10 µL with distilled water. Reactions were carried out at the optimum temperature for the enzyme/s, typically 37 °C, for 2 hours. Agarose gel electrophoresis was carried out to confirm the results of digestion.

2.2.1.4.2 Restriction digestion of pCWORI

Restriction digests of the plasmid DNA (pCWORI) were carried out in a total volume of 10 µL, containing 1 µL of NEBuffer2, 2.5 µg of DNA and 10 units of enzyme/s (Nde I, Hin dIII or a mix of Nde I + Hin dIII, typically 0.5 µL). The volume was made up to 10 µL with distilled water. Reactions were carried out at the

Chapter 2 – Materials and methods optimum temperature for the enzyme/s, typically 37 °C, for 2 hours. Agarose gel electrophoresis was carried out to verify the results of digestion.

2.2.1.5 Agarose gel electrophoresis

Agarose gels (0.8 % w/v; 11.5 cm x 12.5 cm) containing 0.6 µg/mL ethidium bromide were cast. 10 µL DNA samples were mixed with 2 µL loading dye (10 mM

Tris/HCl, pH 7.5, 50 mM EDTA, 30 % v/v glycerol, 0.25 % bromophenol blue) prior to loading on a gel. A 1 kb DNA ladder was used giving the following band sizes (in kb): 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, and 10.0. The gel was run in 1x

TAE Buffer (40 mM Tris acetate, 10 mM EDTA) until the dye front had run two thirds of the length of the gel (typically 80 volts for 60-70 minutes). The resulting bands were visualised by exposing the gel to long wavelength UV radiation.

2.2.2 Isolation of nNOS proteins

2.2.2.1 nNOSred expression and purification

For nNOSred protein expression TB media was used (Section 2.1.1). Plasmid pCRNNR was transformed into BL21(DE3) E. coli competent cells (Section

2.2.1.2.2) and single colonies were picked to inoculate 5 mL LB broth supplemented with 100 µg/mL ampicillin. These were incubated with shaking at 200 rpm for 8-10 hours at 37 °C and used to inoculate 2 x 250 mL of LB media, each supplemented with 100 µg/mL ampicillin (or 100 µg/mL carbenicillin). After no more than 12 hours at 37 °C with shaking at 200 rpm, 5 mL of these were used to inoculate 2-litre flasks each containing 500 mL TB media supplemented with 100 µg/mL carbenicillin. The cells were grown at 37 °C until they reached an OD600 of 0.4, and were then grown at the decreased temperature of 28 °C until they reached an OD600

Chapter 2 – Materials and methods of 0.8- 1.0. nNOSred expression was induced by the addition of 1 mM isopropyl-thio-

β-D-galactoside (IPTG) and cells were then grown for a further ≈24 hours at 25 °C.

Cells were harvested by centrifuging at 7000 rpm (rotor JLA 8.1; centrifuge

Beckman Coulter, Avanti Centrifuges, J-26 XP) for 15 minutes. The cell pellet was kept frozen at -20 °C until further use.

Purification procedures were based on previous methods 193,194. After being frozen at

-20 °C cells were allowed to thaw overnight at 4 °C. The freeze-thaw cycle helped to fracture the cells prior to sonication. Cells were resuspended in lysis buffer (50 mM

TM Tris/HCl, pH 7.4, 1 mM CaCl2 and 10 % (v/v) glycerol), containing Complete

EDTA-free protease inhibitor tablets (1 per ≈200 mL of resuspended cell pellet).

When purifying nNOSred it was necessary to include CaCl2 in the buffer and to use

EDTA-free tablets to ensure that Ca2+-CaM complex remained bound to the enzyme

194. Approximately 3 cell pellet volumes of lysis buffer were added during resuspension (≈200 mL) as this amount of buffer was found to increase the yield obtained from the cell pellet. The cells were left to stir at 4 °C for 15-20 minutes to ensure complete resuspension. Cells were then sonicated on ice at a pulsed setting of

50 % power for 20 seconds. This was repeated 20-25 times with a gap of 59 seconds between pulses to allow the sonicator to cool. The extract was then clarified by high- speed centrifugation at 19000 rpm (rotor JA 25.50; centrifuge Beckman Coulter,

Avanti Centrifuges, J-26 XP) for 45 min at 4 °C.

All chromatography was performed at 4 °C. The cell extract (200 mL) was first loaded onto a 2’5’- adenosine diphosphate (ADP) - Sepharose affinity column (15 mL volume), which was equilibrated with lysis buffer. Unbound protein was removed by washing with lysis buffer, typically 5 column resin volumes. The bound protein was eluted with about 20 mL of the lysis buffer containing 0.5 M NaCl and

Chapter 2 – Materials and methods

25 mM 2’- & 3’-adenosine monophosphate (2’, 3’- AMP). Fractions were assayed for flavin content by using UV-visible spectrophotometry and scanning the sample in the wavelength range 300-800 nm. In order to remove all tightly bound 2’, 3’- AMP and excess NaCl, the protein was dialysed overnight against 4 L of lysis buffer at 4

°C. The protein (≈20 mL) was then loaded onto a Q-Sepharose anion exchange column pre-equilibrated with lysis buffer (≈25 mL volume). The column was washed with 5 column volumes of lysis buffer and the bound protein was eluted with a salt linear gradient of 0 to 700 mM KCl (200 mL). All the fractions (2 mL) were collected and monitored at 456 nm wavelength using UV-vis spectrophotometer. The samples which absorbed at 456 nm were expected to contain nNOSred so they were separated and chosen for further dialysis. The protein was again dialysed overnight against 4 L of lysis buffer at 4 °C. The sample was then concentrated to ≈400 µM using Amicon and VivaSpin protein concentrators (30 000 kDa MW cut off), before being flash frozen in liquid nitrogen in lysis buffer containing 20-30 % (v/v) glycerol and stored at -80 °C.

2.2.2.2 FL nNOS expression and purification

Plasmid pCWORI was transformed into BL21(DE3) E. coli competent cells

(Section 2.2.1.2.2) and a single colony was picked to inoculate 5 mL LB medium cultures supplemented with 100 µg/mL ampicillin. These were incubated with shaking at 200 rpm for 8-10 hours at 37 °C and used to inoculate 250 mL of LB medium each supplemented with 100 µg/mL ampicillin (or 100 µg/mL carbenicillin). After no more than 12 hours at 37 °C with shaking at 200 rpm, these were used to inoculate 2-litre flasks each containing 500 mL TB medium supplemented with 100 µg/mL carbenicillin. Cells were grown at 37 °C until they reached an OD600 of 0.4 and were then grown at the decreased temperature of 28 °C

Chapter 2 – Materials and methods until they reached an OD600 of 0.8- 1.0. FL nNOS expression was induced by the addition of 1 mM isopropyl-thio-β-D-galactoside (IPTG) and supplemented by the addition of 0.45 mM δ-aminolevulinic acid (δ-ALA). Cells were then grown for a further ≈48 hours at 25 °C. Cells were harvested by centrifugation at 7000 rpm (rotor

JLA 8.1; centrifuge Beckman Coulter, Avanti Centrifuges, J-26 XP) for 15 minutes.

The cell pellet was kept frozen at -20 °C.

After being frozen at -20 °C cells were allowed to thaw overnight at 4 °C. The freeze-thaw cycle helped to fracture the cells prior to sonication. Cells were resuspended in lysis buffer (40 mM HEPES, pH 7.6 at 4 °C, containing 150 mM

NaCl, 1 mM L-arginine, 10 µM H4B, 3 mM ascorbic acid and 10 % (v/v) glycerol) supplemented with CompleteTM EDTA-free protease inhibitor tablets (1 per ≈200 mL of resuspended cell pellet), 10 µg/mL DNaseI, 10 mM MgCl2 and 5 mg/mL lysozyme. When purifying FL nNOS it was necessary to include 1 mM CaCl2 in all buffers and to use EDTA-free tablets to ensure that Ca2+-CaM complex remained bound to the enzyme 194. Approximately 3 cell pellet volumes of lysis buffer were added during resuspension (≈200 mL) as this volume of buffer was found to increase the yield obtained from the cell pellet. The cells were left to stir at 4 °C for 15-20 minutes to ensure complete resuspension. They were then placed on ice and sonicated at a pulsed setting of 40 % power for 10 seconds. This was repeated 35 -

40 times with a gap of 50 seconds left between pulses to allow the sonicator to cool.

The extract was then clarified by high-speed centrifuging at 19000 rpm (rotor JA

25.50; centrifuge Beckman Coulter, Avanti Centrifuges, J-26 XP) for 45 min at 4 °C.

All chromatography steps were performed at 4 °C. The cell extract (≈200 mL) was first loaded onto a Ni-IDA Metal Chelate resin column (≈25 mL) which was equilibrated with lysis buffer. Unbound protein was removed by washing with lysis

Chapter 2 – Materials and methods buffer, typically 5 column volumes. After that the column was washed with 5 column volumes of lysis buffer containing 35 mM imidazole, to remove weakly bound contaminating proteins. The bound protein of interest was eluted with lysis buffer containing 250 mM imidazole (≈50 mL). Fractions were assayed by UV- visible spectrophotometry for flavin content. In order to remove all tightly bound imidazole and excess NaCl, the protein was dialysed at 4 °C overnight against 4 L of dialysis buffer (40 mM HEPES, pH 7.6 at 4 °C, containing 1 mM L-arginine, 10 µM

H4B, 25 mM DTT, 3 mM ascorbic acid and 10 % (v/v) glycerol). The protein was then loaded onto a 2’, 5’-adenosine diphosphate (ADP)-Sepharose affinity column

(≈20 mL), which was pre-equilibrated with lysis buffer. Unbound protein was removed by washing with lysis buffer, typically 5 column volumes. The bound protein was eluted with lysis buffer (25 mL) containing 0.5 M NaCl and 25 mM 2’-

& 3’-adenosine monophosphate (AMP). In order to remove all tightly bound AMP and excess NaCl, the protein was dialysed overnight against 4 L of dialysis buffer at

4 °C. The sample was then concentrated to ≈400 µM using VivaSpin protein concentrators (30 000 kDa MW cut off), before being flash frozen in liquid nitrogen in lysis buffer containing 20-30 % (v/v) glycerol and stored at -80 °C.

Chapter 2 – Materials and methods

2.2.3 Methods of protein analysis

2.2.3.1 Determination of protein concentration

The concentration of proteins was calculated spectroscopically using the

Beer-Lambert law.

2.2.3.1.1 nNOSred

Absorbance measurements were performed using a Cary UV-50 Bio UV- visible spectrophotometer at 25 °C in a total volume of 1 mL, using a 1 cm light path length cuvette. The concentration of purified nNOS reductase was calculated based on the absorbance spectra of the flavins present. Protein concentration was determined by absorption measurements at 454 nm using an extinction coefficient of

-1 -1 66 Ɛ454 = 21.6 mM cm , based on two flavins per protein monomer. Purity of protein was assessed by SDS-PAGE.

2.2.3.1.2 FL nNOS

Absorbance measurements were performed using a Cary UV-50 Bio UV- visible spectrophotometer at 25 °C in a total volume of 1 mL, using a 1 cm light path length cuvette. The ferrous heme-CO adduct absorbing at 444 nm was used to

-1 -1 measure hemoprotein content with an extinction coefficient of Ɛ444 = 74 mM cm

195. Purity of the protein was assessed by SDS-PAGE.

2.2.3.2 SDS-PAGE

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the GeBaGel protocol, using 12 % acrylamide gels (in a

GeBaRunner apparatus). Protein samples (volume: 20 µL) were mixed with the 10

µL of 3 x SDS loading buffer and boiled for 5 minutes at 95 °C before being loaded

Chapter 2 – Materials and methods on to the gel. The gel was run in 1 x SDS running buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS, pH 8.3) at a constant voltage of 160 V until the dye front had reached the bottom of the gel (up to 1 hour). The gel was stained using Coomassie blue stain (50 % v/v methanol, 10 % v/v acetic acid, 0.25 % w/v Coomassie blue R-

250 in water) for 30 minutes with gentle agitation and then destained using warm distilled water in the same way for a few hours.

For fast protein staining InstantBlue (Novexin), ready-to-use, proprietary Coomassie stain (containing Coomassie dye, ethanol, phosphoric acid and solubilising agents in water) was used, according to the Novexin protocol.

2.2.3.3 HPLC identification and quantification of FAD and FMN

All the solvents and samples solutions used in this method were prepared using CHROMASOLV for HPLC water from Sigma Aldrich. Methanol (Sigma

Aldrich) used in this method was CHROMASOLV for HPLC grade as well. All solvents were filtered and degassed before being used in this method.

A modified procedure based on 196 was used to identify and quantify FAD and FMN in FL nNOS solution. 5 mM ammonium acetate, pH 6.5 and methanol were used as the solvent system. The eluate was monitored continuously by absorbance at 264 nm, where the absorbance of FAD and FMN solutions were maximal.

Before any measurements were taken, the column (Phenomenex, Luna5 µm C18 100

Å, LC Column 30 x 4.6 mm) was equilibrated at a flow rate of 1 mL/min for 30 min with 85 % solvent A (5 mM ammonium acetate buffer, pH 6.5) and 15 % methanol.

A calibration curve was constructed by injecting 50 µL aliquots of solutions containing known amounts of FAD and FMN (0-80 µM each). After sample

Chapter 2 – Materials and methods injection the column was washed with 85 % solvent A and 15 % methanol. The concentration of methanol was increased linearly from 15-75 % in 20 min and from

75-100 % in 5 min. Under these conditions FAD elutes as a sharp peak at 10.4 min, and FMN as a sharp peak at 11.9 min. Injecting known amounts of FAD and FMN obtained peaks and by using integration of areas corresponding to those peaks a standard curve was constructed.

FL nNOS samples were diluted to a final concentration of 0.2 mM. Protein solutions were incubated at 100 °C for 10 minutes, then denatured protein was removed by centrifugation and the supernatant was filtered. The absorbance spectrum of each solution was measured and 150 µL aliquot was injected onto the HPLC. After sample injection the column was washed with 85 % solvent A and 15 % of methanol. The concentration of methanol was increased linearly from 15-75 % in 20 min and then from 75-100 % in 5 min. Under these conditions, flavins from FL nNOS eluted after similar time to commercial FMN and FAD samples used for the calibration curve. Integration of areas corresponding to obtained FMN and FAD peaks, were compared with the standard curve, and based on that, accurate values for

FAD and FMN concentration were determined.

2.2.3.4 Steady state kinetics

All assays were carried out using a Cary UV-50 Bio UV-visible spectrophotometer at 25 °C in a total volume of 1 mL, using a 1 cm light path length cuvette. The concentration of NADPH consumed was determined using the

-1 -1 197 extinction coefficient of Ɛ340 = 6.22 mM cm . Measurements were repeated at each substrate concentration in triplicate. Cytochrome c reduction studies were carried out for FL nNOS as well as for nNOSred (both WT and R1400E variant).

Chapter 2 – Materials and methods

NADPH oxidation and calmodulin (CaM) dependence assays were carried out only for FL nNOS.

2.2.3.4.1 Cytochrome c reduction

The steady-state activities of nNOS were followed using cytochrome c as an electron acceptor. The assay depends upon determining the rate of cytochrome c reduction by monitoring absorbance at 550 nm 198 . Measurements were carried out in reaction buffer containing 50 mM Tris/HCl, pH 7.4 (nNOSred) or 40 mM HEPES, pH 7.6, 150 mM NaCl (FL nNOS). For CaM+ forms of nNOSred and FL nNOS, reaction buffers were supplemented with 1 mM CaCl2. For the CaM- form of nNOSred, the reaction buffer contained 1 mM EDTA instead of 1 mM CaCl2. The reaction rate was monitored at 550 nm for over a minute by following the reduction

-1 -1 199 of cytochrome c using the extinction coefficient of Ɛ550 = 21.1 mM cm .

Reaction mixtures contained variable concentrations of NADPH and cytochrome c.

Reactions were initiated by the addition of NADPH and 0.050 µM nNOS. Saturating concentrations of NADPH (100 µM) were used to determine the Km for cytochrome c, and likewise, a saturating concentration of cytochrome c (10 µM) was used to determine the Km for NADPH. To derive the Km values, obtained rates were fitted to the Michaelis-Menten hyperbolic function using Origin software (OriginLab,

Northampton, MA).

2.2.3.4.2 NADPH oxidation

This assay was based on the method of Onufriev and Gulyaeva 200. Samples contained 50 nM of protein, 100 µM H4B, 5 mM L-arginine, 4 µM FMN, 4 µM FAD in 40 mM HEPES, pH 7.6 buffer containing 150 mM NaCl and 10 % (v/v) glycerol.

In cases when NADPH oxidation was measured for FL nNOS+ (calmodulin-bound form), calmodulin (100 nM) and CaCl2 (1 mM) were added to the sample. The

100

Chapter 2 – Materials and methods reaction was triggered by addition of 100 µM NADPH. The absorbance change was followed at 340 nm for over a minute and the rate of NADPH oxidation was

-1 -1 197 calculated using the extinction coefficient of Ɛ340 = 6.22 mM cm .

2.2.3.4.3 FL nNOS activity - calmodulin dependence

The assay was based on the NADPH oxidation assay (described in Section

2.2.3.5.2) with the difference that the concentration of calmodulin in the sample was changed from 0 to 150 U/mL (0 to 220 nM). The reaction was triggered by the addition of 100 µM NADPH. NADPH oxidation was followed at 340 nm for over a

-1 -1 197 minute using the extinction coefficient of Ɛ340 = 6.22 mM cm . Measurements were repeated at least three times.

2.2.3.5 Hydrostatic pressure stopped-flow kinetics

All hydrostatic high-pressure stopped-flow assays were performed using a

HPSF-56 high-pressure stopped-flow system (TgK Scientific, Bradford on Avon,

UK) operating in a single wavelength mode. With UV-vis absorption measurement capability, the system had an empirical dead time of less than 10 ms and allowed stopped-flow determinations to be made at pressures from 0 up to 2000 bar. The sample handling system was mounted in a frame and pneumatic press was mounted on a track to allow it to be moved whilst loading the pressure vessel. System pressure was generated by a pressure intensifier with a pneumatic pump for large variations of pressure and a manual pump used to more accurate adjustments of pressure to the desired values. The pressure stainless steel (17-4PH) vessel had an integral thermostated jacket to ensure temperature accuracy. Distilled water was used as a hydraulic fluid, as it is less compressible than for example n-hexane or n- pentane. All reactions were performed at 25 °C in 40 mM HEPES buffer (pH 7.6)

101

Chapter 2 – Materials and methods containing 150 mM NaCl and 10% (v/v) glycerol. The concentration of used

-1 NADPH used was determined using the extinction coefficient of Ɛ340 = 6.22 mM cm-1 197. Measurements were repeated at each pressure at least three times.

Kinetic data in most cases were complex and hence best analysed by computational methods that make no assumptions in fitting data, except the chosen model. The kinetic traces obtained from high-pressure stopped-flow experiments were fitted to a linear, a single-exponential or to a sum-of-exponentials model. It was not known beforehand which model will be best and how many, if any, exponentials will be required to describe adequately a particular kinetic curve, so several separate fits were performed on the data set, each using different models and numbers of exponentials. The standard criteria, which were used to choose which model best describes the data, included: residuals, autocorrelation function values and the R2 value (statistical goodness-of-fit parameter) 201.

2.2.3.5.1 Flavin reduction

The assay was based on the method studied by Knight and Scrutton 194.

Reactions were performed under anaerobic conditions, and for this purpose the sample-handling unit of the stopped-flow instrument was filled with sample and sealed within an anaerobic glove box. Buffer was made oxygen-free by evacuation and extensive bubbling with N2 and equilibration in an anaerobic glove box overnight before use. Flavin reduction by NADPH was measured in single wavelength studies by following the absorbance decrease at 458 nm. Transients were fitted using Kinetic Studio (TgK Scientific) software and the standard two exponential equation. Syringe 1 contained protein (20 µM) and syringe 2 contained

NADPH (0.2 mM). For single turnover reactions performed with nNOSred

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Chapter 2 – Materials and methods

+ complexed with calmodulin (nNOSred ), CaCl2 (1 mM) was included in the buffer

- and for reactions performed with nNOSred CaM- form (nNOSred ), EDTA (5 mM) was included in the buffer (40 mM HEPES, pH 7.6 buffer with 10 % (v/v) glycerol and 150 mM NaCl). For single turnover reactions with the FL nNOS+, calmodulin

(40 µM), L-arginine (10 mM) and H4B (200 µM) were included in syringe 1 and

CaCl2 (1 mM) was included in both syringes. 100 µL of solutions were then rapidly driven from syringes into a mixer to initiate the reaction.

2.2.3.5.2 NADPH oxidation

High pressure stopped-flow assay was based on a method studied by

Onufriev and Gulyaeva 200. The reaction was observed in a single wavelength study

-1 -1 197 at 340 nm using the extinction coefficient of Ɛ340 = 6.22 mM cm . Transients were fitted using Kinetic Studio (TgK Scientific) software and the standard linear equation model. Syringe 1 contained protein (200 nM), L-arginine (10 mM) and tetrahydrobiopterin (200 µM) and syringe 2 contained NADPH (0.2 mM). FAD and

FMN (both 4 µM) were included in the buffer (40 mM HEPES, pH 7.6 buffer with

10 % (v/v) glycerol and 150 mM NaCl). For reactions with FL nNOS+, CaM (200 nM) was included in syringe 1 and CaCl2 (1 mM) was included in both syringes. 100

µL of solutions were then rapidly driven from syringes into a mixer to initiate the reaction.

2.2.3.5.3 NO synthesis

Oxyhaemoglobin was prepared according to the method of 202. 0.1 g of haemoglobin (Hb) was dissolved in 4 mL of buffer (40 mM HEPES, pH 7.6 buffer with 10 % (v/v) glycerol and 150 mM NaCl), in 100-mL flat-bottom flask. The flask was swirled gently to dissolve haemoglobin completely, and then 8 mg of sodium

103

Chapter 2 – Materials and methods dithionite powder was added to promote haemoglobin reduction. The flask was swirled, sealed on the top with Sigma-Parafilm and a light stream of O2 was blown into the flask for at least 5 minutes. The resulting HbO2 solution was desalted and purified by passing it through a Sephadex D-25 column (equilibrated with at least 5 column volumes of 40 mM HEPES, pH 7.6 buffer with 10 % (v/v) glycerol). The

HbO2 solution was carefully layered on top of the column and allowed to enter the

Sephadex resin without dilution and was then followed by buffer. The main band of

HbO2 was collected as it eluted, with leading and tail ends being discarded. During assays, HbO2 was stored on ice in dim light. The purity and concentration of the desalted HbO2 stock solution was determined spectrophotometrically at 415 nm

-1 -1 202 using the extinction coefficient of Ɛ415 = 131 mM cm . For further use, HbO2 was aliquoted and stored at - 20 °C.

The NO synthesis assay was based on the method described by Doyle and Hoekstra

203 and Salter and Knowles 202. The NO synthesis assay is measuring the reaction between the produced NO and the oxygenated, ferrous form of haemoglobin (HbO2), which yields methaemoglobin (MetHb) in its ferric form and nitrate. The assay mixture was identical to the NADPH oxidation measurements (Section 2.2.3.5.2) except that oxyhaemoglobin (20 µM) was included in stopped-flow syringe no 1. For reactions with FL nNOS+, CaM (200 nM) was included in stopped-flow syringe no 1 and CaCl2 (1 mM) was included in both syringes. Small volumes of solutions were then rapidly driven from syringes into a mixer to initiate the reaction. NO-mediated conversion of oxyhaemoglobin to methaemoglobin was monitored in single wavelength study at 401 nm for over a minute using the extinction coefficient of Ɛ401

= 38 mM-1 cm-1 204.

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Chapter 2 – Materials and methods

2.2.3.6 Redox potentiometry

Redox titrations were performed in a Belle Technology glovebox under a dinitrogen atmosphere, maintained at less than 2 ppm oxygen. All buffers and solutions were degassed by being bubbled with N2 prior to entering the glove box to ensure removal of all traces of oxygen. External redox mediators (1 µM benzyl viologen, 7 µM hydroxynaphthoquinone, 0.3 µM methyl viologen and 2 µM phenazine methosulfate) were added to the sample to mediate in the range of 100 to -

480 mV and to facilitate electrical communication between the enzyme and electrode. The electrochemical potential was measured by a Mettler Toledo

SevenEasy meter coupled to a combined Platinum and Ag/AgCl-reference electrode

(Thermo Scientific Orion, Loughborough, UK). A factor of 244 mV was used to correct relative to the standard hydrogen electrode. Anaerobic protein solutions were titrated chemically using sodium dithionite as the reductant 205. Sodium dithionite was taken into the glove box in small powder aliquots. Spectra (220-800 nm) were recorded using a Cary UV-50 Bio UV-visible scanning spectrophotometer. For titration of the nNOSred, absorbance values at 456 nm (near the absorption maximum

- + for the oxidised flavins in both nNOSred and nNOSred ) and 592 nm (near the absorption maximum for the blue semiquinone form of the flavins) were analysed.

Protein samples were prepared in 40 mM HEPES, pH 7.6 buffer with 30 % (v/v) glycerol. Samples (200-250 µL) of enzyme were withdrawn for EPR spectroscopic analysis. The samples were placed in standard 4.8 mm quartz EPR tubes and sealed with Sigma-Parafilm inside the glove box, where they were immediately removed and frozen in liquid nitrogen. Samples were stored in liquid nitrogen to prevent reoxidation until they were analysed. The concentrations of nNOSred and FL nNOS, in CW EPR samples, were typically 70 µM. The concentrations of nNOSred and

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Chapter 2 – Materials and methods

R1400E nNOSred in PELDOR samples, both CaM+ and CaM-, were typically 350

µM. The concentrations of FL nNOS in PELDOR samples, both CaM+ and CaM-, were 410 µM and 290 µM respectively. The concentration of NADP+, if it was added, was ≈400 µM. The concentration of ADP, if it was added, was ≈400 µM.

2.2.3.7 Electron paramagnetic resonance spectroscopy (EPR)

All CW EPR and PELDOR samples were prepared as described in Section

2.2.3.6. All EPR spectra were collected by Dr Stephen Rigby and Dr Karl Fisher

(Manchester Institute of Biotechnology, The University of Manchester). EPR spectra were obtained using a Bruker ELEXSYS E500/E580 spectrometer operating at X- band. Temperature control was effected using an Oxford Instruments ESR900, for continuous wave EPR, or ESR935, for pulsed measurements, cryostat connected to an ITC503 temperature controller. Continuous wave spectra were obtained at 80 K using the parameters given in the figure captions. Four pulse PELDOR spectra were recorded using a π/2-T-π-T+τ-π-τ-acquire sequence with T = 200 ns and τ = 960 ns.

The π pulse length was 32 ns. The ‘fourth’ pulse, a π pulse applied at the pump microwave frequency, was incremented in 16 ns steps during the T+τ period starting at 100 ns after the second π pulse. The interval τ was limited to 960 ns due to the spin-spin relaxation, phase memory time TM, of the flavosemiquinones which precluded detection of the refocused echo at longer values of τ. If the timing of the

‘fourth’ pulse was indicated as t = 0 at 100 ns after the second π pulse and t is incremented thereafter, the modulation of the four pulse PELDOR echo was given by the Equation 1.13 (Chapter 1, Section 1.4.2). All pulse experiments were recorded at 80 K and at a repetition frequency of 200 Hz due to the long electron spin lattice relaxation time of the flavosemiquinone radical. Inter-electron dipolar couplings were determined using Fourier transform of the baseline corrected four pulse

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Chapter 2 – Materials and methods

PELDOR data to produce dipolar spectra. The raw PELDOR data were baseline corrected using a third order polynomial, then a Hamming window function was applied in order to increase signal to noise following the subsequent Fourier transform and drive the decay function to completion, thus avoiding truncation effects. Each data set was then zero filled to 1024 point prior to Fourier transformation.

2.2.3.8 Protein mass spectroscopy

The SDS-PAGE gel was prepared as described in Section 2.2.3.2 and submitted for identification which was carried out by the Protein Mass Spectrometry Core Facility

(The University of Manchester) using Mascot software (version 2.2.06; produced by

Matrix Science). The UniProt (version rat) and UniProt (version E. coli) databases were used. The results were analysed using statistical validation software Scaffold

(version 3.0.04; produced by Proteome Software).

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Chapter 3 – Characterisation of the nNOS reductase domain

Chapter 3 Characterisation of the nNOS reductase domain

108

Chapter 3 – Characterisation of the nNOS reductase domain

3.1 Introduction

The reductase domain of NOS (nNOSred) belongs to a large protein family which includes NADPH-dependent CPR, sulphite reductase flavoprotein and novel protein-1. These reductases share a conserved organisation of FMN, FAD and

55–57 NADPH-binding domains . NOSred has been expressed and purified

60 independently of its oxygenase domain (nNOSoxy) and it can transfer electrons from NADPH to FAD to FMN and on to artificial acceptors (for example cytochrome c), however, it is unable to complete the full catalytic cycle of NOS and it is unable to convert L-arginine to citrulline or produce nitric oxide. Electrons for nitric oxide synthesis are supplied by NADPH from where the hydride ion is directly transferred to the N5 of the FAD in the nNOSred. During the electron transfer, FMN oscillates between the hydroquinone and semiquinone forms, whilst the FAD passes

64,65 2+ through three oxidation states . Binding of Ca -calmodulin (CaM) to NOSred activates substrate-independent NADPH oxidation and is thought to promote internal

62 electron transfer from the flavins in nNOSred to the heme (nNOSoxy) .

The aim of this part of the project was to study the effect of CaM on the cytochrome c reductase activity, flavin blue semiquinone formation and interflavin distance distributions in nNOSred. To satisfy the overall aims of this project, namely to carry out kinetic analysis and EPR spectroscopic studies of the nNOSred, it was necessary to optimise an effective expression and purification protocol for the various forms of the nNOSred. It was also necessary to determine the steady-state kinetic parameters

(kcat, Km and Vmax) for each form of the enzymes to confirm the presence of the CaM

109

Chapter 3 – Characterisation of the nNOS reductase domain binding region in the purified form and to allow comparison with previously published values in order to measure how a specific mutation affects the interflavin electron transfer and the activity of this enzyme.

The major goal was to determine novel interflavin distance distributions within the nNOSred. It was important to study the conformational landscape of nNOSred despite its crystallographic structure being available. The protein crystallographic structure provides information about single interflavin distances existing in the tertiary structure of the protein from a solid crystal, while PELDOR spectroscopy provides information about a number of interflavin distances existing in the protein in its various structural conformations. The PELDOR spectroscopy was used for nNOSred for the first time, providing novel and important data about the distances existing between the flavins in the nNOSred under various conditions (CaM+/-, NADP+/- and/or ADP bound). The use of PELDOR spectroscopy is a novel approach as this has never been used on any form of nNOS or nNOSred before.

The determined interflavin distances provide an idea about various possible structure conformations which may exist at different steps of the catalytic cycle of nNOS. This is very important and meaningful information as it may be used to design specific inhibitors and control the nNOS activity and catalytic cycle in the future. Controlling nNOS activity is crucial, as uncontrolled NO production has been related to various diseases which lack a treatment (Chapter 1, Section 1.3).

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Chapter 3 – Characterisation of the nNOS reductase domain

3.2 Results

3.2.1 Overexpression and purification of WT and R1400E nNOSred

The plasmid used for the co-expression of the rat nNOSred and bovine CaM, pCRNNR, was successfully transformed into BL21(DE3) E. coli competent cells.

The presence of the genes encoding for nNOSred and CaM was confirmed by performing a combination of Eco RI-HF and Sac I-HF restriction enzyme digestions

(Figure 3.1). Bovine serum albumin (BSA) was added during digestion with Sac I-

HF and double digestion (the presence of BSA in the reaction enhanced stability of the restriction enzyme and its performance). In addition, agarose gel electrophoresis was used to verify the size of the purified plasmid DNA. An Eco RI-HF restriction digest of pCRNNR generated DNA fragments of expected sizes: 2240 and 5160 base pairs. An Eco RI-HF, Sac I-HF double digest generated DNA fragment sizes of 590,

2240 and 4570 base pairs as predicted (Figure 3.2).

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Chapter 3 – Characterisation of the nNOS reductase domain

5100 bp 5900 bp

bla

Sac I (6850 bp)

pCRNNR CaM insert Eco RI 7400 bp (40 bp)

nNOSred insert

Eco RI (2280 bp)

Figure 3.1 Map of the vector pCRNNR. Map of the vector pCRNNR co- expressing the rat nNOSred and bovine calmodulin. The vector contains restriction sites for Eco RI and Sac I. The total size of the vector is 7400bp, where 2830bp is an insert (genes for: nNOSred and CaM) and 4570bp is a plasmid. The other highlighted feature is bla - the β-lactamase gene to confer ampicillin resistance.

112

Chapter 3 – Characterisation of the nNOS reductase domain

10 kb 8 kb 6 kb 5 kb 4 kb 3 kb

2 kb 1.5 kb

1 kb

0.5 kb 1 2 3 4 5

Figure 3.2 DNA gel of the restriction enzyme digests carried out on vector pCRNNR. Lane 1, DNA Ladder, bands and band sizes are indicated with green arrows. Lane 2 is empty. Lane 3, vector pCRNNR cut with Sac I-HF. Lane 4, pCRNNR cut with Eco RI-HF. Lane 5, pCRNNR cut with Sac I-HF and Eco RI-HF. The expected band sizes were as follows: single digest with Sac I-HF linearised vector (7400 base pairs), single digest with Eco RI-HF: 2240 and 5160 base pairs, double digest with Eco RI-HF and Sac I-HF: 590, 2240 and 4570 base pairs.

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Chapter 3 – Characterisation of the nNOS reductase domain

Purification of the nNOSred protein according to the protocols (described in Chapter

2, Section 2.2.2.1) resulted in high yields of the CaM bound form of the enzyme

+ (nNOSred ). Transformed cells were grown in Terrific Broth media which resulted in large cell mass production and high protein yields. It is important to note that CaCl2 was included in all of the purification buffers to ensure the tight binding of CaM to the enzyme. The cell pellet was resuspended in lysis buffer (composition of lysis buffer described in Chapter 2, Section 2.2.2.1) to which CompleteTM EDTA-free protease inhibitor tablets, effective in inhibiting serine and cysteine proteases, were added according to the manufacturer’s instructions. Because of the nitric oxide synthase being temperature sensitive all chromatography was performed at 4°C. Also all the buffers and glassware were pre-chilled as this was found to increase the yield of the purification (no precipitation occurred compared to the purification which was performed using the room temperature glassware). A 2’5’-adenosine diphosphate

(ADP) Sepharose affinity column was used as the first chromatography step of purification. If the purity of the protein was sufficient to carry out detailed kinetic analysis and electron paramagnetic resonance spectroscopic measurements (verified by SDS-PAGE), a further step with a Q-Sepharose anion exchange column was avoided as it was found that longer purification procedures resulted in a higher degree of proteolysis and lower efficiency of the purification. Using this method the

+ purification yield was high enough to allow purification of nNOSred with the subsequent addition of EDTA to form the CaM- form of the enzyme, as opposed to

- growing/purifying nNOSred directly. EDTA, added to the protein, acts as a chelator

2+ for Ca thus facilitating the separation of NOSred and CaM. Use of the 2’5’-ADP sepharose resin allows NOS to remain bound, whilst CaM is eluted. This way, it was possible to selectively purify the CaM+ or CaM- form of NOS. The protein

114

Chapter 3 – Characterisation of the nNOS reductase domain concentration in the purified sample was assayed by UV-visible spectrophotometry

(following the flavin maximum absorption at 456 nm, Figure 3.3) and calculated using the Lambert-Beer law. The protein was verified by running SDS-PAGE

(Figure 3.4).

+ Purification of the R1400E nNOSred protein according to the purification protocol

+ used for WT nNOSred (described in Chapter 2, Section 2.2.2.1) resulted in equally high yields of the CaM bound form of enzyme, so no modifications in the expression and purification procedures were necessary. The protein concentration in the purified sample was assayed by UV-visible spectrophotometry (following the flavin absorption at the 456 nm, Figure 3.5) and also calculated using the Lambert-Beer law while the protein was verified by running SDS-PAGE (Figure 3.6).

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Chapter 3 – Characterisation of the nNOS reductase domain

+ Figure 3.3 UV-visible absorbance spectrum of purified WT rat nNOSred . The spectra for nNOS+ and nNOS- are the same and show a typical flavin absorption peak at 456nm (indicated with a red arrow). The protein is in an oxidised form, which is evident by the lack of absorbance at long wavelengths (550-700 nm). Conditions: 50 mM Tris/HCl, pH 7.4, 20% (v/v) glycerol and 1 mM CaCl2, protein concentration: 44 µM.

1 2 3 4 5 6 7 200 kDa 130 kDa 100 kDa

70 kDa 55 kDa 35 kDa

25 kDa 15 kDa 10 kDa

+ Figure 3.4 12% SDS-PAGE gel showing the WT rat nNOSred . Lane 1, Prestained Protein Molecular Weight Marker, bands and band sizes are indicated with green arrows. Lanes 2-7 show the 2’,5’-ADP Sepharose eluate fraction in different concentrations, after overnight dialysis. The protein band is indicated with a blue arrow.

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Chapter 3 – Characterisation of the nNOS reductase domain

Figure 3.5 UV-visible absorbance spectrum of purified of the R1400E rat + + - nNOSred . The spectra for nNOS and nNOS are the same and both show a typical flavin absorption peak at 456nm (indicated with a red arrow). The protein is in the oxidised form, which can be seen by the lack of absorbance at long wavelengths (550-700 nm). Assay conditions as follows; 50 mM Tris/HCl, pH 7.4, 20% (v/v) glycerol and 1 mM CaCl2, protein concentration: 3.4 µM.

1 2 3 4 5 6 200 kDa 130 kDa 100 kDa 70 kDa 55 kDa 35 kDa

25 kDa

15 kDa 10 kDa

+ Figure 3.6 12% SDS-PAGE gel showing the R1400E rat nNOSred . Lane 1, Prestained Protein Molecular Weight Marker, bands and band sizes are indicated with green arrows. Lanes 2-6 show the 2’,5’-ADP Sepharose eluate fraction in different concentrations, after overnight dialysis. The protein band is indicated with a blue arrow.

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Chapter 3 – Characterisation of the nNOS reductase domain

3.2.2 Steady-state kinetics of cytochrome c reductase activity of WT and

R1400E nNOSred

There are various methods for ascertaining whether CaM associates with nNOSred, but the clearest method is to look at the stimulation of the cytochrome c reductase activity upon CaM binding. The cytochrome c reductase activity of the nNOSred was measured as described in Chapter 2, Section 2.2.3.4.1 and used to obtain kinetic constants for both CaM+ and CaM- nNOSred forms. By varying the concentration of NADPH (0-125 µM) at a fixed concentration of cytochrome c (10

µM), the kinetic constants (Km,Vmax, kcat and kcat/Km, Table 3.1) for NADPH were calculated. The concentration of WT nNOSred in both kinetic measurements, for

CaM+ and CaM-, was 100.7 nM, while the concentration of R1400E nNOSred for both CaM+ and CaM- measurements was 100 nM. Measurements were repeated at least three times for each NADPH concentration. Kinetic parameters were determined by fitting the data using a standard (Michaelis-Menten) hyperbolic function (Figure 3.7-3.8) using Origin software (OriginLab, Northampton, MA).

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Chapter 3 – Characterisation of the nNOS reductase domain

+ - Figure 3.7 Cytochrome c reductase activities of WT nNOSred and WT nNOSred + . Figure presents cytochrome c reductase activities of WT nNOSred (A) and WT - nNOSred (B). 100.7 nM protein concentration was used at 25 °C. Reduction of cytochrome c at varying concentrations of NADPH was followed at 550 nm using an extinction coefficient of ε=21.1 mM-1cm-1 199. Data were fitted to a standard Michaelis-Menten hyperbolic function using Origin software (OriginLab, Northampton, MA) with the curve fitting represented by the red solid lines. Kinetic + - parameters determined for WT nNOSred and WT nNOSred can be found in Table 3.1.

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Chapter 3 – Characterisation of the nNOS reductase domain

+ Figure 3.8 Cytochrome c reductase activities of R1400E nNOSred and R1400E - + nNOSred . Figure presents cytochrome c reductase activities of R1400E nNOSred - (A) and R1400E nNOSred (B). 100 nM protein concentration was used at 25 °C. The reduction of cytochrome c occurring at varying concentrations of NADPH was followed at 550 nm using an extinction coefficient of ε=21.1 mM-1cm-1 199. Data were fitted to a standard Michaelis-Menten hyperbolic function using Origin software (OriginLab, Northampton, MA) with the curve fitting represented by the + red solid lines. Kinetic parameters determined for R1400E nNOSred and R1400E - nNOSred can be found in Table 3.1.

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Chapter 3 – Characterisation of the nNOS reductase domain

Table 3.1 Kinetic parameters obtained for WT and R1400E nNOSred in the presence and absence of CaM. Values were obtained from assays carried out as described in Chapter 2, Section 2.2.3.4.1 at 25 °C.

Km Vmax kcat kcat/Km (µM) (µM s-1) (s-1) (µM-1 s-1) + WT nNOSred 3.14 ± 0.43 18.72 ± 0.54 185.90 ± 5.36 59.20 - WT nNOSred 2.41 ± 0.23 1.43 ± 0.03 14.20 ± 0.30 5.89 + R1400E nNOSred 10.00 ± 0.63 18.99 ± 0.32 189.90 ± 3.20 18.99 - R1400E nNOSred 9.01 ± 0.69 6.09 ± 0.12 60.90 ± 1.20 6.76

The obtained values for Km increase by 30% for WT nNOSred and by 11% for

R1400E nNOSred, when CaM is added. The Km for R1400E is 3-3.7-fold higher than for the WT, in both CaM+ and CaM forms, which indicates the affinity of the protein to the substrate (NADPH) is less in the R1400E variant (which is expected considering the R1400E mutation is within the NADPH binding site). In the presence of CaM, Vmax and kcat values for both WT and R1400E are very much alike

-1 -1 (Vmax ≈18 µM s , kcat ≈185-190 s ). In the absence of CaM, the R1400E nNOSred kcat

-1 -1 is 4 times higher (≈60 s ) than the kcat calculated for the WT (≈14 s ). The calculated kinetic parameters suggest that the CaM is present within the protein (WT and

+ R1400E nNOSred ) and that its presence is positively affecting the interflavin electron transfer in WT nNOSred more than in the R1400E variant (after binding

CaM: 13-fold increase of Vmax and kcat for WT and only 3-fold increase for the

R1400E, Table 3.1). In case of either WT or R1400E, after binding CaM, no significant changes have been observed for Km which suggests that the lack of CaM

121

Chapter 3 – Characterisation of the nNOS reductase domain is reducing the rate of electron transfer between FAD and FMN or between FMN and cytochrome c but it is not affecting the protein/NADPH affinity itself.

The specificity constant, kcat/Km, shows that in the absence of CaM, the catalytic efficiencies of WT and R1400E are very similar (≈6 µM-1 s-1). When the CaM is

-1 -1 present, the kcat/Km for WT nNOSred (≈59 µM s ) is 3 times higher than for R1400E

-1 -1 (≈19 µM s ). This shows that when CaM is present WT nNOSred is more efficient and has a higher affinity for the substrate than the R1400E variant. This also suggests that the effect of the R1400E mutation on the electron transfer through the nNOSred to the cytochrome c is not significant unless the CaM is bound within the protein.

3.2.3 EPR redox titration

After successful expression, purification and kinetic analysis of the nNOSred, EPR spectroscopic studies of this domain were performed to meet the next aim of the project – namely to investigate the nature and control of electron transfer in the context of the proposed “domain shuffle hypothesis” for intraprotein electron

+ transfer inferred from the crystal structure of the nNOSred. NADP and 2’5’-ADP were also used to help with the identification of conformational changes and to map the important regions of the protein in respect of NADPH binding. Redox titrations were performed as described in Chapter 2, Section 2.2.3.6. UV-visible (Figure 3.9 and 3.10) and CW-EPR spectra of the flavosemiquinone state were used to determine whether the semiquinoid form of the flavins was formed. For titration of the nNOSred, absorbance values at 456 nm (near the absorption maximum for the

+ - oxidised flavins in both nNOSred and nNOSred ) and 592 nm (near the absorption maximum of the blue semiquinone form of the flavins) were analysed. Figures 3.9

122

Chapter 3 – Characterisation of the nNOS reductase domain and 3.10 show absorbance spectra collected during anaerobic titration with sodium dithionite. All the spectra show a typical flavin absorption peak at 456 nm. The lack of absorbance at long wavelengths in the region of 550-800 nm indicates that titrated proteins were in their oxidised forms. The formation of the reduced form of the protein can be followed by studying the appearance of a blue semiquinone peak at

592 nm and the reduction of the flavin absorption peak at 456 nm (the directions of the changes are indicated in Figure 3.9 and 3.10 by blue arrows). Because of the high concentrations of protein needed for redox potentiometry and EPR studies, the recorded UV-vis absorbances may be considered as off-scale and out of the linear range, however the redox potentiometry studies were predominantly concerned with the semiquinone signal monitored at 592 nm, which was within the range of the instrument, the provided traces should be considered as correct.

The UV-visible transients obtained during anaerobic redox titration, the blue semiquinone absorption changes at 592 nm and the redox potentials published by

Dunford et al. 206 were used to recognise the samples with the highest possible blue semiquinone together with the strongest EPR signal. The samples (≈200 µl) of enzyme were withdrawn for further EPR spectroscopy studies and prepared under strict anaerobic conditions (described in detail in Chapter 2, Section 2.2.3.7). The samples were placed in standard 4.8 mm diameter quartz EPR tubes and closed with

Suba-Seals (number 9) inside the glove box, where they were immediately removed and frozen in liquid nitrogen. Samples were stored in liquid nitrogen to prevent reoxidation until they were analysed.

The Figures 3.9 and 3.10 show the absorbance spectra collected during anaerobic chemical titration of the WT and R1400E rat nNOSred. Each transient (from dark blue to dark red) in the spectrums represents the UV-vis spectrum of the protein after

123

Chapter 3 – Characterisation of the nNOS reductase domain an addition of another aliquot of the sodium dithionite playing the role of the reductant. The proteins in general start in their oxidised form (purple/blue transients), which can be seen by the lack of absorbance at long wavelengths, 550-

800 nm, and they gradually become their reduced forms (red/wine red transients), with the flavin absorption peak at 456 nm decreasing and the blue semiquinone peak at 592 nm appearing.

In Figure 3.10, the absorbance spectrum of the R1400E nNOSred in the absence of

CaM (spectrum B) does not show its full oxidised state at the start of the redox titration (first, purple spectrum recorded before any aliquot of sodium dithionite was added, show increased absorbance in region 550-800 nm).This could have been caused either because the protein has been purified in a partially reduced form or because some amount of reductant (sodium dithionite) being present in the free form inside the glovebox. Either way, despite all the dispersion present in the spectrum and despite being partially reduced, R1400E nNOSred is still characterised by an increasing blue semiquinone peak at 592 nm and decreasing flavin peak at 456 nm

(both indicated by blue arrows, Figure 3.10, spectrum B).

Because the studies dedicated to obtaining the midpoint potentials for nNOSred have been successfully performed and published before 206, no midpoint potentials have been determined during an EPR redox titration presented in this thesis as it was neither novel research nor the main aim of this part of the project.

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Chapter 3 – Characterisation of the nNOS reductase domain

Figure 3.9 UV-visible absorbance spectra collected during anaerobic chemical titration of WT rat nNOSred. The spectra show a typical flavin absorption peak at 456 nm recorded for the WT rat nNOSred in the presence (A) and absence (B) of CaM. Each transient represents the UV-vis spectrum of the protein (from oxidised to fully reduced) after addition of another aliquot of the reductant (sodium dithionite). The protein starts in its oxidised form (purple/blue transients), which can be seen by the lack of absorbance at long wavelengths 550-800 nm, this gradually becomes the reduced form (red/wine red transients), where the flavin absorption peak at 456 nm decreases and the blue semiquinone peak at 592 nm appears (the direction of the changes are indicated by the arrows). Conditions: 50 mM Tris/HCl, pH 7.4, 30 % (v/v) glycerol.

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Chapter 3 – Characterisation of the nNOS reductase domain

Figure 3.10 UV-visible absorbance spectra collected during anaerobic chemical titration of R1400E rat nNOSred. The spectra show a typical flavin absorption peak at 456 nm recorded for the R1400E rat nNOSred in the presence (A) and absence (B) of CaM. Each transient represents the UV-vis spectrum of the protein (from oxidised to fully reduced) after addition of another aliquot of the reductant (sodium dithionite). The protein starts in its oxidised form (purple/blue transients) and gradually becomes the reduced form (red/wine red transients), where the flavin absorption peak at 456 nm decreases and the blue semiquinone peak at 592 nm appears (the direction of the changes are indicated by the arrows). Conditions: 50 mM Tris/HCl, pH 7.4, 30 % (v/v) glycerol.

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Chapter 3 – Characterisation of the nNOS reductase domain

3.2.4 EPR Spectroscopic studies

Electron paramagnetic resonance (EPR) spectroscopy is used to study molecular systems with unpaired electrons. EPR spectroscopy can be used to identify molecules within a sample and to characterise the environment of the paramagnetic electrons. The advantage of the EPR spectroscopy is that it is very sensitive (approx.

1000x more sensitive than NMR) and it has got high specificity as it is looking only at the region that contains paramagnetic centres. In continuous wave EPR (CW EPR) molecules are irradiated continuously by low-power microwaves, while in pulsed

EPR (PELDOR) microwaves are applied in very short and high-power pulses. Both methods, CW EPR and PELDOR, have been explained in detail in Chapter 1,

Section 1.4. For the first time in this thesis, both CW EPR and PELDOR techniques have been involved together to study WT and R1400E nNOSred.

The samples for the both CW EPR and PELDOR studies have been prepared under strict anaerobic conditions. During anaerobic redox titration (Chapter 2, Section

2.2.3.6 and Chapter 3, Section 3.2.3), samples (≈200 µl) of enzyme were withdrawn for further EPR spectroscopic analysis (conditions described in detail in Chapter 2,

Section 2.2.3.7). The maximal absorbance of blue semiquinone at 592 nm was followed in the UV-vis spectrophotometer and used to estimate and predict if the level of the flavosemiquinone in the sample is high enough to provide a strong EPR signal, although, the flavin midpoint potentials obtained for WT nNOSred by Dunford et al. 206 were also considered.

3.2.4.1 CW-EPR studies of nNOSred

The stable X-band EPR signal of the flavin cofactor, in its neutral radical form, is presented for WT nNOSred (Figure 3.12) and R1400E nNOSred (Figure 3.13), both in the presence and absence of CaM and/or NADP+/ADP. The EPR signals (all

127

Chapter 3 – Characterisation of the nNOS reductase domain collected at close to 9.4 GHz frequency) are presented in the form of a single-line first derivatives of the observed absorbance (Figure 3.12 and 3.13), characterised by ge values of 2.0032 and the peak-to-peak line widths (ΔB) and intensity (I) collated in Table 3.2, show that studied paramagnetic species have the same origin. The signals are in the range of approx. 3330-3360 G and are similar for all the samples what proves that it was possible to form neutral flavin semiquinone in the WT or

+ R1400E nNOSred, in the presence or absence of various ligands (CaM, NADP ,

ADP).

ΔB = 20.2 G

ge = 2.0032

Figure 3.11 An example of an EPR spectrum for WT rat nNOSred in the presence of CaM. The figure shows an example of an EPR spectrum of WT rat nNOSred in the one-electron-reduced state in the presence of CaM. The g-factor value (ge), intensity (I) and the line width (ΔB) have been indicated with grey arrows. Experimental conditions: 40 mM HEPES, pH 7.6 buffer containing 30 % (v/v) glycerol, temperature was 80 K.

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Chapter 3 – Characterisation of the nNOS reductase domain

Figure 3.12 EPR spectra of WT rat nNOSred. The figure shows EPR spectra of + WT rat nNOSred in the one-electron-reduced state in the presence of NADP or ADP, - + both in the absence of the CaM (nNOSred ) and in its presence (nNOSred ). Experimental conditions: 40 mM HEPES, pH 7.6 buffer containing 30 % (v/v) glycerol, temperature was 80 K. The protein concentration was ≈300 µM. Concentration of NADP+ was ≈350 µM. Concentration of ADP was ≈350 µM.

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Chapter 3 – Characterisation of the nNOS reductase domain

Figure 3.13 EPR spectra of R1400E rat nNOSred. The figure shows EPR spectra + of R1400E rat nNOSred in the one-electron reduced state in the presence of NADP or ADP, both in the absence of CaM (CaM-) and in its presence (CaM+). Experimental conditions: 40 mM HEPES, pH 7.6 buffer containing 30 % (v/v) glycerol, temperature was 80 K. The protein concentration was ≈300 µM. Concentration of NADP+ was ≈350 µM. Concentration of ADP was ≈350 µM.

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Chapter 3 – Characterisation of the nNOS reductase domain

Table 3.2 The ge, line widths and intensity data determined from CW EPR studies on WT and R1400E nNOSred, in the presence and absence of CaM and various ligands.

ge ΔB (G) Intensity Intensity change (a. u.) factor + WT nNOSred 2.0032 20.0 3783850 0.51 + WT nNOSred /ADP 2.0032 20.0 3971940 0.53 + + WT nNOSred /NADP 2.0032 19.6 4201630 0.56 - WT nNOSred 2.0032 20.0 7468410 1.00 - WT nNOSred /ADP 2.0032 19.5 6910940 0.93 - + WT nNOSred /NADP 2.0032 20.0 5570260 0.75

+ R1400E nNOSred 2.0032 18.7 3329200 0.96 + R1400E nNOSred /ADP 2.0032 19.7 4056600 1.17 + + R1400E nNOSred /NADP 2.0032 20.0 3446660 1.00 - R1400E nNOSred 2.0032 20.0 3452770 1.00 - R1400E nNOSred /ADP 2.0032 20.0 4146000 1.20 - + R1400E nNOSred /NADP 2.0032 19.7 3937370 1.14

The same ge, and very similar line width values and signals also suggest that for nNOSred, neither R1400E mutation nor CaM or ligands presence/absence, can prevent flavosemiquinone radical formation. However, in the presence of CaM or ligands, the double integrals of the EPR signal for WT nNOSred (proportional to the number of unpaired electrons contributing to the signal) are changed by the intensity change factor (Table 3.2) and the line widths have also changed with the ge value constant at 2.0032.

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Chapter 3 – Characterisation of the nNOS reductase domain

In the CW EPR spectroscopy, anionic and neutral flavin semiquinones have the same g-factor value, but can be distinguished by different line widths of the spectra 207.

The line width is defined as the distance between the positive and negative extrema in the first derivative presentation and reflects different hyperfine interaction constants in these two flavin species 207. A ≈19-20 G line width is characteristic for a neutral semiquinone, whereas ≈15 G line width is characteristic for an anionic semiquinone, and intermediate values are indicative of a mixture of the two types of semiquinone 207. The values of the line width determined in this thesis show that the flavin semiquinone formed in the WT and R1400E nNOSred, is fully or mainly the neutral flavin semiquinone form (majority of line widths in the range 19-20 G). The line widths of flavosemiquinone radical EPR spectra are determined by hyperfine coupling between the unpaired electron of the radical state and magnetic nuclei

(proton or nitrogen atoms) attached to the cofactor. A change in the distribution of the unpaired electron over the flavosemiquinone and/or the loss of a proton through ionisation results in a change in the spectrum line width due to the effect on the hyperfine coupling 206. The binding of CaM and/or ligands introduces modification in the environment of the flavin cofactor. The yield of flavosemiquinone is often reduced, presumably because of the modification of the midpoint potentials for the cofactor. And the environment is perturbed in a way that it modified the distribution of the unpaired electron within the flavosemiquinone that is formed. Comparing the intensity obtained for WT nNOSred in the absence of CaM with intensity values observed after the CaM and/or ligands are bound, the decrease in the intensity has

+ been observed in all cases. It has also been observed that, for WT nNOSred, NADP binding is affecting the line width only if CaM is bound within the protein, a similar observation has been made before by Dunford et al. 206 . When the nucleotide

132

Chapter 3 – Characterisation of the nNOS reductase domain fragment 2’5’-ADP is bound to the protein and the CaM is present, the ≈50 % reduction in the intensity is observed with the line width being increased.

Along with binding CaM and/or additives, less significant changes have been observed in the double integrals of the EPR signal for R1400E nNOSred, than was

+ observed for WT nNOSred. When CaM and/or ADP/NADP are bound to the

R1400E variant, there is almost no change in the intensity recorded, also the line widths change only slightly, and the biggest change for line width has been observed when only the CaM is bound within the R1400E nNOSred. The R1400E variant is less sensitive than WT in terms of the changes occurring after CaM and/or

ADP/NADP+ binding, which suggests that the mutation of the Arg1400, which interacts with the 2’-phospahte group of NADP+, is not only affecting the protein’s affinity to the NADPH but also preventing the proper binding of NADP+/ADP and controlling the environmental changes related to this binding.

3.2.4.2 PELDOR studies of nNOSred

Pulsed electron-electron double resonance (PELDOR, also sometimes called double electron-electron resonance or DEER) can determine, from measurement of the dipolar interaction, the nanometer long distances and orientations between two paramagnetic centres. The theoretical background of the PELDOR spectroscopy experiment has been explained in Chapter 1, Section 1.4 and the PELDOR experiment conditions have been provided in detail in Chapter 2, Section 2.2.3.7.

Despite the crystallography structure of the rat WT nNOSred being available (1TLL), it is a static crystal representation of the protein and it doesn’t provide more than one interflavin distance, whereas PELDOR spectroscopy used in this project provides information about the possible distance distributions in the various forms of

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Chapter 3 – Characterisation of the nNOS reductase domain

nNOSred. It is very important information as it suggests possible conformation changes during the catalytic cycle and it may be used in the future to design specific inhibitors aimed at controlling NO production on the reductase domain level. The interflavin distances in nNOSred which have been presented in this thesis are novel results; they have not been obtained before and are unpublished so far.

In this project, the X-band four-pulse PELDOR spectroscopy has been utilised to obtain the interflavin distances in nNOSred in the presence or absence of CaM and/or

NADP+. While the data can be taken comparatively as a measure of relative interflavin distances within nNOSred, the actual measured distance should also be considered. The unpaired electron is delocalised over all three rings of the flavosemiquinone structure. Because of this, the measured distances do not represent distances between the points, but rather the distances between a weighted mean of the unpaired electron spin density distribution over the flavosemiquinone 208. The centre of such a distribution lays essentially at C(4a) 209,210 (Chapter 1, Figure1.5).

Furthermore, even where the unpaired electron spin density distribution of a flavosemiquinone varies with the binding of substrates, inhibitors or other effectors close to it, the centre of the distribution remains at C(4a) 208. This approximation has therefore been used in previous studies involving PELDOR of flavin semiquinones

208,211.

In four-pulse PELDOR, as it is suggested by its name, four short and intense microwave pulses, either π/2 or π, have been applied on two spins to obtain EPR signals (Chapter 1, Section 1.4.2 and Figure 1.23). The signal coming from the sample is in the form of the modulation pattern of the refocused echo and can be described by Equation 1.13 (presented in Chapter 1, Section 1.4.2) as a function of time. When the modulation pattern of the refocused echo is obtained, it is necessary

134

Chapter 3 – Characterisation of the nNOS reductase domain to use the Fourier Transform to transform it from the function of time into the function of frequency to obtain a frequency-domain spectrum. The Fourier transformation step is essential in the PELDOR analysis as it allows dipolar coupling values to be obtained which then can be used to determine the distances between two flavins in nNOSred. The interspin distances can be calculated from the dipolar coupling (νDD) values after Equation 1.14 is rearranged into Equation 1.15 (both presented in Chapter 1, Section 1.4.2). The Fourier transforms of the four-pulse

PELDOR echo decays produced by two-electron reduced (one on each flavin, forming two flavosemiquinones) WT and R1400E nNOSred in the presence or absence of CaM and/or NADP+ have been presented in Figure 3.14 and 3.15. In these figures, the x-axis provides a measure of the dipolar coupling, νDD, between two flavosemiquinones, while purple numbers, provided over the peaks, represent interflavin distances (in nm) calculated based on the dipolar coupling values given by the respective peaks.

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Chapter 3 – Characterisation of the nNOS reductase domain

3.5

2.8 2.4

3.6

2.6 2.2 2.0

3.3

2.5

3.7 2.6 2.9 2.1 2.0

Figure 3.14 Fourier transforms of the X-band 4 pulse ELDOR echo decays produced by two electron reduced (dithionite) WT nNOSred in the presence and absence of CaM and/or NADP+. Experimental parameters and pulse sequence given in Chapter 2, Section 2.2.3.7; temperature was 80 K.

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Chapter 3 – Characterisation of the nNOS reductase domain

3.3

2.5

3.3

2.2

Figure 3.15 Fourier transforms of the X-band 4 pulse ELDOR echo decays produced by two electron reduced (dithionite) R1400E WT nNOSred in the presence of CaM and presence or absence of NADP+. Experimental parameters and pulse sequence given in Chapter 2, Section 2.2.3.7; temperature was 80 K.

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Chapter 3 – Characterisation of the nNOS reductase domain

Table 3.3 Distances between the FAD and FMN flavin semiquinones in the two- + electron reduced state of WT nNOS, in the presence (WT nNOSred ) or absence - (WT nNOSred ) of CaM, determined from PELDOR spectroscopy.

νDD (MHz) Distance, r Relative (nm) integral - WT nNOSred 1.3, 2.4, 3.6 3.5, 2.8, 2.4 0.52, 0.28, 0.20 - + WT nNOSred /NADP 1.1, 3.2, 4.6, 6.2 3.6, 2.6, 2.2, 0.62, 0.20, 0.08, 2.0 0.10 + WT nNOSred 1.4, 3.6 3.3, 2.5 0.70, 0.30 + + WT nNOSred /NADP 1.0, 2.2, 3.0, 5.7, 3.7, 2.9, 2.6, 0.36, 0.14, 0.21, 6.8 2.1, 2.0 0.18, 0.11 + R1400E nNOSred 1.4, 3.6 3.3, 2.5 0.70, 0.30 + + R1400E nNOSred /NADP 1.4, 4.6 3.3, 2.2 0.87, 0.13

Several inter-flavin distances were determined from each of the spectra in Figure

3.14 and 3.15; these distances are collated in Table 3.3 along with the respective dipolar coupling values and relative integrals. It is important to consider the relative integrals of the spectral lines which provide an indication of the number of molecules in each conformation, although this could only be an approximation since the complete extent of each line is not visible in the spectrum. Taken together, the distances and relative integrals show that while there are always multiple conformations of nNOS present, these conformations and their relative proportions vary with CaM and NADP+ binding. Analysing the distance distribution obtained for the WT nNOSred in the presence and absence of CaM it can be seen that when CaM

+ is bound, fewer inter-flavin distances are observed (3.3 and 2.5 nm for WT nNOSred

- instead of 3.5, 2.8 and 2.4 nm for WT nNOSred ) with the dominating 3.3 nm distance

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Chapter 3 – Characterisation of the nNOS reductase domain

- (relative integral of 0.7) not observed for the WT nNOSred . This may suggest that

CaM binding is promoting a more rigid conformation with flavins being pushed closer together (domination of shorter distance of 3.3 nm) to effect and stimulate the internal electron transfer within the nNOSred. Even more distances can be determined

+ + for WT nNOSred when NADP is bound. In the presence of NADP , previously unobserved distances have been detected: short, 2.0-2.2 nm distances and longer 3.6-

3.7 nm distances. This suggests that, regardless of CaM being present or absent,

NADP+ binding is promoting more possible conformations with flavins being either pushed closer together (presence of new shorter distances) or separated (new longer

+ + distances). The WT nNOSred and R1400E nNOSred have been characterised by exactly the same distance distributions (3.3 and 2.5 nm); which suggests that this mutation may affect the conformation of the NADPH binding site but it is not affecting the structure of the nNOSred or inter-flavin distance. No significant effect has been observed for the R1400E variant when NADP+ is bound within the protein.

The distance distributions obtained for R1400E in the presence and absence of

NADP+ are very similar; with a dominating 3.3 nm distance (having a relative integral of either 0.7 or 0.87 respectively) and a secondary shorter one (either 2.2 or

2.5 nm). Considering the lack of effect of the NADP+ binding on the R1400E

+ nNOSred , no PELDOR studies have been performed for the R1400E nNOSred in the absence of CaM.

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Chapter 3 – Characterisation of the nNOS reductase domain

3.3 Discussion

To meet all the aims of this project, it was necessary to first establish an effective purification protocol for the various forms of WT and R1400E variant nNOSred in the presence and absence of CaM. Expression and purification of

194 nNOSred were based on successful protocols established by Knight & Scrutton

206 212,213 and Dunford et al. . Despite some groups working on nNOSred using CaM-

Sepharose affinity resin to purify the protein and provide even higher separation, the purification methods used in this thesis were found to be more than adequate and the introduction of another column to the purification process and elongating it

(especially when the protein is temperature sensitive) was found to be an unnecessary extra step.

The cytochrome c reductase activity of nNOSred was used to obtain kinetic parameters for NADPH for both CaM+ and CaM- forms of WT and R1400E variant of nNOSred (Figure 3.7 and 3.8, kinetic parameters values collated in Table 3.1).

When CaM was bound within the protein, a 13-fold increase in the Vmax was

-1 - -1 observed for WT nNOSred (from ≈1.4 µM s for WT nNOSred to ≈18.7 µM s for

+ WT nNOSred ). A very similar difference in the rate of reaction (≈11-fold) has been previously reported by Gachhui et al. 101, however the turnover reported for WT

+ 101 -1 - nNOSred by Gachhui et al. is lower (≈48 s ) than reported in this thesis (≈185 s

1); which may be caused by using different purification protocols or by a different organism in which rat nNOSred has been generated (E. coli in case of this thesis and the Pichia pastoris yeast in the case of Gachhui et al 101). This 13-fold increase in the rate of reaction confirms that CaM binding is fully activating the nNOSred and promoting the internal electron transfer between flavins and the artificial electron acceptor (cytochrome c). When CaM is bound to R1400E nNOSred, a 3-fold increase

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Chapter 3 – Characterisation of the nNOS reductase domain

in Vmax was observed, which is significantly smaller than the 13-fold increase observed for WT nNOSred, although the final Vmax for WT and R1400E are comparable (≈18.5-19 µM s-1) which implies that the presence of Ca2+ and CaM has a smaller effect on the R1400E variant compared to the WT, than has been

70,212 previously mentioned by Garcin et al. or Tiso et al. . The Km for NADPH observed for the R1400E variant is 4-fold higher than Km observed for WT, although in both cases, no significant changes in Km have been observed after binding the

CaM within the respective protein, which suggests that CaM binding is promoting the internal electron transfer between flavins or FMN and cytochrome c but it has no effect on the protein’s affinity for NADPH. The obtained specificity constant, kcat/Km, shows that, in the absence of CaM, the catalytic efficiencies of WT and

-1 -1 R1400E are very much alike (≈6 µM s ), but when CaM is present, the kcat/Km for

-1 -1 -1 -1 WT nNOSred (≈59 µM s ) is 3 times higher than for R1400E (≈19 µM s ); which shows that the effect of the R1400E mutation on the electron transfer from the nNOSred to the cytochrome c is not significant unless the CaM is bound within the protein.. The specificity constants (kcat/Km) obtained also suggest that the introduction of Glu1400 in the place of Arg1400 repels rather than attracts NADPH, destabilises the C-terminal tail but still supports internal electron transfer (very similar Vmax to WT) despite decreased affinity for NADPH (Km higher than for WT) along with lower efficiency. The data observed in this chapter provide important information about an effect of CaM binding on the activity and efficiency of nNOSred along with an effect of the R1400E mutation on the nNOSred efficiency and its affinity for NADPH.

The EPR redox titration was performed to allow determination of the presence of blue flavosemiquinone in the protein sample and investigate the nature of CaM

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Chapter 3 – Characterisation of the nNOS reductase domain

effects on the radical formation and internal electron transfer within nNOSred. To identify the best ratio of FMN/FAD blue semiquinone radical in the sample, correlated with strong EPR signal in the further studies, the UV-visible transients have been obtained and compared with the UV-visible data and the redox potentials published by Dunford et al. 206 and used as an indication.

In the case of diflavin proteins, flavin semiquinone formation during reduction of flavins have been studied using EPR spectroscopy only a few times before for either nNOS 206 or P450 BM3 125,207. In the case of P450 BM3, it has been published by

Munro et al. 125 and Murataliev et al. 207 that a characterisation of flavin semiquinone by EPR spectroscopy could be performed only after short exposure of the protein to

NADPH (when the protein has been pre-reduced), and after that, only one of the two flavins in the diflavin P450 BM3 was in a semiquinone form when the other has been fully reduced. It was also stated that by analogy with the eukaryotic CPR, the semiquinone is more likely to reside on the FMN flavin 125. The CW EPR spectroscopy studies presented in this thesis, which has hardly been used for nNOS before, provides important information about the blue flavosemiquinone formed in the nNOSred during anaerobic redox titration. The WT and R1400E variant of rat nNOSred have been studied along with the possible effect of CaM on the neutral flavin radical formation. The effect of two ligands, NADP+ and ADP has also been analysed to identify the possible conformational changes occurring during catalysis and to map important regions of the protein in respect of NADPH binding.

According to the observed CW EPR signals, all of the studied forms of nNOSred were characterised by the same ge values of 2.0032 and very similar peak-to-peak line widths (Table 3.2) proving that the studied paramagnetic centres have the same origin, and that neither CaM nor NADP+/ADP addition can prevent the

142

Chapter 3 – Characterisation of the nNOS reductase domain flavosemiquinone radical formation. It suggests that the Arg1400 residue may not only affect the nNOS/NADPH affinity but may also control and prevent the proper

NADP+/ADP binding and related possible conformational changes occurring considering no significant changes have been observed in line width or intensity after

CaM/NADP+/ADP binding.

The first derivatives of EPR signals and obtained ge and line width values observed in this thesis have been in agreement with CW EPR first derivative plots and data

101 206 published so far by Gachhui et al. or Dunford et al. . Although WT nNOSred has been studied using CW EPR before 206, the CW EPR studies on R1400E variant presented in this thesis are novel and they have not been published so far. Results presented in this thesis suggest that the Arg1400 residue may play a key role in controlling the nNOS activity at the structural level. Thus far, not many people have

70 studied R1400E nNOSred and for those that have , the studies were only related to the kinetic performance of the protein but never in relation to the possible effect of the mutation on the protein structure and conformational changes.

After confirmation that it was possible to produce the one-electron reduced form of the nNOSred, further studies by PELDOR spectroscopy have been performed.

PELDOR spectroscopy of two-electron nNOSred has been used to measure distances between the FAD and FMN centres and to investigate the effect of ligand binding on the possible domain motion, since the influence of the cofactors on the kinetics of interflavin electron transfer has been previously demonstrated 214,215. PELDOR experiments similar to those presented in this thesis have only been recorded three times before, two of them were performed on diflavin proteins: CPR 208 and homodimeric FAD-dependent sulfhydryl oxidase 211 (wherein each monomer contained one flavosemiquinone, rather than two semiquinones being present in the

143

Chapter 3 – Characterisation of the nNOS reductase domain same domain). To date, there is no report of any PELDOR experiment involving interflavin distances measurements determined for any form of NOS. This chapter showed that low-temperature PELDOR spectroscopy can be applied to study dynamic events in the diflavin NOSred and provide exclusive information about

CaM/NADP+-dependent changes in interflavin distance and the possible

+ conformational landscape in nNOSred existing after the CaM and/or NADP is bound.

Figure 3.14 and 3.15 show the X-band four pulses PELDOR spectra and several inter-flavin distances determined for two-electron reduced WT and R1400E nNOSred in the presence and absence of CaM and NADP+. The obtained distances and relative integrals show that while there are always multiple conformations of nNOS present, these conformations and their relative proportions vary with CaM and NADP+

+ binding. The binding of NADP to nNOSred favours conformations with widely distributed distances, mostly with one longer distance (36-37 Å) and a few shorter

(20-21 Å) interflavin distances relative to the unliganded protein. The unliganded

R1400E nNOSred is characterised by only two interflavin distances, exactly the same that can be observed in WT, although after binding the NADP+, the equilibrium distribution states in R1400E favour more compact conformations compared to those

+ exhibited by WT nNOSred in the presence of NADP . It seems that introduction of the R1400E mutation in nNOSred can keep the structure rigid and prevent forming the possible open conformation which can be observed and triggered by binding

+ NADP in the WT nNOSred.

Two CPR structures, “open” 216 and “closed” 55, have been published before, showing that FAD- and FMN-binding domains of CPR are separated by a hinge domain (residues 232-244 55). The open conformation appears to be formed when the

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Chapter 3 – Characterisation of the nNOS reductase domain

FMN domain rotates about this hinge, increasing the separation between the FAD and FMN C(4a) atoms from 15.4 Å in the closed conformation to 31.6 Å in the open

208 conformation . nNOSred has a very similar domain arrangement and landscape to

CPR, and possibly because of that, the interflavin distances measured in this chapter are very similar to those determined from CPR crystal structures as well as values measured for CPR using PELDOR 208. The interflavin distances measured by Hay et al. using a very similar PELDOR experiment were in excellent agreement with distances observed in the available CPR crystal structures, so it is very possible the same thing has occurred in the case of nNOSred and the distances of the open conformation structure have been successfully observed, even if the crystal structure of the “open” form of nNOSred is currently missing. Based on that, it can be concluded that it is very possible for nNOSred to form a few different open/closed conformations, as is happening for CPR 208.

69 Considering the available crystal structure obtained for WT rat nNOSred (1TLL) present the interflavin distance of 15.24 Å (C(4a) from FAD to C(4a) from FMN), and that the distances obtained in this thesis are slightly longer than 15.24 Å (they are in a range of 20-37 Å) it can be concluded that the available 1TLL crystal structure is presenting only the “closed” form of nNOSred when the distances determined in this project may be characterising the possible “open” forms of the protein. The PELDOR data provide information about several interflavin distances available for nNOSred which suggests that an equilibrium distribution of multiple extended open and compact closed conformations is found in solution.

This is a remarkable result as no crystal structure of either nNOSred or full length nNOS in its open form is available to date. This information could also be used in the future to design specific inhibitors which could be affecting only nNOS in its

145

Chapter 3 – Characterisation of the nNOS reductase domain open conformation. The design of inhibitors is a very complex process and drugs often have a range of side-effects since, to some degree, they will also bind to other human proteins with a similar structure. With the information available herein showing the domain movements of NOS it could be envisaged that in the future drugs could be developed to only bind to a specific conformation. This could be tailored so that only NOS predominately resides in this conformation/binding mode and so drastically limit the degree to which the inhibitor will bind to other proteins in the body.

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Chapter 4 – Characterisation of full length nNOS

Chapter 4

Characterisation of full length nNOS

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Chapter 4 – Characterisation of full length nNOS

4.1 Introduction

In order to produce nitric oxide (NO), full length nitric oxide synthase (FL) utilises L-arginine (L-arg) as the substrate, molecular oxygen and NADPH as co- substrates, and FAD, FMN and H4B as necessary cofactors (Chapter 1, Section 1.1.2 and 1.1.3). The functional NOS transfers electrons from NADPH, via the flavins,

FAD and FMN in the carboxy-terminal reductase domain, to the heme in the amino- terminal oxygenase domain (nNOSoxy). In order to synthesise NO, the NOS enzyme goes through two steps. Firstly, NOS hydroxylates L-arginine to Nω-hydroxy-L- arginine which remains largely bound to the enzyme, then NOS oxidises Nω- hydroxy-L-arginine to L-citrulline and NO 217. A fully active NOS needs to have

Ca2+-calmodulin (CaM) bound in the calmodulin binding site, however, its dependence is not fully understood. It is known that CaM binding is brought about by an increase in intracellular Ca2+ (half-maximal activity between 200 and 400 nM)

217, so the effect of calmodulin on NOS needs to be investigated under a variety of conditions. H4B is also essential for fulfilling the NOS catalytic cycle and producing

NO because it stabilises the quaternary structure of the active dimeric form of NOS

3,81 and it also acts as an electron donor during oxygen activation and recapturing of an electron from the ferrous nitrosyl complex in order to trigger NO release 37.

In order to study FL nNOS it was first necessary to determine and optimise an effective expression and purification protocol. Pulsed electron electron double resonance (PELDOR) spectroscopy was then employed to elucidate the conformational landscape of that protein at low temperature, as was achieved with

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the nNOSred in Chapter 3. The key R1400E mutation (whose significance is discussed in Chapter 1) within the NADPH binding site was also generated in the FL enzyme. The steady-state kinetic parameters (Km and Vmax) for each form of the FL enzyme were determined to allow comparison with previously published values as well as with the values calculated for the nNOSred (both WT and the R1400E variant) as listed in Chapter 3.

4.2 Results

4.2.1 Overexpression and purification of FL WT and R1400E rat nNOS

The plasmid used for the expression of the FL rat nNOS, pCWORI, was successfully transformed into BL21(DE3) E. coli competent cells. FL rat nNOS was then expressed and purified. Purification procedures were based on the methods of 193,194 as described in Chapter 2, Section 2.2.2.2. The presence of the gene encoding for FL nNOS was confirmed by the use of Nde I and Hin dIII restriction enzyme digests.

The plasmid map in Figure 4.1 shows the expected positions for cutting by these restriction enzymes. In addition, agarose gel electrophoresis was used to verify the correctness of purified plasmid DNA. A single Nde I restriction digestion of pCWORI linearised the vector and generated a DNA fragment whose size, according to the gel, is greater than 10 kb and consistent with the expected size of 10.1 kb. A single Hin dIII restriction digest generated three DNA fragments of expected sizes

1000, 1100 and 8000 base pairs. A Nde I and Hin dIII double digest generated four

DNA fragments of expected sizes 1000, 1100, 3000 and 5000 base pairs as predicted

(Figure 4.2). The presence of the gene encoding FL NOS was also confirmed by

DNA sequencing (Eurofins MWG Operon, Germany).

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Chapter 4 – Characterisation of full length nNOS

FL nNOS insert Hin dIII (8454 bp)

Hin dIII (9451 bp) Nde I (5477 bp) pCWORI 10100 bp 10100 bp

lacI reg Hin dIII (529 bp)

bla

Figure 4.1 Vector map of pCWORI. The map of vector pCWORI with the insert for FL rat nNOS expression. The restriction sites of Nde I and Hin dIII are shown to highlight their cutting positions. The total size of the vector is 10100 bp, where 4623 bp is the FL nNOS insert and 5477 bp is the plasmid. Other highlighted features are bla - the β-lactamase gene to confer ampicillin resistance and lacI – the regulatory lac repressor gene.

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Chapter 4 – Characterisation of full length nNOS

1 2 3 4

10 kb 8 kb 6 kb 5 kb 4 kb

3 kb

2.5 kb

1.5 kb

1 kb

Figure 4.2 DNA gel of the restriction enzyme digests carried out on vector pCWORI.

Lane 1, DNA Ladder, bands and band sizes are indicated with green arrows. Lane 2 is empty. Lane 2, vector pCWORI cut with Nde I. Lane 3, vector pCWORI cut with Hin dIII. Lane 4, vector pCWORI cut with Nde I and Hin dIII. The expected band sizes were as follows: single digest with Nde I linearised vector (10100 base pairs), single digest with Hin dIII: 1000, 1100 and 8000 base pairs, double digest with Nde I and Hin dIII: 1000, 1100, 3000 and 5000 base pairs.

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Chapter 4 – Characterisation of full length nNOS

Figure 4.3 UV-visible absorbance spectra of purified FL WT rat nNOS-. The UV-visible spectra recorded for FL WT nNOS+ and FL WT nNOS- are the same (the spectrum for WT nNOS+ is not shown). The green line shows oxidised FL WT nNOS- with the nNOS maximum peak indicated by the green arrow, while the red line shows FL WT nNOS- pre-reduced with sodium dithionite and with CO bound to its ferrous heme and a typical sharp low spin ferrous heme-CO absorption peak at 444 nm (indicated with a red arrow). Conditions: 7.57 µM protein 40 mM HEPES, pH 7.6, 10 % (v/v) glycerol, 150 mM NaCl, 1 mM L-arg.

The concentration of the FL WT nNOS was calculated spectroscopically using the

Beer-Lambert law (described in Chapter 2, Section 2.2.3.1.2). The ferrous heme-CO adduct absorbing at 444 nm (indicated with red arrow in Figure 4.3) was used to

-1 -1 measure hemoprotein content with an extinction coefficient of ε444 = 74 mM cm

195. SDS-PAGE was then used to verify the correct molecular weight of the purified

FL WT nNOS and to determine the purity of the isolated form (Figure 4.4).

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Chapter 4 – Characterisation of full length nNOS

1 2 3 4

250 kDa 170 kDa 130 kDa 130 kDa 100 kDa 100 kDa 70 kDa 70 kDa

55 kDa 55 kDa 40 kDa

35 kDa 35 kDa 25 kDa 25 kDa

15 kDa 15 kDa

10 kDa 10 kDa

Figure 4.4 12% SDS-PAGE gel showing the FL WT rat nNOS+ and FL WT rat nNOS-. Lane 1, PageRuler Plus Prestained Protein Ladder, bands and band sizes (in kDa) are indicated with green arrows. Lane 2, FL WT nNOS CaM+ (indicated with a red arrow), CaM (indicated with a blue arrow). Lane 3, PageRuler Prestained Protein Ladder, bands and band sizes (in kDa) are indicated with green arrows. Lane 4, FL WT nNOS CaM- (indicated with a red arrow).

Following the purification protocol used for FL WT nNOS- (described earlier in this chapter and also in Chapter 2, Section 2.2.2.2), the same procedures have been applied to FL R1400E nNOS-. The concentration of the FL R1400E nNOS was calculated as described in Chapter 2, Section 2.2.3.1.2. Isolated and purified protein was assayed by UV-visible spectrophotometry (Figure 4.5) to confirm the presence of ferric heme within the protein. The SDS-PAGE (Figure 4.6) was also used to confirm the ability of the protein to bind CaM as well as its purity.

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Chapter 4 – Characterisation of full length nNOS

Figure 4.5 UV-visible absorbance spectra of purified FL R1400E rat nNOS-. The UV-visible spectra recorded for FL R1400E nNOS+ and FL R1400E nNOS- are the same (the spectrum for FL R1400E nNOS+ is not shown). The blue line shows oxidised FL R1400E nNOS- with the nNOS maximum peak indicated by the blue arrow, while the red line shows FL WT nNOS- pre-reduced with sodium dithionite and with CO bound to its ferrous heme and a typical sharp low spin ferrous heme- CO absorption peak at 444 nm (indicated with a red arrow). Conditions: 3.47 µM protein, 40 mM HEPES, pH 7.6, 10 % (v/v) glycerol, 150 mM NaCl, 1 mM L-arg.

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Chapter 4 – Characterisation of full length nNOS

1 2 3 4

170 kDa 130 kDa

100 kDa 70 kDa

55 kDa

40 kDa

35 kDa 25 kDa

15 kDa

10 kDa

Figure 4.6 12% SDS-PAGE gel showing the FL R1400E rat nNOS+.

Lane 1, PageRuler Prestained Protein Ladder, bands and band sizes (in kDa) are indicated with green arrows. Lanes 2-4, FL R1400E nNOS CaM+ (indicated with a red arrow) and CaM (indicated with a blue arrow), each lane represents different concentrations of the same sample (Lane 2: 5 µM, Lane 3: 10 µM, Lane 4: 1 µM).

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Chapter 4 – Characterisation of full length nNOS

4.2.2 Protein mass spectroscopy

It was impossible to obtain a single protein band on the SDS-PAGE gel, so protein mass spectroscopy was carried out as described in Chapter 2, Section 2.2.3.8 to confirm if the presence of additional bands was related to the nNOS domains falling apart during preparation of the SDS-PAGE gel or if was it related to the presence of impurities in the sample. Eight different bands from SDS-PAGE gel were chosen to be identified (chosen bands are marked and numbered in Figure 4.7).

1 2 3 4

250 kDa 3 1 170 kDa 4

130 kDa 130 kDa 51 6 100 kDa 7 100 kDa 70 kDa 70 kDa 8

55 kDa 55 kDa 40 kDa

35 kDa 35 kDa 25 kDa 25 kDa 2 15 kDa 15 kDa

10 kDa 10 kDa

Figure 4.7 12% SDS-PAGE gel showing the FL WT rat nNOS+ and FL WT rat nNOS-. Lane 1, PageRuler Plus Prestained Protein Ladder (band sizes (in kDa): 250, 130, 100, 70, 55, 35, 25, 15, and 10; exact bands are indicated with green arrows in Figure 4.4). Lane 2, FL WT nNOS CaM+. Lane 3, PageRuler Prestained Protein Ladder (band sizes (in kDa): 170, 130, 100, 70, 55, 40, 35, 25, 15, and 10; exact bands are indicated with green arrows in Figure 4.4). Lane 4, FL WT nNOS CaM-.

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Chapter 4 – Characterisation of full length nNOS

The mass spectroscopy protein identification results confirmed, with a high degree of

certainty, bands present in SDS-PAGE gels after purification are those of rat FL WT

nNOS (bands 1 and 3-8) and calmodulin (band 2) (Table 4.1). The excised bands 1

and 2 show FL rat nNOS and calmodulin, respectively, and show that they remain

bound together during purification. It is only the denaturing effect of the SDS-PAGE

that separates the two proteins. The excised bands 3 to 8 are all identified as NOS. It

is highly probable that purification in the absence of calmodulin leads to ‘clipping’

of the FL protein with loss of amino acids at the terminal end, hence shortening the

protein and leading to fragments of varying size. Trials (not shown) have

demonstrated that the longer the time taken to carry out the purification steps, the

greater the levels of fragmentation.

Table 4.1 Mass spectroscopy results searched against the generic UniProt (version rat) database. The results were searched against a set of sequences limited to the specified sample species (rat). The number of matched peptides is the number of unique peptides that have been matched to the identified protein in the sample. The greater the number of matches the more certain the identification. 1 match is a possible identification; 2-3 matches a probable identification; 4 and over means almost certain identification.

Identified Band Band Band Band Band Band Band Band Proteins 1 2 3 4 5 6 7 8 Nitric oxide synthase, brain 92 2 96 81 56 53 66 58 OS=Rattus norvegicus Calmodulin OS=Rattus norvegicus 2 5

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Chapter 4 – Characterisation of full length nNOS

4.2.3 HPLC identification and quantification of FAD and FMN

The number of proteins known to contain flavins is constantly increasing thanks to the increasing power of separation techniques and molecular biology tools.

When characterising flavin-containing enzymes, it is essential to recognise the presence of flavin cofactor(s), identify and then quantify them. Literature describes a number of methods for doing this, however, in this thesis only high-performance liquid chromatography (HPLC) was used because of its very high sensitivity and accuracy (described in detail in Chapter 2, Section 2.2.3.3). In particular, it was important to identify and quantify flavins in FL nNOS in order to confirm that flavins remain bound during the purification procedure. Measuring flavin concentration of the intact protein via the usual UV-visible spectrophotometric method is not feasible in this instance due to the high heme absorbance peak around

400 nm which masks all the flavin peaks. HPLC separates out the signals from the heme and the flavins enabling their independent measurement.

To identify and quantify FAD and FMN in FL nNOS solution a modified HPLC procedure based on Aliverti et al. 196 was used. Ammonium acetate, 5 mM, pH 6.5, and methanol were used as the solvent system. After a few trial experiments, an ammonium acetate and methanol gradient used for this study was slightly changed, in comparison to Aliverti et al.’s 196 procedure, to ensure the highest performance of the available column and equipment. The eluate was monitored continuously at 264 nm where the absorbance of FAD and FMN solutions were maximal. It was possible to measure eluate absorbance at shorter wavelengths; however, varying the eluent composition could affect quantitation by baseline absorbance changes. A calibration curve was constructed using aliquots of solutions containing known amounts of FAD and FMN. Injecting known amounts of FAD and FMN the peaks in Figure 4.8 at

158

Chapter 4 – Characterisation of full length nNOS reproducible elution times were observed. Using integration of areas corresponding to those peaks, construction of a standard curve was possible. The experiment has been repeated at least three times for each FMN/FAD concentration to minimise the errors.

FAD FMN

Figure 4.8 An example of flavin identification, quantification and saturation HPLC data recorded for FL WT nNOS-. 5 mM ammonium acetate, pH 6.5, and methanol were used as the solvent system. The eluate was monitored continuously by absorbance at 264 nm, where the absorbance of FAD and FMN solutions were maximal. Calibration curves (Figure 4.9 and Figure 4.10) were constructed by injecting 50 µL aliquots of solutions containing known amounts of FAD and FMN (0-80 µM each). Injecting known amounts of FAD and FMN and by integrating areas corresponding to the relevant peaks a standard curve was constructed. Values of FAD and FMN saturation level can be found in Table 4.2.

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Chapter 4 – Characterisation of full length nNOS

y = 96.87x R2= 0.9971

Figure 4.9 FAD calibration curve determined by HPLC. 5 mM ammonium acetate, pH 6.5, and methanol were used as the solvent system. The eluate was monitored continuously by absorbance at 264 nm, where the absorbance of FAD and FMN solutions were maximal. A calibration curve was constructed by injecting 50 µL aliquots of solutions containing known amounts of FAD (0-80 µM each). Under conditions described in Chapter 2, Section 2.2.3.3, FAD elutes as a sharp peak at 10.4 min. The standard calibration curve was constructed using integration of areas corresponding to FAD peaks and known FAD concentration in the injected sample. Error bars represent one standard deviation calculated from the average of at least three reductive transients. The red line represents the best linear fit.

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Chapter 4 – Characterisation of full length nNOS

y = 68.72x R2= 0.9993

Figure 4.10 FMN calibration curve determined by HPLC. 5 mM ammonium acetate, pH 6.5, and methanol were used as the solvent system. The eluate was monitored continuously by absorbance at 264 nm, where the absorbance of FAD and FMN solutions were maximal. A calibration curve was constructed by injecting 50 µL aliquots of solutions containing known amounts of FMN (0-80 µM each). Under conditions described in Chapter 2, Section 2.2.3.3, FMN elutes as a sharp peak at 11.9 min. The standard calibration curve was constructed using integration of areas corresponding to FMN peaks and known FMN concentration in the injected sample. Error bars represent one standard deviation calculated from the average of at least three reductive transients. The red line represents the best linear fit.

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Chapter 4 – Characterisation of full length nNOS

The nNOS concentration used for identification and quantification was 21.3 µM

(WT) or 20.7 µM (R1400E). For FL WT nNOS, the peak area for FAD was 2080.2 and for FMN it was 1387.3. For FL R1400E nNOS, the peak area for FAD was

1966.5 and for FMN it was 1360.6. The standard calibration curves (Figure 4.9 and

4.10) were used to calculate the concentration of FAD and FMN in the nNOS sample. Results are presented in Table 4.2.

Table 4.2 The results of identification, quantification and saturation of flavin observed using HPLC and calculated for FL WT and R1400E nNOS. 5 mM ammonium acetate, pH 6.5, and methanol were used as the solvent system. nNOS concentration has been calculated according to the method described in Chapter 2, Section 2.2.3.1.2. The flavin saturation data were calculated based on calibration curves determined for FAD and FMN earlier.

FL WT nNOS FL R1400E nNOS nNOS concentration [µM] 21.3 20.7 FAD concentration [µM] 21.5 ± 0.7 20.3 ± 0.5 FAD saturation [%] 100.9 ± 2.2 98.0 ± 2.4 FMN concentration [µM] 20.2 ± 0.8 19.8 ± 0.5 FMN saturation [%] 94.9 ± 3.7 95.6 ± 2.4

The accurate concentration of the FL nNOS was calculated based on the ferrous heme-CO adduct method (described in Chapter 2, Section 2.2.3.1.2). The results in

Table 4.3 show the high percentage of flavin saturation for both FAD and FMN and that during the purification procedure both FAD and FMN remain firmly bound to the protein at full saturation, for either WT or R1400E FL nNOS.

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4.2.4 Steady-state kinetics of cytochrome c reduction

A number of steady-state kinetic assays have been used so far to measure

NOS activity. To monitor electron transfer efficiency from the flavins to an exogenous electron acceptor, the NADPH-dependent reduction of cytochrome c activity was studied by monitoring the cytochrome c reduction rate

100,218 spectrophotometrically at 550 nm . The flow of electrons through the nNOSred is NADPH to FAD to FMN. It is the NOS hydroquinone that donates electrons to cytochrome c 219; the NOS FMN semiquinone or a NOS in which FMN has been removed cannot reduce cytochrome c 220. The rate-limiting step in this process is the transfer of electrons from FAD to FMN 66, therefore, cytochrome c reduction can be

221 used as a measure of electron flux through the entire nNOSred . The cytochrome c reductase activity of the FL rat nNOS was used to obtain kinetic constants for both nNOS forms, CaM-bound (CaM+) and CaM-free (CaM-). By varying the concentration of NADPH (0-100 µM) at a fixed concentration of cytochrome c (10

µM) (conditions of the assay have been given in Chapter 2, Section 2.2.3.4.1), the kinetic parameters (Km, Vmax, kcat and kcat/Km) for NADPH were calculated for FL

WT and R1400E nNOS (Table 4.3). The concentration of FL WT nNOS, for both

CaM+ and CaM- forms, was 98 nM, while the concentration of FL R1400E nNOS

(again for both CaM+ and CaM- forms), was 100 nM. Assay conditions were as described in Chapter 2, Section 2.2.3.4.1. Data were fitted to a standard Michaelis-

Menten hyperbolic function using Origin software (OriginLab, Northampton, MA).

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Chapter 4 – Characterisation of full length nNOS

Figure 4.11 Cytochrome c reductase activities of FL WT nNOS+ and FL WT nNOS-. Figure shows the cytochrome c reductase activities of FL WT nNOS+ (A) and FL WT nNOS- (B). Assays contained 98 nM NOS protein at 25 °C. Reduction of cytochrome c at varying concentrations of NADPH was followed at 550 nm using an extinction coefficient of ε=21.1 mM-1cm-1 199. Data were fitted to a standard Michaelis-Menten hyperbolic function using Origin software (OriginLab, Northampton, MA) with the curve fitting represented by the red solid lines. Values + - Vmax and Km determined for FL WT nNOS and FL WT nNOS can be found in Table 4.3.

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Chapter 4 – Characterisation of full length nNOS

Figure 4.12 Cytochrome c reductase activities of FL R1400E nNOS+ and FL R1400E nNOS-. Figure shows the cytochrome c reductase activities of FL R1400E nNOS+ (A) and FL R1400E nNOS- (B). 100 nM protein concentration was used at 25 °C. Reduction of cytochrome c was followed at 550 nm in assays containing varying concentrations of NADPH and using an extinction coefficient of ε=21.1 mM-1cm-1 199. Data were fitted to a standard Michaelis-Menten hyperbolic function using Origin software (OriginLab, Northampton, MA) with the curve fitting represented by the red solid lines. Values Vmax and Km determined for FL R1400E nNOS+ and FL R1400E nNOS- can be found in Table 4.3.

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Chapter 4 – Characterisation of full length nNOS

Table 4.3 Kinetic parameters obtained for FL WT and R1400E nNOS in the presence and absence of CaM. Values were obtained from assays carried out as described in Chapter 2, Section 2.2.3.4.1 using 98 nM (WT) or 100 nM (R1400E) protein concentrations at 25 °C at 25 °C.

Km Vmax kcat kcat/Km (µM) (µM s-1) (s-1) (µM-1 s-1) FL WT nNOS+ 0.88 ± 0.09 14.25 ± 0.32 145.41 ± 3.27 165.24 FL WT nNOS- 0.70 ± 0.10 1.54 ± 0.03 15.71 ± 0.31 22.45 FL R1400E nNOS+ 2.04 ± 0.19 20.10 ± 0.73 201.00 ± 7.30 98.53 FL R1400E nNOS- 1.68 ± 0.15 9.56 ± 0.38 95.60 ± 3.80 56.90

The value obtained for Km changed slightly for WT (from 0.70 to 0.88 µM) and similarly for R1400E (from 1.68 to 2.04 µM) after CaM was bound. In terms of the effect that CaM has on the Vmax and kcat, a 9-fold increase has been observed for WT

(from ≈1.5 to ≈14 µM s-1) when only ≈2-fold increase has been observed for the

R1400E variant after binding the CaM. This, similarly to the nNOSred, suggests that the internal electron transfer is affected by CaM binding more in the case of WT than it is in the case of the R1400E variant. Also, in the presence of CaM, the Vmax and

-1 -1 kcat observed for FL R1400E nNOS (Vmax: ≈20 µM s ; kcat: ≈200 s ) are 30 % higher

-1 -1 than those observed for WT (Vmax: ≈14 µMs ; kcat: ≈145 s ) which suggests that despite the lowering of the affinity for NADPH by the introduction of the mutation, the internal electron transfer is still maintained at a very high level. In the absence of

CaM, the interflavin electron transfer rate in R1400E (9.56 µM s-1) is still 6 times faster than it is in WT (1.54 µM s-1).

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Chapter 4 – Characterisation of full length nNOS

The specificity constants kcat/Km calculated for FL WT nNOS show that when CaM is bound, the kcat/Km for WT is almost 8 times higher than when CaM is absent. In

R1400E, kcat/Km for CaM+ is also higher than for CaM-, only the difference between those values is smaller than in the case of WT (≈2-fold). This proves that nNOSred is always more efficient in CaM+ than in CaM- form.

4.2.5 NADPH oxidation

Although it is important to be able to measure the activity of FL nNOS, it is not an easy task to measure the amount of NO produced by NOS. The NO production assay (based on oxyhemoglobin binding NO 203) is very difficult to perform using a simple UV-visible spectrophotometer because the NO produced is released into the atmosphere very quickly; as a result, the measured NO production rate is inconsistent and not very accurate. For this reason, as an alternative, NADPH oxidation activity was measured to study the overall NO production activity of NOS.

NADPH is consumed during the nNOS reaction so NADPH oxidation was monitored by following the reduction in absorbance at 340 nm (Figure 4.13). In the absence of substrate, NADPH oxidation by nNOS results in superoxide formation

221. In the absence of CaM, superoxide formation occurs via the flavins of the

221 nNOSred because electrons are not passed to the heme . The assay was based on a method developed by Onufriev and Gulyaeva 200. Assay conditions are as described in Chapter 2, Section 2.2.3.5.2. Measurements were repeated at least three times.

The results are presented in Table 4.4.

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Chapter 4 – Characterisation of full length nNOS

Figure 4.13 UV-vis visualisation of NADPH oxidation activity of FL WT nNOS+. NADPH oxidation (decrease in absorbance shown in colours from purple to light green) was followed at 340 nm using an extinction coefficient of ε340 = 6.22 mM-1 cm-1 197. Time interval between each spectrum is 30 s. Heme and flavin absorbances at longer wavelengths (400 - 700 nm) are also visible.

Table 4.4 NADPH oxidation rates determined for FL WT and R1400E variant nNOS (both CaM+ and CaM- forms). Values were calculated from assays carried out as described in Chapter 2, Section 2.2.3.5.2, using 86 nM (FL WT nNOS) and 92 nM (FL R1400E nNOS) protein concentrations at 25 °C.

FL WT FL WT FL R1400E FL R1400E nNOS+ nNOS- nNOS+ nNOS- -1 kcat (s ) 1.00 ± 0.03 0.15 ± 0.01 0.98 ± 0.05 0.32 ± 0.01

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Chapter 4 – Characterisation of full length nNOS

The NADPH oxidation rates determined for FL WT and R1400E variant nNOS

(both CaM+ and CaM- forms) show that the R1400E mutation has an NADPH oxidation rate 2x higher than WT nNOS when the proteins are both in their CaM- forms. However, when CaM is added to the solution FL and R1400E nNOS NADPH oxidation rates are increased to a similar level (≈1.00 s-1).

4.2.6 FL nNOS activity - calmodulin dependence

The assay measures how nNOS activity depends on calmodulin concentration and it was based on the NADPH oxidation assay described earlier, with the difference that the concentration of calmodulin in the sample was varied from 0 to

220 nM rather than being constant. FL WT nNOS- (50 nM) was used to measure the

NADPH oxidation rate by following the change in absorbance at 340 nm, using an

-1 -1 197 extinction coefficient of ε340 = 6.22 mM cm . Assay conditions are described in

Chapter 2, Section 2.2.3.5.2. Measurements were repeated at least three times.

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Chapter 4 – Characterisation of full length nNOS

Figure 4.14 FL WT nNOS+ activity – calmodulin dependence. 50 nM protein concentration was used at 25 °C. NADPH oxidation rate was determined by following the change in absorbance at 340 nm using an extinction coefficient of ε340 = 6.22 mM-1 cm-1 197.

The aim of this experiment was to establish if the FL nNOS activity depends on the calmodulin concentration and in what manner. If we consider a concentration of 50 nM of the FL WT nNOS was used and that FL WT nNOS reaches its maximal

NADPH oxidation activity with 50 nM calmodulin or more, it was concluded that at least an equal concentration of the FL nNOS- and the calmodulin is necessary for the full nNOS performance.

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Chapter 4 – Characterisation of full length nNOS

4.2.7 EPR redox titration

It is important to determine mid-point potentials (Em, the driving force required for electron transfer) of the redox centres (FAD, FMN and heme) in NOS as knowledge about the Em’s can help us to further understand the role of the flavins in electron flow through the enzyme. The main approach for determining the Em is redox titration using sodium dithionite as the reductant. Potentiometric titration of nitric oxide synthase isoforms provides information about the thermodynamics of electron transfer and associated protonation/deprotonation events as well as information about the coupling of redox processes to ligand binding. Understanding the thermodynamics of electron transfer in NOS is necessary to understand and describe the enzyme’s regulation 222. In the redox titration approach, titrations are generally monitored in situ optically with a UV-vis spectrophotometer, or by measuring EPR spectra of samples withdrawn from the cuvette at certain solution potentials 206. Mediators are added to facilitate electron transfer between enzyme and electrode 205.

Optical redox titrations under strict anaerobic conditions were performed to analyse

206 flavin cofactors in nNOSred in the presence and absence of calmodulin and the

193 isolated FAD domain of nNOS . In nNOSred studies, the mid-point reduction potentials were determined and a considerable change in redox properties of the nNOS flavins was observed upon binding of CaM 206. In the studies on the isolated

FAD domain of nNOS, the neutral, blue semiquinone was observed during the course of the redox titration, with absorption maximum close to 600 nm. The mid- point reduction potentials for the oxidised/semiquinone and semiquinone/hydroquinone couples were determined and compared for the FAD cofactor in the wild-type nNOS FAD domain and the F1395A/S/W variants 193.

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The main goal of this experiment was to determine the redox potential at which the

FAD/FMN semiquinone ratio of the sample is at its optimum (closest to 1) and that this sample can be expected to give a very strong PELDOR signal during EPR studies. It was not possible to estimate the amount of the semiquinone in the protein sample using UV-vis spectroscopy (as was done for the nNOSred by following the change in absorbance at about 600 nm) as the heme peak (absorbing at about 410 nm) being much bigger, was successfully masking the FMN/FAD semiquinone peak at about 600 nm. Using redox titration and sodium dithionite as the reductant, it was possible to estimate the redox potential of the ideal EPR sample, which could be used for the future PELDOR studies. The effects of ADP and NADP+ on the protein’s redox potential were also studied. Mediators were added to facilitate electrical communication between enzyme and electrode, prior to titration. The CW

EPR spectroscopy has been explained in detail in Chapter 1, Section 1.4 while the detailed conditions of the experiment were as described in Chapter 2, Section

2.2.3.6.

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Figure 4.15 Relative integrals of the flavosemiquinone spectrum from CW EPR collected for FL WT nNOS. The figure represents relative integrals of the flavosemiquinone spectrum from CW EPR collected for FL WT nNOS CaM+ (A) and CaM- (B) form. Experimental conditions: 40 mM HEPES, pH 7.6 buffer with 30 % (v/v) glycerol, temperature was 80 K. The protein concentration was 70 µM. Data were analysed by fitting to the Nernst equation describing a 2x2 one-electron reduction process. Fittings are represented by red solid lines. Data manipulation and analysis were performed using Origin 8.5 software (OriginLab, Northampton, MA).

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Figure 4.16 Relative integrals of the flavosemiquinone spectrum from CW EPR collected for FL WT nNOS CaM- form in the presence of ADP or NADP+. The figure represents relative integrals of the flavosemiquinone spectrum from CW EPR collected for FL WT nNOS CaM- form in the presence of ADP (A) and NADP+ (B). Experimental conditions: 40 mM HEPES, pH 7.6 buffer with 30 % (v/v) glycerol, temperature was 80 K. The protein concentration was 70 µM and the NADP+ concentration was 100 µM. Data were analysed by fitting to the Nernst equation describing a 2x2 one-electron reduction process. Fittings are represented by red solid lines. Data manipulation and analysis were performed using Origin 8.5 software (OriginLab, Northampton, MA).

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From the EPR-redox titrations above, the results are collated in Table 4.5 below. The main goal of this work was to produce samples for PELDOR in which the flavin semiquinones are at their highest levels. Because high concentrations of protein are required, coupled with a large sample volume in which to record the UV-visible spectra alongside the potentials, it was not possible to record more data points in the titrations. Nevertheless, it is still possible to fit reduction potentials to the data collected, albeit the errors are larger than would be desired compared to a dedicated study into the mid-point potentials. Despite the larger than expected errors, this is the first time that the mid-point potentials have been obtained for FL WT nNOS.

Table 4.5 Mid-point potentials for the four-electron reduction of FL WT nNOS. Kinetic parameters determined for FL WT nNOS in the presence and absence of calmodulin, ADP or NADP+. Mid-point potentials were obtained by fitting redox potential data derived from spectroelectrochemical redox titrations for the neutral blue semiquinone form of the flavins (592nm) using Origin 8.5 software (OriginLab, Northampton, MA). Values were calculated from assays performed as described in Chapter 2, Section 2.2.3.6, containing 70 µM NOS protein at 25 °C.

FMN FAD

EMS1[mV] EMS2[mV] EMS1[mV] EMS2[mV] FL WT nNOS+ -89 ± 11 -158 ± 45 -158 ± 72 -259 ± 6 FL WT nNOS- -51 ± 17 -86 ± 15 -165 ± 15 -250 ± 5 FL WT nNOS- + ADP 74 ± 7 -103 ± 23 - 92 ± 22 -326 ± 3 FL WT nNOS- + NADP+ 92 ± 5 -224 ± 54 -206 ± 29 -211 ± 69

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The results show that calmodulin binding to the FL WT nNOS decreased the FMN mid-point potentials but had no significant effect on the FAD mid-point potentials.

Binding ADP to the CaM- form of FL WT nNOS increases the observed redox potential, while binding NADP+ to the same form of the protein caused a decrease of the observed redox potentials. Binding of both cofactors (ADP and NADP+) affected the values of the calculated FMN and FAD mid-point potentials in various ways. A major disadvantage of the PELDOR experiment is how time consuming and particularly protein consuming the process is. The high concentrations coupled with the large volumes needed mean there is a limit to the amount of work that can be performed. Because of this and due to the limited time constraints of the project the effect of the cofactors binding to the CaM- and CaM+ forms was not studied further.

4.2.8 PELDOR spectroscopy

The double electron-electron resonance (DEER) technique, alternatively called pulsed electron double resonance (PELDOR) separates pairwise dipolar couplings between electron spins from other electron spin interactions. The interactions are observed in the time domain by an approach reminiscent of the spin- echo double resonance experiment used in nuclear magnetic resonance (NMR).

Decay of the time domain signal owing to transverse electron spin relaxation is factored out by applying an observer echo sequence of constant duration. This approach was later extended to a four-pulse sequence for measuring the signal without dead time on commercial pulse electron paramagnetic resonance (EPR) spectrophotometers 223,224. The resulting four-pulse PELDOR experiment has become the most widely used approach for measuring distances between electron spins in biomacromolecules in the range of approximately 1.8-8 nm 189,223,225. The more detailed theoretical background of the PELDOR spectroscopy experiment has

176

Chapter 4 – Characterisation of full length nNOS been explained in Chapter 1, Section 1.4 and the PELDOR experiment conditions have been provided in detail in Chapter 2, Section 2.2.3.7.

The main aim of the X-band four-pulse PELDOR studies was to determine the distances between the FMN and FAD within FL WT nNOS in the presence and absence of CaM. The conditions of the experiment were as described in Chapter 2,

Section 2.2.3.7. Despite that the crystallographic structures of separated reductase

(1TLL) and oxidase domains (1ZVL) are available, there is no crystal structure established for FL nNOS so far. That is why it is so important to determine the distance between the flavins in the FL nNOS, as this will provide an idea how the FL conformation is affected by the presence of CaM as well as protein domains.

Additionally, PELDOR spectroscopy is able to provide not only a single interflavin distance, but a distances distribution characterised by possible multiple distances where each of them is describing slightly different protein conformation. The ability to provide a distance distribution by PELDOR also suggests that the protein is able to exist, within the sample, in various conformational states. Knowledge about possibility of existence of different conformational structures of FL nNOS can be used in the future to find new ways to control NO production, which is very important information, considering that uncontrolled NO production has been linked to various diseases with lacking treatment (Chapter 1, Section 1.3). The X-band four- pulse PELDOR spectroscopy studies presented in this chapter provided novel information about interflavin distances observed in FL WT nNOS (no attempts to determine them in FL nNOS have been published so far) with CaM being present or absent within the protein. Although it is usually said that PELDOR spectroscopy is measuring the distance between two unpaired electrons, in the case of interflavin distances, the measured distance is only an approximation, because of the unpaired

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Chapter 4 – Characterisation of full length nNOS electron in flavins not being localised on single atom but rather it is delocalised over all three rings of the flavosemiquinone structure. Because of this, the measured distance does not illustrate the exact distance between two points, but rather the distance between a weighted mean of the unpaired electron spin density distributed over the flavosemiquinone 208 with the centre of this spin density distribution being located at C(4a) 209,210 (Chapter 1, Figure1.5). This approximation has therefore been used in previous studies involving PELDOR of flavin semiquinones 208,211.

To obtain an EPR signal using four-pulsed PELDOR spectroscopy, four short microwave pulses have been applied on two unpaired spins (described in detail in

Chapter 1, Section 1.4.2 and Figure 1.23). The signal obtained from the PELDOR studies on the flavosemiquinone was in the form of the modulation pattern of the refocused echo (it is a function of time; Chapter 1, Section 1.4.2, Equation 1.13) which was then Fourier transformed into the function of frequency. The frequency- domain spectrum, obtained via Fourier transform, allowed obtaining of dipolar coupling values, used later on to calculate the interflavin distances in FL nNOS

(Chapter 1, Section 1.4.2, Equation 1.14 and 1.15). The Fourier transforms of the four-pulse PELDOR echo decays produced by two-electron reduced (one on each flavin, forming two flavosemiquinones) WT nNOS in the presence and absence of

CaM have been presented in Figure 4.17. In Figure 4.17, the x-axis provides a measure of the dipolar coupling, νDD, between FAD and FMN semiquinones, while purple numbers, provided over the peaks, represent interflavin distances (in nm) calculated based on the dipolar coupling values given by the respective peaks. The dipolar coupling values, distances distribution obtained for FL WT nNOS in the presence and absence of CaM along with the respective relative integrals have been presented in Table 4.6 below.

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Chapter 4 – Characterisation of full length nNOS

3.7 2.6 2.2 2.0

5.1

2.5 2.1 2.2

Figure 4.17 Fourier transforms of the X-band 4 pulse PELDOR echo decays produced by two electron reduced nNOS. The figure shows the distance distributions for FL WT nNOS- and for FL WT nNOS+ . Experimental parameters and pulse sequence given in Chapter 2, Section 2.2.3.7; temperature was 80 K; 400 scans were co-added.

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Table 4.6 Distances between the FAD and FMN flavin semiquinone in the two- electron-reduced state of FL WT nNOS, in the presence (FL WT nNOS+) or absence (FL WT nNOS-) of calmodulin, determined by PELDOR spectroscopy.

νDD (MHz) Distance, r (nm) Relative integral FL WT nNOS/CaM+ 0.5, 3.5, 4.7, 5.8 5.1, 2.5, 2.2, 2.1 0.69, 0.12, 0.05, 0.14

FL WT nNOS/CaM- 1, 3.1, 4.7, 6.2 3.7, 2.6, 2.2, 2.0 0.46, 0.18, 0.20, 0.16

Four different interflavin distances have been determined from each of the spectra in

Figure 4.17 (obtained distances have been collated in Table 4.6). From the distance distribution obtained it can be seen that there are always multiple conformations existing in FL WT nNOS and that they differ with CaM being present or absent within the protein. When CaM is absent, the observed distances are spread over the range from 2.0 to 3.7 nm, with the domination of shorter interflavin distances in a range of 2.0-2.6 nm. The distance distribution observed for FL WT nNOS- is very

- + similar to that observed for the WT nNOSred when NADP was bound within.

However, when CaM is bound within FL nNOS, the observed distances distribution is dominated by a very long interflavin distance of 5.1 nm (not observed in any form of either FL nNOS or nNOSred previously) along with the shorter 2.0-2.5 nm distances observed earlier in FL WT nNOS-.

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Chapter 4 – Characterisation of full length nNOS

4.3 Discussion The primary goal of this chapter was to further our understanding of FL rat nNOS. To meet this aim, it was necessary to first optimise an expression and purification protocol to ensure high yields. In comparison to previous purification protocols (for example the protocol established by Roman et al. 226) a few changes have been introduced to ensure higher efficiency of the purification procedure, for example during E. coli growth and expression of the protein the volume of the

Terrific Broth was reduced from 1 L in 2L-flasks to 0.5 L in the same flasks to ensure higher and more homogenous aeration of the culture. Also, in those 0.5 L cultures, considering that expression of nNOS takes up to 48 h, to ensure even antibiotic selection and prevent antibiotic degradation during that time, the ampicillin was substituted with the more stable carbenicillin (as they share the same

β-lactamase gene (bla), which confers resistance to both, ampicillin and carbenicillin, a semi-synthetic ampicillin analogue). Additionally, during the purification procedure, 1 mM of L-arg and 10 µM H4B were added to all the purification buffers, including the first step resuspension buffer, while in the previous protocols, L-arg and H4B were added only to the buffer at the final purification step. All these changes caused the expression and purification of the FL nNOS WT and R1400E variant to be successful, yielding suitably high concentrations of reasonably pure protein for use in subsequent experiments.

In order to identify FL nNOS and calmodulin (in the case of the CaM+ form) mass spectrometry was employed. This technique identified the bands from a SDS-PAGE gel and confirmed with a high probability the presence of FL nNOS and also calmodulin. This is an important result as it confirms that the purification protocol is reliable and yields FL nNOS which is able to tightly bind calmodulin within the

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Chapter 4 – Characterisation of full length nNOS protein. In a similar fashion, it also confirms the lack of calmodulin in the CaM- form of the protein (as would be expected in the absence of CaM during expression) and confirms the identification of CaM from SDS-PAGE and MS.

High-pressure liquid chromatography (HPLC) has been used to identify and quantify flavins present in the protein sample. Due to the high heme absorbance peak at about

400 nm masking the flavins peak at about 456 nm, it was very important to recognise the presence of the flavin cofactors and compare the quantity of the FMN and FAD to the concentration of the protein in the examined sample. The FMN and FAD eluted as sharp peaks at the expected times provided by Aliverti et al. 196. Based on the calculations from the calibration curves, it was revealed that either WT or

R1400E FL nNOS examined by HPLC were fully saturated with FMN and FAD.

These results prove that the expression and purification protocols were reliable and highly efficient and did not cause one or both flavins to fall out of the FL nNOS.

The cytochrome c reductase activity of FL nNOS was used to obtain the kinetic parameters for WT and the R1400E variant, both in their CaM+ and CaM- forms. By varying the concentration of NADPH at fixed concentration of cytochrome c, the Km and Vmax for FL nNOS were calculated. Collected data fitted well to the standard

Michaelis-Menten equation. Comparing the kinetic parameters established for WT and the R1400E , in the presence and absence of calmodulin, it can be concluded that the presence of calmodulin affects electron transfer from NADPH via the nNOSred to the cytochrome c more in the WT than in the R1400E (9-fold Vmax difference for WT and 2-fold difference for the R1400E). When the Vmax values for the CaM- forms of

WT and R1400E are compared, it can be seen that the R1400E is about 6-fold faster than the WT in the absence of calmodulin. The maximal velocity of the cytochrome c reduction by WT and R1400E show that electron transfer through the nNOSred of

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Chapter 4 – Characterisation of full length nNOS the R1400E variant is faster than it is in WT despite R1400E being thought to affect an affinity of the protein for the NADPH (due to the mutation being located in the

NADPH binding site). Additionally it was shown that CaM binding to the FL nNOS stimulates its electron transfer to the cytochrome c by the expected magnitude as in the data published for the FL nNOS before 63,195,227,228.

In general, the FL WT nNOS herein catalyses electron transfer from NADPH to the cytochrome c at rates that are up to 2 times faster than most of those obtained and

+ -1 published by other groups (kcat observed in this thesis for FL WT nNOS is ≈145 s

-163,195,227,228 - in comparison to ≈80-100 s , when kcat observed for FL WT nNOS is ≈16 s-1 in comparison to ≈7.0-7.5 s-1 63,195,227,228). There are a number of factors to help explain why the rates differ between groups; natural activity of the enzyme batches, purity of the protein and cofactors/substrates used. Another major contributing factor may be how groups measure the “initial” rates from the slopes.

The Km values observed for FL (both CaM forms) are very much alike (≈0.7-0.9

µM), while Km values observed for R1400E (CaM+ and CaM-) is approx. 2-fold higher (≈2 µM) which shows the expected effect of the R1400E mutation (lower affinity of the protein to NADPH). The specificity constants kcat/Km observed for FL

WT and R1400E nNOS show that when CaM is bound, the kcat/Km is higher than when CaM is absent which suggests that CaM presence is causing some major conformational changes which are promoting internal electron transfer and increasing efficiency of the protein activating it.

Despite the fact that quite a few groups have reported the cytochrome c reductase activity of FL nNOS 63,195,227,228 all of them have reported the reaction rates based on the single activity measurements (measurements at a single concentration of

183

Chapter 4 – Characterisation of full length nNOS reactants), but not based on detailed steady-state kinetic studies, as has been performed in this project. The R1400E data presented in this chapter are novel and, along with the results for WT, are consistent with the cytochrome c reduction rates for the nNOSred presented in Chapter 3, Section 3.2.2.

The electron transfer rates into the FAD domain were measured by following the

NADPH oxidation activity of FL WT and R1400E variant of nNOS at 340 nm.

According to the results obtained, the NADPH oxidation rates for FL WT and

R1400E nNOS are very much alike when calmodulin is bound within the protein.

When calmodulin is absent, the observed rate reaches the level of 15% (FL WT) or

30 % (FL R1400E) of the maximum NADPH oxidation rate for the respective CaM+ form. This suggests that the R1400E mutation may affect protein/NADPH affinity but does not affect the NADPH oxidation activity of the CaM+ forms. Conversely, the NADPH oxidation rate for the CaM- form of R1400E is 2x faster than the same rate measured for the WT which implies that the R1400E protein is less sensitive to the lack of calmodulin. Under the same conditions, it is possible to achieve a higher level of performance than the WT whilst still being dependent on the calmodulin presence. The NADPH oxidation rates obtained and presented in this chapter

-1 (kcat≈1.0 s ) are consistent with data previously published by Stuehr or Adak

-1 63,195,218,229 (kcat≈1.2-2 s ) . Following analysis of the results from the NADPH oxidation and calmodulin dependence experiments, it can be concluded that calmodulin has an obvious and immediate effect on the FL nNOS activity. The FL nNOS activity is enhanced with the increase in concentration of the calmodulin in the sample. When nNOS reaches its full calmodulin saturation point, the activity of nNOS is maximal and it stays the same even when the calmodulin concentration is increased up to 5 times. This shows that calmodulin is essential for maximal NO

184

Chapter 4 – Characterisation of full length nNOS production by nNOS. In all FL nNOS studies performed so far, because no data were available for the nNOS/calmodulin dependence, it was assumed that for optimum activity, the protein to calmodulin stoichiometry should be 1:1.

Another aim of the project was the EPR redox titration of FL WT nNOS and the estimation of the FAD/FMN ratio in the protein samples. Redox titrations under strict anaerobic condition were performed to analyse flavin cofactors in the FL WT nNOS in the presence and absence of calmodulin. It was essential for the preparation of the PELDOR samples to identify the concentration of the blue semiquinone in the protein. This was carried out using an EPR-redox titration rather than the usual optical method because of the difficulty of observing the blue semiquinone absorption peak with a maximum close to 600 nm due to the heme. The heme, present in the FL nNOS sample at all times, has strong signals at ~400 nm from the

Soret peak, along with signals at 500-600 nm from the α and β peaks which mask the semiquinone peak of the flavins. Using redox titration apparatus and sodium dithionite as the reductant, it was possible to estimate the redox potential of the concentrated EPR sample which was then used for further PELDOR studies. The

EPR spectra of the one-electron-reduced state for stated redox potentials were recorded showing that it was possible to form the semi-reduced blue semiquinone state. The EPR redox titration curves were constructed based on the relative integrals obtained during the CW EPR studies.

The EPR redox titration mid-point potentials for FL WT nNOS in its CaM- and

CaM+ forms shows that calmodulin binding to the protein induces a decrease in the calculated FMN mid-point potentials, while not changing the FAD potentials significantly. The rat FL WT nNOS mid-point potentials are novel results that have not been obtained or published so far. The results obtained in this thesis for FMN

185

Chapter 4 – Characterisation of full length nNOS mid-point potentials are consistent with data published by Dunford et al. 206 for the rat nNOSred. However, calculated FAD mid-point potentials show a different trend of changes to those observed by Dunford et al. 206. This suggests that calmodulin binding affects the FAD mid-point potentials differently in the FL nNOS than it is in the nNOSred, although further dedicated studies should be performed to support this hypothesis. When ADP or NADP+ are bound to the CaM- form of FL WT nNOS, the calculated values of the mid-point potentials were affected (increased or decreased) although the errors were larger than expected when compared to a dedicated study.

The cofactor/nNOS binding mid-point potentials study needs further exploration, which unfortunately has not been feasible during research for this thesis.

Pulsed EPR spectroscopy, despite being a very powerful approach, has not been widely used to study nitric oxide synthase structure and internal electron transfer. In

2010, Astashkin et al. 230 used the RIDME technique and obtained and published data presenting the experimentally determined distance distributions between paramagnetic FMN and heme iron centres in the human form of iNOS, but since then, pulsed EPR spectroscopy data have rarely been presented and never for the rat form of the FL WT nNOS.

In NOS, large domain movements are proposed to play an important role in

22,70,206 facilitating internal electron transfer between the nNOSred and heme . Despite the crystal structure for FL WT nNOS remaining unsolved, the available crystal structure for nNOSred (1TLL) shows that the enzyme is “locked” in a conformation that prevents electron transfer between the domains, which means that catalysis, by the active site of heme, is inhibited. Binding of the activator protein, CaM, is proposed to release the FMN domain from its locked position and ensure that the

22,70,206,221 FMN cofactor is able to deliver electrons to the nNOSoxy . This is assumed

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Chapter 4 – Characterisation of full length nNOS to involve large-scale conformational movements of the entire FMN domain and allows the domain to engage in two distinct protein-protein interactions with the

22,70,206,221 FAD and nNOSoxy .

Various kinetic studies have suggested that conformational change affects electron transfer in nNOS, although this evidence was based largely on inference from a variety of kinetic experiments rather than by direct observation. Here, the pulsed electron-electron double resonance (PELDOR) spectroscopy has been used to gain an important and novel insight into the conformational landscape of the FL WT nNOS. Obtained distances showed that the presence and absence of calmodulin strongly influenced the distance distribution between the FAD and FMN semiquinones in the protein. PELDOR results represent the conformational distributions exhibited by the protein just before motion ceased as a result of a lack of thermal energy (samples are frozen at 80 K straight after the flavosemiquinone is formed and remain frozen during measurement). Using this method, the interflavin distances in FL WT nNOS, in the absence of CaM, were found to be very similar to

+ the distances that were observed for the WT nNOSred in the presence of NADP , and were characterised by evenly contributing distances of 20-37 Å representing four discrete energetic minima (four distinct conformational substates). It is though that when CaM is absent, the nNOS structure conformation is dispersed with the FMN domain being locked and FMN-heme distance being too far to allow electron transfer and protein activity, and the overall structure of FL NOS- seems to be quite broad.

Binding of CaM causes a redistribution across the conformational landscape with four interflavin distances, one very long dominating distance of 51 Å and a population of the shorter flavin-flavin distances (21-25 Å), similar to those which

- have been seen before in the FL WT nNOS and nNOSred. This 51 Å distance is

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Chapter 4 – Characterisation of full length nNOS thought to be an inter-monomer distance, indicating the major changes in the structure conformation after the CaM is bound. It suggests that, after CaM binding, the two dimers are pushed closer together to allow the electron transfer from FMN to heme to occur.

The observation of interflavin distance distribution in FL nNOS using PELDOR spectroscopy required the role of the heme domain to be considered. The nNOSoxy might not only influence the equilibrium between nNOSred conformations, but also the magnetic properties of the enzyme that impact on the outcome of the PELDOR experiment. The heme domain is cytochrome P450-like with a thiolate proximal ligand to the heme iron. However, unlike cytochromes P450, the iron is in the high spin state, even at low temperature 231. Furthermore, reduction of the heme iron during the formation of the diflavosemiquinone leaves it in the paramagnetic, although EPR ‘invisible’, S = 2 high spin ferrous state, rather than creating a diamagnetic state. This high spin iron will decrease the TM (electron spin phase memory time) of the flavinsemiquinones as they get closer to the heme making their contributions to the PELDOR data weaker and more difficult to observe.

The results presented in this chapter show a complex energy landscape with multiple conformation states and provide a novel insight in control of internal electron transfer in nNOS. The domination of the 51 Å distance suggests a solid and stable

FL nNOS conformation in the conditions used for the PELDOR studies. The crystal structure of the intact FL NOS protein still remains elusive but the knowledge about the distances obtained from PELDOR spectroscopy, which suggests an inflexible structure when CaM is bound, could be used in the future to set up the conditions for a successful FL nNOS crystallisation.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Chapter 5 High-pressure stopped-flow kinetic studies of nNOS

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

5.1 Introduction

Use of pressure as an experimental parameter for the study of biological and chemical reactions is growing. Chemical reactions are often highly pressure dependent, so use of high pressure is an elegant way to perturb, reversibly or not, chemical and biological equilibriums and reactions. Another advantage of using the pressure parameter is that reactions can be slowed down or accelerated depending on the type of interaction involved 232. For example, pressure weakens electrostatic interactions, but increases the stability of some hydrophobic interactions, such as stacking between aromatic residues 233,234.

High-pressure stopped-flow techniques have also been widely used to monitor fast reactions with gaseous fluid as reactant. For example, in Bailey’s laboratories a high- pressure stopped-flow apparatus was developed and used to follow the reaction of dioxygen with bovine cytochrome c oxidase 235. This apparatus and technique were also applied to study the reaction of nitric oxide synthase with oxygen, which was fast and was thought to take place within several steps separated by ephemeral intermediates 233. These experiments under high-pressure actually proved that oxygen binding occurred in more than one step 233. The use of extreme experimental conditions, such as low temperatures or high pressure, associated with rapid kinetic analysis, has proven to be a convenient tool to study complex reactions 201.

So far in this thesis, the flexibility of NOS has been presented and it was shown that domain movement is possible, and even necessary, in order to synthesise nitric

190

Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS oxide. Herein, the aim is to artificially control the external environment of the protein, in terms of pressure, to see how this affects electron transfer and NO production. Other researchers, such as Hay et al. 208, Jung 236 and others 237, have studied the effects of pressure on other proteins but this is the first time pressure perturbation has been used to study NADPH oxidation, flavin reduction or NO formation in NOS. It was shown in Chapter 3 and Chapter 4 by PELDOR spectroscopy that there is domain movement upon binding of CaM and also NADP+ due to the changes in the intra- and inter-flavin distances in NOS. As NOS has been shown to be flexible, applying pressure to the protein might be expected to perturb this flexibility and domain movement. In this chapter I aimed to observe how changes in atmospheric pressure affect NOS in terms of the rate of electron transfer and NO synthesis.

The oxygenase domain (NOSoxy) of nNOS receives electrons from the reductase domain (NOSred) in a mechanism similar to that catalysed by CPR and the cytochrome P450 enzymes (mentioned in Chapter 1, Section 1.2). Like CPR, the nNOSred must be reduced beyond a one-electron-reduced state to enable efficient

66 2+ electron transfer to the NOSoxy . Ca -calmodulin (CaM) binding facilitates electron transfer from the NOSred to the NOSoxy and is thought to enhance the rate of interflavin electron transfer as well (Chapter 1, Section 1.1.3.5). The CaM-binding effect was also studied by comparing the reactions performed in the presence and absence of CaM. More background to the catalytic cycle and effects of CaM are described in Chapter 1, Section 1.1.2 and 1.1.3.5. Catalytic turnover, starting with

NADPH oxidation and ending with NO production, involves complex reactions and analysis, which can provide information about the overall NOS catalytic cycle.

Breaking down the steps in the cycle, Knight and Scrutton 194 looked at the rat

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

nNOSred in order to measure the individual phases in flavin reduction. However, this analysis has never been carried out for FL nNOS (FL nNOS) and never under any pressure other than atmospheric (≈1 bar). Hence, the reduction by NADPH of the

FAD and FMN redox centres in the isolated nNOSred and the FL version of rat nNOS have now been studied by anaerobic stopped-flow spectroscopy using single wavelength absorption detection. These studies aimed to establish the mechanism of electron flow, the role of bound CaM, and the effect of high-pressure on the overall flavin reduction in nNOSred together with FL nNOS. The high-pressure approach was also used to study the effect of high hydrostatic pressure on NADPH oxidation and NO production in nNOS. The flavin reduction and nNOS activity were also studied for the R1400E variant (an Arg1400 mutation in the NADPH-binding site mentioned in Chapter 1, Section 1.1.3.2) to examine if and how the mutation affects electron transfer through the protein under high pressure.

With chemical reactions often being highly pressure dependent, a perturbation of the elementary steps of electron transfer within nNOSred by pressure therefore offers the possibility for a detailed characterisation of the enzyme mechanism. High hydrostatic pressure has not been extensively used in nitric oxide synthase in the past. This may be because of the complexity of the reaction catalysed by nNOS, the number of substrates and cofactors essential for the maximum protein performance and various other conditions, which need to be considered and which may greatly affect the obtained results.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

5.2 Results

It was not known beforehand which model would fit best and how many, if any, exponentials would be required to describe adequately a particular kinetic curve, so several separate fits were performed on the data set, each using different models and numbers of exponentials. The standard criteria, which were used to choose which model best describes the data, included: residuals, autocorrelation function values and the R2 value (statistical goodness-of-fit parameter) 201.

5.2.1 NADPH oxidation

In the NOS-catalysed reaction, formation of NO and L-citrulline in the presence of molecular oxygen is accompanied by the oxidation of NADPH, which provides a convenient assay for measuring NOS activity. NADPH oxidation has been followed fluorimetrically by Onufriev and Gulyaeva 200 to measure NOS activity but the effects of high pressure on the rate of NADPH oxidation, catalysed by FL nNOS, has never been studied before.

Steady-state reactions were performed under aerobic conditions as molecular oxygen is necessary for the entire NOS catalytic cycle to occur (detailed background of the technique and assay conditions are described in Section 2.2.3.5 and 2.2.3.5.2).

Saturating concentrations of NAPDH and L-arginine were used so these were not limiting the rate of reaction. Air-saturated (200 µM O2) buffer was used to ensure that the lack of oxygen was not the limiting factor of the reaction. The rate of

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NADPH oxidation was measured under steady-state turnover conditions in a high- pressure stopped-flow apparatus by following the decrease in absorption at 340 nm.

The decrease in absorption at 340 nm was fitted to the standard linear equation model using Kinetic Studio (TgK Scientific) software to extract the apparent reaction velocity.

The NADPH oxidation rates for WT and R1400E nNOS, in the presence (CaM+) and absence of CaM (CaM-), are represented by very similar values (≈1 s-1) (Figure

5.1 and 5.2). In both cases the rate increased after binding CaM (≈7-fold increase for

-1 WT and ≈3-fold increase for R1400E) and reached a similar Vobs ≈1 s activity level.

The rate of NADPH oxidation increased when the high pressure was applied and

CaM was absent and decreased when CaM was present, just to reach a similar

-1 NADPH oxidation rate (Vobs ≈0.5-0.6 s ) at 1750 bar, independently of the CaM presence. The NADPH oxidation rates observed for the R1400E variant under different pressures are very similar to the WT rates. Based on that, it can be concluded, that the R1400E mutation, despite lowering the nNOS/NADPH affinity in different forms of nNOS (shown previously in Chapter 4, Section 4.2.5), does not have a big impact on the nNOS rate limiting step or the overall NADPH oxidation rate.

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Figure 5.1 The steady-state rates of NADPH oxidation as a function of hydrostatic pressure for the FL WT nNOS in the presence and absence of CaM. The dependence of the rate of NADPH oxidation on hydrostatic pressure is shown in the presence (A) and absence of CaM (B). The blue points represent the rates measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rates in the depressurised sample at the pressure of 0 bar. NADPH oxidation rates were measured in a high-pressure stopped-flow apparatus at 340 nm. Error bars represent one standard deviation calculated from the average of at least three reductive transients. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.2.

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Figure 5.2 The steady-state rates of NADPH oxidation as a function of hydrostatic pressure for the FL R1400E nNOS in the presence and absence of CaM. The dependence of the rate of NADPH oxidation on hydrostatic pressure is shown in the presence (A) and absence of CaM (B).The blue points represent the rates measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rates in the depressurised sample at the pressure of 0 bar. NADPH oxidation rates were measured in a high-pressure stopped-flow apparatus at 340 nm. Error bars represent one standard deviation calculated from the average of at least three reductive transients. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.2.

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5.2.2 Flavin reduction

The pre-steady state reduction of the FAD and FMN in the nNOSred and in

FL nNOS by NADPH (in excess) has been studied in the presence and absence of

CaM as a function of pressure by following the absorption change at 458 nm in a high-pressure stopped-flow spectrometer. The 458 nm wavelength has been chosen because it allows one to follow the flavin reduction signal with no interference from the heme reduction signal (even if heme is converted from ferric to ferrous, no change in absorption at 458 nm is observed; as can be seen in Chapter 4, Section

4.2.1, Figure 4.3 and 4.5). Reactions were performed under anaerobic conditions

(detailed assay conditions in Section 2.2.3.5 and 2.2.3.5.1) and transients were fitted to the standard two exponential equation model using Kinetic Studio (TgK

Scientific) software.

It was previously observed by Knight and Scrutton194, that at least three resolvable steps are observed in stopped-flow studies of nNOSred flavin reduction with

NADPH. The first reductive step represented the rapid formation of an equilibrium between an NADPH-enzyme charge transfer species and two-electron-reduced enzyme bound to NADP+. In the work of Knight and Scrutton the first rate constant was in a range of >500 s-1, and it has not been observed in this thesis due to the equipment limitations (5 ms instrument deadtime). The second and third steps observed by Knight and Scrutton represented further reduction of the enzyme flavins and NADP+ release (those rate constants were possible to observe in this thesis and they are presented in this chapter, in the section below).

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5.2.2.1 The single turnover flavin reduction in WT and R1400E nNOSred.

The decrease in absorbance observed at 458 nm for the WT nNOSred can be fitted using a two exponential equation, providing two rate constants, k1 and k2. The flavin reduction first rate constant, k1, in WT nNOSred does not appear to be

- significantly affected by CaM binding (k1 for either CaM+ or CaM- form was ≈30 s

1 ), however a 5-fold decrease in second rate constant, k2, can be observed (k2 was ≈1 s-1for CaM+ and ≈5 s-1 for CaM-) (Table, 5.1, Figure 5.3-5.5). The rates constants for both steps have also been measured at a range of hydrostatic pressures, which revealed different effects for k1 and k2. The rate constant of the first step for the WT nNOSred, with CaM+ decreases at higher pressure and exhibits approximately a 2- fold decrease at 1750 bar compared to 0 bar (Table 5.1, Figure 5.4). However, the rate constant of the second step increases at higher pressure and is approximately 5- fold higher at 1750 bar (Table 5.1, Figure 5.4). Hence, the high pressure positively affects one of the flavin reduction steps and negatively affects the other, suggesting that it may be influencing conformational change within the nNOS structure.

However, in the absence of CaM the first rate constant remains relatively unchanged at higher pressures whereas the second rate constant actually decreases at higher pressures (Table 5.1, Figure 5.5). This may suggest that CaM binding is most affected by high pressure.

For the R1400E variant, studied earlier in this thesis and again here, the first flavin reduction rate constants are very similar (100-140 s-1, Figure 5.7 and 5.8) in both the presence and absence of CaM. This first rate constant is about five-fold faster than the first rate constants for the WT nNOSred in either the presence of absence of CaM.

This may suggest that the R1400E mutation is promoting one of the steps in the interflavin electron transfer within the nNOSred resulting in a faster rate of transfer.

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Similar values for k1 for both CaM+ and CaM- versions of the R1400E nNOSred suggest that there is no significant effect of CaM on the first step of electron transfer within the NOSred. The second step (k2) in the presence of CaM is initially about 5- fold slower than k1 and displays a linear dependence on pressure. In the absence of

CaM however, the second flavin reduction rate constant starts lower than in the presence of CaM (≈9 s-1 compared to ≈20 s-1 in CaM+). With increasing pressure the rate constant, in the absence of CaM, also increases to approximately twice its original value and reaches a value of ≈20 s-1, which is the same as the initial rate in the presence of CaM (Table 5.1, Figure 5.7 and 5.8). The k2 rate constant is pressure dependent and the data imply it is possible to increase k2 for the R1400E variant in the absence of CaM to the same magnitude as when CaM is bound using high hydrostatic pressure only. If the transients are now analysed in more detail it is clear that the amplitude decreases as the pressure is increased in all cases (Table 5.1,

Figure 5.3 and 5.6). The transients are monitored at 458 nm and it may not be unexpected that the amplitude of the transients will decrease as the protein environment around the flavins may be altered at higher pressure. There appears to be no clear correlation between the amplitude changes and the changes in the observed rate constants. It can be deduced that the decrease in amplitude as pressure increases is purely due to the presence of the external pressure, probably due to the flavin becoming less exposed or the protein partially denaturing as pressure increases.

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Table 5.1 k1 and k2 flavin reduction rate constants and respective amplitudes observed at different pressures for WT and R1400E nNOSred, in the presence (CaM+) and absence (CaM-) of CaM. ‘0 bar’ rate constants were observed under the atmospheric pressure (when no external pressure was used), ‘1750 bar’ rate constants were observed under 1750 bar hydrostatic external pressure and ‘0 bar DOWN’ rate constants were observed after the sample has been depressurised back to 0 bar (atmospheric pressure) after being pressurised up to 1750 bar pressure earlier. The rate constants and amplitudes values are the average values of at least three measurements.

0 bar 1750 bar 0 bar DOWN -1 -1 -1 -1 -1 -1 k1 [s ] Amp1 k2 [s ] Amp2 k1 [s ] Amp1 k2 [s ] Amp2 k1 [s ] Amp1 k2 [s ] Amp2

WT nNOSred/CaM+ 30.03 -0.076 0.91 -0.014 14.66 -0.049 5.01 -0.007 32.10 -0.067 4.82 -0.007 ± 1.87 ± 0.29 ± 2.97 ± 0.59 ± 2.64 ± 0.49

WT nNOSred/CaM- 29.36 -0.063 5.29 -0.046 22.72 -0.068 2.69 -0.0081 32.05 -0.061 5.90 -0.037 ± 3.01 ± 0.21 ± 1.25 ± 0.33 ± 1.51 ± 0.17

R1400E nNOSred/CaM+ 91.44 -0.054 20.67 -0.043 80.23 -0.055 4.84 -0.005 78.62 -0.053 12.80 -0.012 ± 3.50 ± 1.23 ± 2.87 ± 0.82 ± 2.59 ± 0.67

R1400E nNOSred/CaM- 140.93 -0.058 8.60 -0.027 100.28 -0.038 15.06 -0.006 89.23 -0.056 9.81 -0.006 ± 5.83 ± 1.62 ± 4.55 ± 1.52 ± 3.62 ± 1.12

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.3 Examples of flavin reduction transients observed for the WT nNOSred in the presence and absence of CaM. Flavin reduction transients for WT nNOSred in its CaM+ (A) and CaM- (B) form. The blue-green transients represent the absorption change measured at increasing pressures from 0 to 1750 bar whereas the red transient shows the absorption change in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.4 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the WT nNOSred in the presence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The navy points represent the rate constants measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rate constants in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1.Error bars represent one standard deviation calculated from the average of at least three reductive transients.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.5 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the WT nNOSred in the absence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The navy points represent the rate constants measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rate constants in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1.Error bars represent one standard deviation calculated from the average of at least three reductive transients.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.6 Examples of flavin reduction transients observed for the R1400E nNOSred in the presence and absence of CaM. Flavin reduction transients for R1400E nNOSred in its CaM+ (A) and CaM- (B) form. The blue-green transients represent the absorption change measured at increasing pressures from 0 to 1750 bar whereas the red transient shows the absorption change in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.7 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the R1400E nNOSred in the presence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The navy points represent the rate constants measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rate constants in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1. Error bars represent one standard deviation calculated from the average of at least three reductive transients.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.8 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the R1400E nNOSred in the absence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The navy points represent the rate constants measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rate constants in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1. Error bars represent one standard deviation calculated from the average of at least three reductive transients.

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5.2.2.2 The single turnover flavin reduction in FL WT and R1400E nNOS.

The decrease in absorbance at 458 nm for the FL WT NOS can also be fitted to two observed rate constants in the absence and presence of CaM (Table 5.2,

Figure 5.9-5.11). As in the nNOSred, increasing the pressure leads to a decrease in the amplitude change. The first rate constant (k1) is relatively unchanged over the whole pressure range in the presence of CaM at approximately 80 s-1 (Table 5.2, Figure

5.10). However, in the absence of CaM k1 shows irreversible pressure dependence, with an initial rate constant of ≈16 s-1 which increases with the pressure to ≈28 s-1, and stays on that level when depressurised (≈30 s-1) (Table 5.2, Figure 5.11).

The second rate constant (k2) for the WT FL protein is much slower than k1 in both the presence and absence of CaM. In the presence of CaM, k2 shows a dependence on the pressure with an initial rate constant of 8 s-1, increasing to ≈15 s-1 as the pressure is gradually increased from 0 to 1750 bar (Table 5.2, Figure 5.10). In the absence of CaM (Table 5.2, Figure 5.11) k2 is lower than in the presence of CaM with a rate constant of ≈3 s-1. The data show a 3-fold decrease in rate constant as the pressure increases to a final value of ≈1 s-1. This suggests that the effect of high pressure on the FL WT nNOS is greater on some of the flavin reduction steps when the CaM is bound within the protein.

It was also observed that the rate constants for the WT protein are highest at a pressure of approximately 500 bar, with a decrease in rate constants or no observed changes when higher pressures were used. This suggests that 500 bar is the top limit of the pressure to be used to study the protein activation and positive effect of the pressure on FL WT nNOS without the protein being denatured or deactivated, which may be happening under pressures higher than 500 bar.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

For the FL R1400E variant the general trend is once again observed in that the amplitude of the absorbance changes decrease as the pressure increases (Table 5.2,

Figure 5.112). The first rate constant (k1) for the FL R1400E variant in the presence of CaM displays a slight dependence on pressure (Table 5.2, Figure 5.13), starting at

-1 -1 ≈65 s and increasing to ≈100 s . The second rate constant (k2) shows no real dependence on pressure with a rate constant of ≈8-9 s-1.

In the absence of CaM (Table 5.2, Figure 5.14) there is a significant dependence of

-1 k1 on the pressure. With an initial rate constant of ≈20 s at 0 bar, this rate constant

-1 increases 6-fold to a final first rate constant of ≈120 s at 1750 bar, and after that k1 doesn’t change significantly when the sample is depressurised. This trend is also

-1 -1 observed for k2, with a 3-fold increase from ≈6 s to ≈18 s , with the difference that

-1 -1 the k2 decreases 2-fold (from ≈18 s to ≈9 s ) when depressurised. This shows that high pressure is positively affecting at least one of the steps of the interflavin electron transfer in the FL R1400E protein. It is another result which suggests that the pressure affects the rate constants of the CaM- forms of the FL protein more than when CaM is bound within the protein. No change was observed for k1 and 2-fold change was observed for k2, when the sample was depressurised from 1750 bar back to 0 bar, suggesting that irreversible changes are taking place at high pressures.

The flavin reduction rate constants measured for the depressurised sample (0 bar

DOWN) in all FL R1400E nNOS cases are higher than the respective initial flavin reduction rate constants studied at the 0 bar pressure, however when the amplitudes and transients were analysed (Table 5.2, Figure 5.9 and 5.12), it was observed that the depressurised samples for FL R1400E nNOS, in the absence of CaM were characterised by faster than initial flavin reduction rate (all observed rate constants higher than initial). This was never observed for either FL WT or R1400E nNOS in

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

the presence of CaM or for any studied form of the nNOSred. It may suggest that major irreversible change in the conformational structure of the protein (CaM- form) is promoting the interflavin electron transfer and flavin reduction has been forced by the high pressure. However, the effect of this change has not been seen under high pressure but only when the sample was depressurised (Table 5.2, Figure 5.9 and

5.12).

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Table 5.2 k1 and k2 flavin reduction rate constants and respective amplitudes observed at different pressures for FL WT and R1400E nNOS, in the presence (CaM+) and absence (CaM-) of CaM. ‘0 bar’ rate constants were observed under the atmospheric pressure (when no external pressure was used), ‘1750 bar’ rate constants were observed under 1750 bar hydrostatic external pressure and ‘0 bar DOWN’ rate constants were observed after the sample has been depressurised back to 0 bar (atmospheric pressure) after being pressurised up to 1750 bar pressure earlier. The rate constants and amplitudes values are the average values of at least three measurements.

0 bar 1750 bar 0 bar DOWN -1 -1 -1 -1 -1 -1 k1 [s ] Amp1 k2 [s ] Amp2 k1 [s ] Amp1 k2 [s ] Amp2 k1 [s ] Amp1 k2 [s ] Amp2 FL WT nNOS/CaM+ 80.28 -0.03 8.18 -0.02 65.09 -0.02 13.34 -0.01 82.53 -0.03 8.13 -0.02 ± 2.16 ± 0.42 ± 7.72 ± 0.67 ± 4.63 ± 0.39 FL WT nNOS/CaM- 16.00 -0.03 4.15 -0.02 27.66 -0.03 1.49 -0.01 29.62 -0.04 3.34 -0.02 ± 0.96 ± 0.28 ± 1.82 ± 0.30 ± 0.69 ± 0.17 FL R1400E nNOS/CaM+ 64.44 -0.02 9.64 -0.01 86.53 -0.01 7.25 -0.01 78.62 -0.02 12.80 -0.01 ± 3.85 ± 0.24 ± 1.51 ± 0.27 ± 4.59 ± 0.57 FL R1400E nNOS/CaM- 20.06 -0.02 5.88 -0.02 126.18 -0.02 18.83 -0.01 121.29 -0.02 9.25 -0.02 ± 1.86 ± 0.72 ± 4.59 ± 0.89 ± 5.93 ± 0.56

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.9 Examples of flavin reduction transients observed for FL WT nNOS in the presence and absence of CaM. Flavin reduction transients for FL WT nNOS in its CaM+ (A) and CaM- (B) form. The blue-green transients represent the absorption change measured at increasing pressures from 0 to 1750 bar whereas the red transient shows the absorption change in the depressurised sample at the pressure

of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.10 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the FL WT nNOS in the presence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The navy points represent the rate constants measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rate constants in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1. Error bars represent one standard deviation calculated from the average of at least three reductive transients.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.11 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the FL WT nNOS in the absence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The navy points represent the rate constants measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rate constants in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1. Error bars represent one standard deviation calculated from the average of at least three reductive transients.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.12 Examples of flavin reduction transients observed for FL R1400E nNOS in the presence and absence of CaM. Flavin reduction transients for FL R1400E nNOS in its CaM+ (A) and CaM- (B) form. The blue-green transients represent the absorption change measured at increasing pressures from 0 to 1750 bar whereas the red transient shows the absorption change in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.13 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the FL R1400E nNOS in the presence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The navy points represent the rate constants measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rate constants in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1. Error bars represent one standard deviation calculated from the average of at least three reductive transients.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.14 Observed rate constants for flavin reduction as a function of hydrostatic pressure for the FL R1400E nNOS in the absence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The navy points represent the rate constants measured by increasing the pressure from 0 to 1750 bar whereas the red points represent the rate constants in the depressurised sample at the pressure of 0 bar. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.1. Error bars represent one standard deviation calculated from the average of at least three reductive transients.

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5.2.3 NO synthesis

In order to study the formation of NO by FL nNOS it was necessary to develop a system to detect small amounts of NO. For this purpose oxyhaemoglobin was prepared from commercial haemoglobin, according to the method of Salter &

Knowles 202. Commercially available crystallised haemoglobin usually consists largely of the ‘met’ form and must be converted to its reduced oxy-form before it is used in the assay. Oxyhaemoglobin was readily prepared by dissolving commercial haemoglobin in distilled water and adding sodium dithionite powder to promote reduction from methaemoglobin (dark brown) to deoxyhaemoglobin (wine red). A light stream of oxygen was then blown over the deoxyhaemoglobin to form oxyhaemoglobin (HbO2, bright red). The resulting HbO2 solution was then desalted, purified, aliquoted and stored at - 20 °C for further use. A description and conditions of the oxyhaemoglobin preparation protocol are described in Section 2.2.3.5.3.

The NO synthesis assay is based on the method described previously 202,203,238 to follow the reaction between the NO produced by NOS and the oxygenated, ferrous form of haemoglobin (oxyhaemoglobin, HbO2). The single turnover reaction was performed under aerobic conditions (detailed assay conditions are described in

Section 2.2.3.5 and 2.2.3.5.3) and the NO-mediated conversion of oxyhaemoglobin

- (OxyHb) to methaemoglobin (MetHb) in its ferric form, with nitrate (NO3 ) as an additional product, was studied in a high-pressure stopped-flow apparatus at 401 nm.

Transients were fitted to the two exponential equation model using Kinetic Studio

(TgK Scientific) software.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.15 UV-visible absorbance spectra of oxyhaemoglobin (HbO2) and methaemoglobin (MetHb). The NO synthesis reaction assay follows the reaction between the NO produced by nNOS, and the oxygenated, ferrous form of haemoglobin (oxyhaemoglobin, HbO2). The reaction was studied in a high-pressure stopped-flow apparatus at 401 nm. Conditions: 40mM HEPES, pH 7.6, 150mM NaCl, 10% (v/v) glycerol and 1mM CaCl2.

The UV-visible absorbance spectrum of oxyhaemoglobin contains three distinctive peaks with the main Soret peak at 418 nm and 2 smaller peaks between 500-600 nm

(approximately at 542 and 577 nm, Figure 5.15). When oxyhaemoglobin is converted to the high spin ferric methaemoglobin, the dominant Soret peak shifts to

401 nm and the peaks within the 500-600 nm region disappear.

The observed NO synthesis assay based on the NO-mediated conversion of OxyHb to MetHb provided two exponential rate constants, k1 and k2. There is little known

218

Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS about the mechanism of the OxyHb-NO reaction but based on what has been published so far, it can be hypothesised that k1 is the NO to OxyHb binding rate

(which in theory should be equal to the NO synthesis rate) whereas k2 is the nitrite release rate. Additionally, the k1, for both WT and the R1400E variant are very similar to the NADPH oxidation rates observed earlier (this chapter and Chapter 4) for the respective forms of the nNOS.

Both observed rate constants, k1 and k2, are very similar for both, FL WT and

R1400E nNOS, in the presence of CaM (Table 5.3). The initial (at 0 bar pressure) absorbance change recorded over 10 seconds for FL WT nNOS is slightly higher than the absorbance change observed for the R1400E variant under the same conditions, with the initial k1 and k2 for FL WT nNOS (Table, 5.3, Figure 5.17)

-1 -1 being faster (significantly – k1 ≈2.3 s or slightly – k2 ≈0.29 s ) than the initial k1

-1 -1 and k2 for the FL R1400E nNOS (k1 ≈1.6 s , k2 ≈0.28 s , Table 5.3, Figure 5.19). At relevant pressures, the rate constants of both, the first and second phases of NO formation, in either WT or R1400E, are very similar and they all decrease significantly with the pressure. All the above suggest that introduction of the

R1400E mutation is not major (slightly lower initial k1) and has no significant effect on the nNOS activity at pressures higher than 0 bar.

What has been also observed is that the NO formation rate constants observed for the depressurised samples (0 bar DOWN) are, in all cases, lower than initial rate constants of this reaction and this suggests that that the application of high pressure is causing partial denaturation of the protein preventing it from restoring its initial activity.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Only assays of FL WT and R1400E nNOS in the presence of CaM showed any formation of NO. In the absence of CaM, no absorbance change was observed for either WT or the R1400E variant of nNOS, confirming that FL nNOS in the absence of CaM is not able to undergo catalysis and produce NO. Moreover, in the absence of CaM there was also no NO synthesis activity observed at higher pressures.

To make sure that the observed rate constants are the actual OxyHb into MetHb formation rates, and that the OxyHb-NO binding rate is not a limiting step, the anaerobic control reaction was observed. In the control reaction, OxyHb (10µM) was mixed with anaerobic buffer (the same which was used for NO formation assay using nNOS) saturated with 25 µM of NO. The transients were fitted using a single exponential equation model. The rate of OxyHb-NO binding were observed at different pressures; at 0-1000 bar the rates were rapid and too fast to be measured due to the 5 ms deadtime of the stopped-flow equipment; when at the pressures higher than 1000 bar, OxyHb-NO biding rates decreased and reached the level of

130-150 s-1 (available to observe), which is still much faster than any of the NO formation rate constants observed in this chapter. This proved that the OxyHb-NO binding rate is very fast and it is not limiting the rate constants observed in this chapter in any way.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Table 5.3 k1 and k2 rate constants and respective amplitudes of NO-mediated OxyHb into MetHb conversion observed at different pressures (0-1500 bar) for FL WT and R1400E nNOS, in the presence (CaM+). The rate constants and amplitudes values are the average values of at least three measurements.

Pressure FL WT nNOS/CaM+ FL R1400E nNOS/CaM+ -1 -1 -1 -1 k1 [s ] Amp1 k2 [s ] Amp2 k1 [s ] Amp1 k2 [s ] Amp2 0 bar 2.37 0.12 0.29 0.29 1.61 0.21 0.28 0.28 ± 0.20 ± 0.01 ± 0.09 ±0.02 500 bar 1.17 0.11 0.14 0.13 1.22 0.11 0.15 0.15 ± 0.02 ± 0.01 ± 0.02 ± 0.01 1000 bar 0.95 0.06 0.10 0.1 1.03 0.06 0.10 0.13 ± 0.04 ± 0.01 ± 0.06 ± 0.01 1500 bar 0.73 0.03 0.08 0.05 0.83 0.02 0.07 0.08 ± 0.01 ± 0.01 ± 0.05 ± 0.01 0 bar DOWN 1.09 0.08 0.17 0.13 1.24 0.11 0.16 0.15 ± 0.10 ± 0.01 ± 0.03 ± 0.02

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.16 Absorption transients for NO-mediated OxyHb into MetHb conversion (NO synthesis by FL WT nNOS) in the presence (CaM+) or absence (CaM-) of CaM. Absorption transients at 0, 500, 1000 and 1500 bar pressure showing the absorption change at 401 nm as oxyhaemoglobin is converted to methaemoglobin upon binding to the NO produced by FL WT nNOS. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.3.

Figure 5.16 shows the absorption changes at 401 nm. In this figure, it can be seen that the absorbance change amplitudes are decreasing significantly at higher pressures, which suggests that the pressure has a negative effect on the observed reaction and that at higher pressures the OxyHb to MetHb conversion is not as efficient as it is when high pressure is not applied. The depressurised sample (0 bar

DOWN) seems to recover ≈30 % of the initial activity. No activity has been observed when CaM is not present in the reaction.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.17 Observed rate constants for NO synthesis as a function of hydrostatic pressure measured for FL WT nNOS in the presence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The red points represent the rate constants in the depressurised sample at the pressure of 0 bar (0 bar DOWN). NO synthesis rate constants were measured in a high-pressure stopped- flow apparatus at 401 nm. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.3. Error bars represent one standard deviation calculated from the average of at least three transients.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.18 Absorption transients for NO-mediated OxyHb into MetHb conversion (NO synthesis by FL R1400E nNOS) in the presence (CaM+) or absence (CaM -) of CaM. Absorption transients at 0, 500, 1000 and 1500 bar pressure showing the absorption change at 401 nm as oxyhaemoglobin is converted to methaemoglobin upon binding to the NO produced by FL R1400E nNOS. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.3.

Figure 5.18 shows the absorption changes transients for NO-mediated OxyHb into

MetHb conversion observed at 401 nm. In this figure, it can be seen that the absorbance change amplitudes decrease significantly at higher pressures, similar to that observed for FL WT nNOS in Figure 5.16. Again, the depressurised sample (0 bar DOWN) seems to recover ≈50 % of the initial activity when no significant absorbance changes can be observed and when CaM is not present in the reaction.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

Figure 5.19 Observed rate constants for NO synthesis as a function of hydrostatic pressure measured for FL R1400E nNOS in the presence of CaM. The dependence of k1 (A) and k2 (B) on hydrostatic pressure is shown. The red points represent the rate constants in the depressurised sample at the pressure of 0 bar (0 bar DOWN). NO synthesis rate constants were measured in a high-pressure stopped- flow apparatus at 401 nm. Assay conditions as described in Chapter 2, Section 2.2.3.5 and 2.2.3.5.3. Error bars represent one standard deviation calculated from the average of at least three transients.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

5.3 Discussion

The work carried out in this chapter aimed to describe the characterisation of rat neuronal nNOS activity under high hydrostatic pressure up to 1750 bar. The effect of the high pressure on flavin reduction, NADPH oxidation and NO synthesis in FL nNOS or nNOSred has been studied by both anaerobic and aerobic stopped- flow spectroscopy. The high-pressure approach was also used to study the possibility of increasing the activity of nNOS in the absence of CaM by using high hydrostatic pressure to perturb the structure of the protein and its complex reaction mechanism.

Previously, high pressure studies have been used to study CO binding to the heme cofactor of NOS and P450 BM3 232, binding of oxygen and CO to the reduced heme of the isolated oxygenase domain of eNOS 239,240 or cytochrome P450 240 and to examine the kinetics of oxygen binding to NOS at low temperatures. However, it has never previously been used to study the kinetics of flavin reduction, NADPH oxidation or NO synthesis.

Steady state NADPH oxidation activities of nNOS were measured at 340 nm. In the absence of substrate, NADPH oxidation by nNOS results in superoxide formation. In the absence of CaM, superoxide formation occurs via the flavins of the NOSred, because electrons are not passed to the heme 221. A number of studies 62,218,241 or

Adak et al. 63,195 have measured the rate of NADPH oxidation in nNOS previously and the NADPH oxidation rates presented in this chapter are very similar to these values (NADPH oxidation rates published before are in range of ≈1-2 s-1, whereas rate constants observed in this chapter are in the range of 1-1.2 s-1). The rate of

NADPH oxidation in the R1400E variant, which is thought to be a key mutation for

NADPH binding, is very similar to the WT. For both the WT and R1400E nNOS the

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NADPH oxidation rates decrease at higher pressures when CaM is present, and increase when CaM is absent. Significantly, at higher pressures (1750 bar) the rate of

NADPH oxidation is very similar in both the absence and presence of CaM (0.5-0.6 s-1). The increase of the NADPH oxidation rate when CaM is absent (from 0.15-0.2 s-1 up to 5.5-6.0 s-1) suggests that the high hydrostatic pressure may cause a conformational change of the nNOS structure that may promote the electron transfer and increase the initial NADPH oxidation rate by 3-4 fold. Similar NADPH oxidation rates observed at the highest used pressures (1500-1750 bar) for both

CaM+ and CaM- forms of WT and R1400E nNOS suggest that, at certain point, under very high pressures, the pressure is mimicking the effect that CaM has on protein and CaM binding does not affect the NADPH-oxidation activity of nNOS anymore, independently on the CaM presence or absence in the sample.

Interestingly, all of the effects of high pressure on the rates of NADPH oxidation reaction were reversible and returned to the circa initial values after depressurisation.

This is a very important finding as it suggests it may be possible to increase slightly the NADPH oxidation activity of nNOS when CaM is absent by perturbing the system only with high pressure; such a conclusion has not been reached or published before.

The flavin reduction kinetics for NOS have previously been studied under various conditions using stopped-flow approaches 38,63,101,194,195,218,241. Several of these studies simplified the flavin reduction kinetics by fitting the spectral changes to a single exponential function 218,241 whereas others required multiple exponential functions to accurately fit the data due to electron transfer between the two flavin cofactors (3 exponential rate constants with the fastest rate constant being in the range over 500 s-1) 194. However, it was not possible to measure the fastest of these

227

Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS rate constant by using the high-pressure stopped flow instrument presented and used in this chapter because of the limitations of the apparatus (5 ms deadtime). As the single exponential rate are likely to be too simplistic for the complex reaction pathway in NOS, the kinetic transients obtained here for WT and R1400E nNOS

(both NOSred and FL, in the presence and absence of CaM) were fitted to two exponentials, which is consistent with other studies 63,195,227. However, it was not possible to compare the flavin reduction rate constants observed under high pressures with any other studies due to the fact that no reports using higher than atmospheric pressure have been published so far. The applications of high pressure to nNOS systems and results presented in this thesis are novel and have never been reported before.

No significant effect on flavin reduction rate constants has been observed when CaM was bound to the WT or R1400E nNOSred, which is consistent with what has been observed by Knight & Scruttion 194 previously. When CaM was bound to the FL WT

-1 nNOS, the flavin reduction rate constants increased significantly (k1: from ≈16 s to

-1 -1 -1 ≈80 s ; k2 from ≈4 s to ≈8 s ), which suggests that CaM is promoting the electron transfer and flavin reduction in the FL more than it is in the nNOSred. High pressure applied to the WT nNOSred caused a decrease in all of the observed rate constants except for the observed k2 value for WT nNOSred/CaM+, which increased from ≈0.9 s-1 to ≈5 s-1. In the FL WT nNOS/CaM+ sample high pressure caused a decrease in

-1 -1 -1 the k1 value (from ≈80 s to ≈65 s ) but an increase in the k2 value (from ≈8 s to

≈13 s-1). When CaM was absent, the high pressure had an opposite effect to that

-1 -1 observed for FL WT nNOS/CaM+ (k1 rate constant increased from ≈16 s to ≈27 s ,

-1 -1 whereas k2 rate constant decreased from ≈4 s to ≈1.5 s ). This suggests that CaM binding affects the FL form of nNOS more than it affects the NOSred. It also shows

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS that binding of CaM within the FL WT nNOS affects the conformational equilibrium within nNOS, allowing for different effects of high pressure on the protein. This supports previous results suggesting that CaM influences the conformational equilibria in nNOS 22,97–99,242.

The introduction of the R1400E mutation has different effects on nNOSred and FL

WT nNOS. In the FL WT nNOS, the mutation caused a decrease in the k1 value

-1 -1 -1 -1 (from ≈80 s to ≈64 s ) and a slight increase in the k2 value (from ≈8 s to ≈9.5 s ) when CaM was present, whereas a slight increase was observed in both k1 and k2 (k1:

-1 -1 -1 -1 ≈16 s to ≈20 s ; k2: ≈4 s to ≈5.5 s ) when CaM was absent. In nNOSred, the mutation increased significantly all of the rate constants (for CaM+: k1 changed from

-1 -1 -1 -1 ≈30 s to ≈90 s , and k2 changed from ≈0.9 s to ≈20 s , when for CaM-: +: k1

-1 -1 -1 - changed from ≈30 s to ≈140 s , and k2 changed from ≈5 s to ≈8.5 s 1) independently of the presence of CaM. This is slightly unexpected considering that the flavin reduction rate constants observed for R1400E nNOSred are much higher than observed for WT. It was previously shown that the R1400E mutation is promoting the internal electron transfer despite lowering the affinity of the protein for NADPH 70. However, it was never observed prior to this that the mutation increases the rate constants. There could be two explanations for this; firstly the

R1400E mutation may be causing conformational changes within nNOSred and promoting flavin reduction and electron transfer within the NOSred. Secondly, it may be that due to the dead time of the available high pressure stopped-flow equipment the first rapid rate constant for WT (which were reported before being as fast as

>500 s-1 194) were missed and that this rate constant is actually decreased to ≈90-140 s-1 when the R1400E mutation is introduced (which can be observed in equipment with 5 ms deadtime). As no studies on flavin reduction in the R1400E variant of

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

nNOSred or nNOS have been published so far, there are currently no data available for comparison. Further dedicated studies on flavin reduction in this nNOS variant are necessary to provide more a detailed explanation of the observations reported here.

The amplitudes for all of the measured flavin reduction rate constants decreased with higher pressures for either WT or R1400E, nNOSred or FL, independently of the

CaM presence. Surprisingly, in FL nNOS, when CaM was absent, the depressurised samples (0 bar DOWN) were characterised by reductive transients with amplitudes higher than initially observed. This has not been observed for nNOSred, and it may suggest that pressure is causing conformational changes and that the initial protein structure is not fully restored when the sample is depressurised, but in case of FL nNOS/CaM- those changes are actually promoting the flavin reduction rather than inhibiting them.

NO formation has previously been studied spectrophotometrically under steady-state conditions 62,243,244, Adak et al. 63,195 37 but has never been measured under single turnover conditions by using a stopped-flow approach. The rate of NO formation was fitted using a two exponential equation model for both FL WT and R1400E nNOS in the presence of CaM. The first rate constants for either WT or the R1400E are quite similar (≈1.6-2.2 s-1) and are likely to represent the rate of NO formation as they are consistent with the steady-state rate constants published elsewhere

62,63,195,227,244. The second rate constants for both, WT and R1400E, are also very much alike (≈0.28-0.29 s-1) and they are very likely to characterise the rate of nitrite release.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

All of the observed rate constants, either k1 or k2, for both WT and R1400E decreased with the higher pressures, reaching very similar values at relevant pressures, despite having slightly different initial rates constants (≈1.6-2.2 s-1). Based on that, it can be concluded, that the R1400E mutation, despite lowering the nNOS/NADPH affinity and having an effect on flavin reduction in some of the forms of nNOS, does not significantly affect the rate of NO formation. This suggest that the rate limiting step for FL nNOS is not changed by the mutation and is still located within the NOSoxy domain, consistent with what has been published previously by Stuehr et al. 37.

The reductive transients observed for WT and R1400E are represented by similar amplitudes and also all decreasing at higher pressure. Based on that, it can be concluded that high pressure promotes the conformational changes which are inhibiting the NO formation catalysed by NOS. However, when the sample is depressurised back to the 0 bar atmospheric pressure (0 bar DOWN) the protein restores at least 50% of its initial activity, so it seems that at least some of the conformational changes caused by high pressure are reversible.

No NO formation could be observed for either FL WT or R1400E nNOS in the absence of CaM. This proves that the presence of CaM is essential for fulfilling the catalytic cycle by nNOS and to produce NO, which is consistent with previous studies 62,63,195,227,244. Also, there was no NO formation increasing pressures in the absence of CaM, which suggests that it may not be possible to use high pressure as a single impact force for conformational changes promoting electron transfer and catalytic activity in FL nNOS when CaM is not bound within the protein.

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Chapter 5 – High-pressure stopped-flow kinetic studies of nNOS

The results presented in this chapter have provided detailed insight into the role of

CaM, the characteristics of the R1400E mutation along with novel observations on pressure-dependent conformational changes in NOS. This is the first time that high pressure has been utilised to measure the effect of pressure on the internal electron transfer and catalytic activity of NOS by perturbing the structure of the protein and its complex reaction mechanism.

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Chapter 6 – Final conclusions and future perspectives

Chapter 6

Final conclusions and future perspectives

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Chapter 6 – Final conclusions and future perspectives

This thesis describes the production and characterisation of nNOSred and FL nNOS in calmodulin replete and calmodulin depleted forms. Electron transfer within these proteins was studied using various spectroscopic and kinetic approaches.

PELDOR spectroscopy has been utilised to provide interflavin distance distributions and novel insights in the nNOS conformational landscape. High hydrostatic pressure has also been used to perturb the nNOS reaction and allow us to observe the effect of the pressure on nNOS and its flavin reduction, NADPH oxidation or NO formation rates.

70 The published nNOSred crystal structure (1TLL) has revealed how certain regulatory elements may affect internal electron transfer. Specifically, the position of the C-terminal tail helix suggests that it physically restrains the FMN module from moving away from the FAD module, which is required for its electron transfer functions. A closer view revealed that the C-terminal tail residue, Arg1400, makes an electrostatic interaction with the negatively charged 2’-phosphate group of bound

NADPH. It was hypothesised that Arg1400 enables an interaction between bound

NADPH, the C-terminal tail and the FMN module that could conceivably link

NADPH binding to repression of FMN electron transfer in CaM- nNOS. To study the importance and role of this Arg1400 residue, in this thesis, the Glu amino acid has

1400 been substituted for Arg in nNOSred and FL nNOS, followed by further kinetic and spectroscopic analysis of this possibly key mutant.

To meet all the aims of this project, firstly it was essential to successfully produce and purify nNOSred and FL nNOS, both in either WT or R1400E form. The use of

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Chapter 6 – Final conclusions and future perspectives

HPLC confirmed that the purification of FL nNOS was successful and that the purified protein had full flavin saturation and the expected heme to flavin (FMN or

FAD) ratio (~1:1).

The cytochrome c reduction activity of nNOSred and FL nNOS (both WT and

R1400E) was used to obtain kinetic parameters for NADPH, for both CaM+ and

CaM- forms. In the presence of CaM, the kcat value observed for WT FL nNOS is

20% lower than kcat observed for WT nNOSred. However, the kcat values in the

R1400E nNOSred and FL R1400E nNOS are very much alike, which suggests that in the presence of CaM, the R1400E mutation may be mimicking the negative effect of presence of the heme domain on the electron transfer through the reductase domain in FL nNOS (which may be observed in WT FL nNOS, but is not observed in

R1400E FL nNOS). On the other hand, in CaM- enzymes, kcat values for the WT forms of nNOS are very similar, while FL R1400E nNOS is 50% faster than R1400E nNOSred, which shows R1400E is promoting electron transfer despite being thought to lower the affinity of the enzyme for NADPH. Additionally, in CaM-, the kcat values for R1400E are 6-fold (FL) or 4-fold (nNOSred) higher than respective WT

- 2+ nNOS kcat values. The R1400E also showed a lower dependence on CaM/Ca compared to the WT (smaller change in kinetic parameters observed after CaM binding).

The observed Km values show that despite binding CaM this does not affect Km significantly, the Km values differs between the different forms of nNOS studied.

The lowest Km was observed for FL WT nNOS and the highest for R1400E nNOSred; considering Km as the rate constant of Michaelis complex decomposition it may be concluded that FL WT nNOS, with the nNOSoxy domain present, is the most stable form of the protein when the high Km value observed for R1400E nNOSred suggests

235

Chapter 6 – Final conclusions and future perspectives that in this form of nNOS decomposition is more favoured and the complex is relatively less stable. This proves that the presence of nNOSoxy in the protein is stabilising and preserving the nNOS structure allowing it to provide its best kinetic performance.

Analysing the specificity constant, kcat/Km, for all of the forms of nNOS studied it can be concluded that all of the FL nNOS forms (independent of CaM presence) are always more efficient (higher kcat/Km) than the respective nNOSred forms (lower kcat/Km). This indicates that the FL form of nNOS, where both domains nNOSred and nNOSoxy are present, is more stable and more efficient at electron transfer to the cytochrome c acceptor than nNOSred by itself. Additionally, in the absence of CaM,

FL R1400E nNOS showed ≈2-fold higher kcat/Km than observed for FL WT nNOS, which shows that the introduction of the R1400E mutation may help to maintain the locked electron-accepting position of the FMN module (allowing it to move closer to

FAD or heme) and support internal electron transfer despite CaM absence and decreased affinity of the protein for NADPH. This is consistent with the views of

Garcin et al. 70.

A number of kinetic studies performed in the past suggested that a major large-scale conformational change is involved in internal electron transfer control in nNOS, however this evidence was based mostly on inference from a variety of kinetic experiments rather than by direct observation. In this thesis, a pulsed EPR spectroscopy approach has been employed to gain important and unique information about the conformational landscape changes in nNOS. PELDOR spectroscopy has been used to measure the distances between the FAD and FMN semiquinones

+ existing in partially reduced nNOSred and FL nNOS. The effects of ligand (NADP ) and CaM binding on the energy landscape were also studied. The PELDOR results

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Chapter 6 – Final conclusions and future perspectives represent the conformational distribution exhibited by the protein just before motion ceased because of the lack of thermal energy at 80K. These “trapped” conformations exist at the bottom of thermodynamic wells or minima.

X-band four-pulsed PELDOR results obtained for WT nNOSred in the absence of

CaM (Chapter 3, Section 3.2.4.2, Figure 3.14 and Table 3.3) show three maxima that are evidently indicating three dipolar coupling (υDD) values and three corresponding interflavin distances of 35, 28 and 24 Å, suggesting quite a ‘rugged’ conformational energy landscape at low temperature (80K), with three energy minima each having a

+ different inter-flavin distance. When NADP was added to WT nNOSred, the conformational energy landscape became even more ‘rocky’ and four υDD values and corresponding interflavin distances were observed (Chapter 3, Section 3.2.4.2,

Figure 3.14 and Table 3.3). When analysing the relative integrals for those four observed υDD values and their conjugate distances, it can be concluded that despite all those distances being evident, their relative integrals (representing spectrum contribution) suggest that the protein conformation with the longest interflavin distance, 36 Å, is the most highly populated. There is also a shortest inter-flavin distance evident in these data, 20 Å, which is shorter than observed without NADP+ but this conformation is not highly populated (relative integral: 0.10). When CaM is bound to nNOS, the internal electron transfer and NO synthesis are promoted. The

PELDOR data produced for nNOSred in the presence of CaM (Chapter 3, Section

3.2.4.2, Figure 3.14 and Table 3.3) represent the simplest and least ‘rugged’ conformational energy landscape observed so far, where only two conformations with the interflavin distances of 33 and 25 Å are present. However, when NADP+ is bound to the nNOSred in the presence of CaM, the conformational energy landscape becomes very rugged again; with five detectible conformations and an equilibrium

237

Chapter 6 – Final conclusions and future perspectives between these conformations favouring shorter interflavin distances (Chapter 3,

Section 3.2.4.2, Figure 3.14 and Table 3.3). The available crystal structure obtained

+ 69 for WT rat nNOSred with NADP bound (1TLL) presents only the “closed” form of nNOSred where the interflavin distance of 15.24 Å (C(4a) from FAD to C(4a) from

FMN) can be observed. This distance is in fair agreement with the observed and

+ presented here shorter interflavin distances obtained for nNOSred CaM+ with NADP bound, however considering that the longer distances are also evident it can be concluded that they may represent the ‘open’ form of nNOSred and suggest that an equilibrium distribution of multiple extended open and compact closed conformations is found in the solution when CaM and NADP+ are present.

The PELDOR data obtained for R1400E nNOSred, in the presence of CaM and

NADP+, show only two conformations thus suggesting a conformational energy landscape almost identical to that of the WT nNOSred CaM+ in the absence of

NADP+. This data is in agreement with that earlier in this chapter about the R1400E variant, its interaction with 2’-phosphate group of bound NADPH and how it is regulating the equilibrium of the FMN module controlling the electron transfer in both CaM+ and CaM- nNOSred. These results also support the belief that the interaction of R1400E with the 2’-phosphate group of bound NADP contributes to the major change in conformational equilibrium.

PELDOR results for FL WT nNOS show a complex energy landscape with multiple conformational states (Chapter 4, Section 4.3.8, Figure 4.17 and Table 4.6). In the absence of CaM, the interflavin distance distribution matches that exhibited by

+ nNOSred CaM- in the presence of NADP (Chapter 3, Section 3.2.4.2, Figure 3.14 and Table 3.3), however there is a marked difference in the relative integrals of the

+ lines present in these two spectra. In NADP -bound nNOSred CaM- the distance

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Chapter 6 – Final conclusions and future perspectives distribution is dominated by the 36 Å interflavin distance, whereas in the FL nNOS

CaM- the distribution between conformations is more even. This may reflect the, mentioned in Chapter 4, influence of the heme iron on TM, leading to a reduction in the amplitude of the contribution from the conformation with a 36 Å inter-flavin distance, as in this conformation the FMN radical would be closer to the heme than in the other conformations observed. Nevertheless, these data suggest that the heme domain does influence the conformational equilibrium within the reductase domain.

When CaM is bound to FL WT nNOS, the distance distribution obtained from

PELDOR is changed in comparison to the FL WT nNOS CaM- form. The FL WT nNOS CaM+ spectrum is unusually broad and weak, which very possibly, is reflecting a change in the redox state of the heme. This spectrum is dominated by a broad line corresponding to a distance of 51 Å which, very likely, is representing an interflavin distance between cofactors on two different monomers within the dimer.

Except for the 51 Å, the rest of the observed distances are shorter than in FL WT nNOS CaM-. Binding CaM is thought to affect the FMN module mobility releasing it and promoting shifting between FAD module and heme (in nNOSoxy) to allow the electron transfer to occur. These observations are consistent with a recent thermodynamic study of the heme reduction kinetics in nNOS 245, which suggested that the FMN-nNOSoxy interaction in the presence of CaM is intermittent and/or transient.

After kinetic and spectroscopic approaches were used to study domain movements and conformational changes effected by CaM and/or ligands binding in nNOSred and

FL nNOS, the next aim of this project was to artificially control the external environment of the protein, in terms of pressure, to see how this affects electron transfer and NO production. A high-pressure stopped-flow technique was used to

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Chapter 6 – Final conclusions and future perspectives perturb an equilibrium distribution of conformational states and study the kinetics of

NADPH oxidation, flavin reduction by NADPH and NO formation.

The steady-state turnover of NADPH oxidation has been studied as a function of high pressure. The NADPH oxidation rate for either FL WT or R1400E CaM- nNOS increased with the pressure, however for CaM+, the opposite effect and a decreased rate with the pressure has been observed. The NADPH oxidation rates for both,

CaM+ and CaM- forms, are very much alike at the highest 1750 bar pressure which suggests that high pressure is perturbing the conformational landscape and mimicking the effect of CaM on the protein, independently of CaM presence or absence in the sample. Additionally, the effect of high pressure on the NADPH oxidation rates is reversible and the depressurised samples are characterised by circa initial values, which suggests that it may be possible to limitedly control the NADPH oxidation rate, especially in the absence of CaM, using only high pressure.

Considering that the NADPH oxidation rate characterises the NADPH-FAD interaction, it may also be concluded that high pressure is effecting large-scale conformational changes which may lead to the FAD being shifted closer to the

NADPH, which would explain the increasing NADPH oxidation rates in the absence of CaM and decreasing when CaM is present. Even if CaM and NADPH release the

FMN module and allow it to move closer to the heme, the distance between FAD and NADPH is thought to be decreasing with the pressure simultaneously increasing the distance between FMN and FAD making it too far to allow the FMN to accept the electrons from FAD and pass them further to the heme, which would be seen in a decreased rate).

The hydrostatic pressure dependence of flavin reduction has been studied by looking at the single turnover rate in nNOSred and FL nNOS, both WT and R1400E, in the

240

Chapter 6 – Final conclusions and future perspectives presence and absence of CaM. Two rate constants for flavin reduction have been obtained, where k1 is thought to represent hydride transfer from NADPH to FAD leading to initial enzyme reduction, whereas k2 is thought to serve as an internal electron redistribution between FAD and FMN 194,246.

When analysing the flavin reduction rate constant it has been observed that CaM binding does not change the rate constants for nNOSred but it does affect the flavin reduction rate constants in FL nNOS, which suggests that CaM binding is affecting and promoting internal electron transfer more in FL nNOS than it is in nNOSred. The application of high pressure affected nNOSred differently than it affected FL WT nNOS flavin rate constants (described in details in Chapter 5, Section 5.2.2 and 5.3) which suggests that the reductase domain, where flavin reduction is happening, is the most affected by the pressure and the conformational changes that followe it. The binding of CaM within the FL WT nNOS also affects the conformational equilibrium within nNOS, allowing for different effects of high pressure on the protein with and without CaM. This supports previous results suggesting that CaM influences the conformational equilibria in nNOS 22,97–99,242.

When flavin reduction is analysed in R1400E nNOS it can be concluded that the effect of the pressure on R1400E is different than the effect of pressure on WT, which again proves that R1400E is affecting electron transfer within nNOSred despite lowering the affinity of the protein for its substrate and that the introduction of this mutation is causing structural changes in the NADPH-FAD-FMN region.

The rate of NO formation as a function of pressure was observed using an assay based on the NO-mediated conversion of oxyhaemoglobin to methaemoglobin. The rate of NO formation was fitted using a two exponential equation model for both FL

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Chapter 6 – Final conclusions and future perspectives

WT and R1400E nNOS in the presence of CaM, while in the absence of CaM no NO formation has been observed. Both of the observed rate constants, either k1 or k2, for both WT and R1400E decreased and reached very similar values at relevant pressures. Based on those results it can be concluded, that high pressure is forcing major changes in the conformational energy landscape of the protein and negatively affecting internal electron transfer and NO formation in FL nNOS. Those changes may either affect the position of the FAD module (if it is shifted closer to NADPH and further from FMN, as it was suggested before in case of NADPH oxidation results) affecting electron transfer from FAD to FMN or they can impair CaM binding and affect the electron transfer from FMN to heme. To find out which step of NO formation is most affected by high pressure, further dedicated studies are necessary.

Moreover, the NO formation results are very similar for FL WT and R1400E nNOS, both in the presence of CaM, where a similar trend in changes with the high pressure has been observed. That suggests again that the R1400E mutation in FL nNOS may be affecting protein/NADPH affinity and flavin reduction but it has no effect on the heme reduction step, which enables O2 binding and substrate oxidation to occur within the oxygenase domain and is thought to be the rate limiting step for nNOS catalysis and NO formation 36,38,66.

Concluding, this thesis provided quite a few remarkable and novel results and took the knowledge about the conformational energy landscape of nNOSred and FL nNOS to the next level providing both spatial and temporal information about the nNOS free energy landscape and its remodelling by CaM and NADP+.

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Chapter 6 – Final conclusions and future perspectives

The results presented in this thesis provide a great platform for additional detailed studies dedicated to further understand the electron transfer mechanism, conformational energy landscape and NO formation control in nNOS. Presented in this thesis PELDOR results may be used to predict unknown possible conformations of nNOSred or FL nNOS which could be used in the future as a basis to design the specific structural inhibitors. Considering also that the crystal structure of the FL nNOS protein still remains unsolved, the knowledge about the distances obtained from PELDOR spectroscopy could be used to set up the conditions for a successful

FL nNOS crystallisation. Additionally, if site directed spin labelling is used to create spin-labelled CaM, using PELDOR conditions set up and used in this thesis, the distances between flavins, CaM and possibly heme could be obtained, which would provide further insight into the nNOS mechanism and conformational changes occurring during catalysis.

243

References

244

References

1. Masters, B. S. S., McMillan, K., Sheta, A., Nishimura, J. S., Roman, J. & Martasek, P. Neuronal formed a cysteine nitric oxide evolution : a modular structure protein heme enzyme studies that of by convergent to produce NO ” as a cellular. FASEB J. 10, 552–558 (1996).

2. Geller, D. A. & Billiar, T. R. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev. 17, 7–23 (1998).

3. Crane, B. R., Arvai, A. S., Ghosh, D. K., Wu, C., Getzoff, E. D., Stuehr, D. J. & Tainer, J. A. Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279, 2121–6 (1998).

4. Fischmann, T. O., Hruza, A., Niu, X. D., Fossetta, J. D., Lunn, C. A., Dolphin, E., Prongay, A. J., Reichert, P., Lundell, D. J., Narula, S. K. & Weber, P. C. Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation. Nat. Struct. Biol. 6, 233–42 (1999).

5. Li, H., Shimizu, H., Flinspach, M., Jamal, J., Yang, W., Xian, M., Cai, T., Wen, E. Z., Jia, Q., Wang, P. G. & Poulos, T. L. Articles the novel binding mode of N-slkyl-N ′-hydroxyguanidine to neuronal nitric pxide synthase provides mechanistic insights into NO biosynthesis. Biochemistry 41, 13868– 13875 (2002).

6. Raman, C. S., Li, H., Martásek, P., Král, V., Masters, B. S. & Poulos, T. L. Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell 95, 939–50 (1998).

7. Daff, S. NO synthase: structures and mechanisms. Nitric oxide : biology and chemistry. Official journal of the Nitric Oxide Society 23, 1–11 (2010).

8. Brenman, J. E., Chao, D. S., Gee, S. H., McGee, A. W., Craven, S. E., Santillano, D. R., Wu, Z., Huang, F., Xia, H., Peters, M. F., Froehner, S. C. & Bredt, D. S. Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell 84, 757–67 (1996).

245

References

9. Salerno, J. C., Harris, D. E., Irizarry, K., Patel, B., Morales, A. J., Smith, S. M., Martasek, P., Roman, L. J., Masters, B. S., Jones, C. L., Weissman, B. A., Lane, P., Liu, Q. & Gross, S. S. An autoinhibitory control element defines calcium-regulated isoforms of nitric oxide synthase. J. Biol. Chem. 272, 29769–77 (1997).

10. Daff, S., Sagami, I. & Shimizu, T. The 42-amino acid insert in the FMN domain of neuronal nitric-oxide synthase exerts control over Ca2+/ calmodulin-dependent electron transfer. J. Biol. Chem. 274, 30589–95 (1999).

11. Cho, K. O., Hunt, C. A. & Kennedy, M. B. The rat brain postsynaptic density fraction contains a homolog of the Drosophila discs-large tumor suppressor protein. Neuron 9, 929–42 (1992).

12. Fanning, A. S. & Anderson, J. M. Protein-protein interactions: PDZ domain networks. Curr. Biol. 6, 1385–8 (1996).

13. Stricker, N. L., Christopherson, K. S., Yi, B. A., Schatz, P. J., Raab, R. W., Dawes, G., Bassett, D. E., Bredt, D. S. & Li, M. PDZ domain of neuronal nitric oxide synthase recognizes novel C-terminal peptide sequences. Nat. Biotech. 15, 336 – 342 (1997).

14. Venema, V. J., Ju, H., Zou, R. & Venema, R. C. Interaction of neuronal nitric- oxide synthase with caveolin-3 in skeletal muscle. J. Biol. Chem. 272, 28187– 28190 (1997).

15. Panda, K., Rosenfeld, R. J., Ghosh, S., Meade, A. L., Getzoff, E. D. & Stuehr, D. J. Distinct dimer interaction and regulation in nitric-oxide synthase types I, II, and III. J. Biol. Chem. 277, 31020–30 (2002).

16. Hemmens, B., Goessler, W., Schmidt, K. & Mayer, B. Role of bound zinc in dimer stabilization but not enzyme activity of neuronal nitric-oxide synthase. J. Biol. Chem. 275, 35786–91 (2000).

17. Nishida, C. R. & Ortiz De Montellano, P. R. Electron transfer and catalytic activity of nitric oxide synthases. J. Biol. Chem. 273, 5566–5571 (1998).

246

References

18. Moncada, S., Palmer, R. M. & Higgs, E. A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109–142 (1991).

19. Dinerman, J. L., Lowenstein, C. J. & Snyder, S. H. Molecular mechanisms of nitric oxide regulation. Potential relevance to cardiovascular disease. J. Am. Heart Assoc. 73, 217–222 (1993).

20. Jr, K. J. F., Jr, L. J. R. & L, F. P. Nitric oxide: a new paradigm for second messengers. J. Med. Chem. 38, 4343–4362 (1995).

21. Nathan, C. Perspectives series : Nitric oxide and nitric oxide synthases inducible nitric oxide synthase : what difference does it make ? J. Clin. Invest. 100, 2417–2423 (1997).

22. Stuehr, D. J., Tejero, J. & Haque, M. M. Structural and mechanistic aspects of flavoproteins: electron transfer through the nitric oxide synthase flavoprotein domain. FEBS Journal 276, 3959–74 (2009).

23. Stuehr, D. J. & Ikeda-Saito, M. Spectral characterization of brain and macrophage nitric oxide synthases. J. Biol. Chem. 267, 20547–20550 (1992).

24. Abu-Soud, H. M., Gachhui, R., Raushel, F. M. & Stuehr, D. J. The ferrous- dioxy complex of neuronal nitric oxide synthase. J. Biol. Chem. 272, 17349– 17353 (1997).

25. Berka, V., Yeh, H. C., Gao, D., Kiran, F. & Tsai, A. L. Redox function of tetrahydrobiopterin and effect of L-arginine on oxygen binding in endothelial nitric oxide synthase. Biochemistry 43, 13137–48 (2004).

26. Ost, T. W. B. & Daff, S. Thermodynamic and kinetic analysis of the nitrosyl, carbonyl, and dioxy heme complexes of neuronal nitric-oxide synthase. The roles of substrate and tetrahydrobiopterin in oxygen activation. J. Biol. Chem. 280, 965–73 (2005).

27. Wei, C. C., Wang, Z. Q., Hemann, C., Hille, R. & Stuehr, D. J. A tetrahydrobiopterin radical forms and then becomes reduced during Nomega-

247

References

hydroxyarginine oxidation by nitric-oxide synthase. J. Biol. Chem. 278, 46668–73 (2003).

28. Wei, C. C., Wang, Z. Q., Tejero, J., Yang, Y. P., Hemann, C., Hille, R. & Stuehr, D. J. Catalytic reduction of a tetrahydrobiopterin radical within nitric- oxide synthase. J. Biol. Chem. 283, 11734–42 (2008).

29. Wei, C. C., Wang, Z. Q., Wang, Q., Meade, A. L., Hemann, C., Hille, R. & Stuehr, D. J. Rapid kinetic studies link tetrahydrobiopterin radical formation to heme-dioxy reduction and arginine hydroxylation in inducible nitric-oxide synthase. J. Biol. Chem. 276, 315–9 (2001).

30. Hurshman, A. R., Krebs, C., Edmondson, D. E., Huynh, B. H. & Marletta, M. A. Accelerated publications formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen. Biochemistry 38, 15689–15696 (1999).

31. Sorlie, M., Gorren, A. C. F., Marchal, S., Shimizu, T., Lange, R., Andersson, K. K. & Mayer, B. Single-turnover of nitric-oxide synthase in the presence of 4-amino-tetrahydrobiopterin: proposed role for tetrahydrobiopterin as a proton donor. J. Biol. Chem. 278, 48602–10 (2003).

32. Boggs, S., Huang, L. & Stuehr, D. J. Formation and reactions of the heme- dioxygen intermediate in the first and second steps of nitric oxide synthesis as studied by stopped-flow spectroscopy under single-turnover conditions. Biochemistry 39, 2332–9 (2000).

33. Assreuy, J., Cunha, F. Q., Liew, F. Y. & Moncada, S. Feedback inhibition of nitric oxide synthase activity by nitric oxide. Br. J. Pharmac. 108, 833–7 (1993).

34. Buga, G. M., Griscavage, J. M., Rogers, N. E. & Ignarro, L. J. Negative feedback regulation of endothelial cell function by nitric oxide. J. Am. Heart Assoc. 73, 808–812 (1993).

248

References

35. Rogers, N. E. & Ignarro, L. J. Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine. Biochem. biophys. Res. Commun. 189, 242–249 (1992).

36. Santolini, J., Meade, A. L. & Stuehr, D. J. Differences in three kinetic parameters underpin the unique catalytic profiles of nitric-oxide synthases I, II, and III. J. Biol. Chem. 276, 48887–98 (2001).

37. Stuehr, D. J., Santolini, J., Wang, Z. Q., Wei, C. C. & Adak, S. Update on mechanism and catalytic regulation in the NO synthases. J. Biol. Chem. 279, 36167–70 (2004).

38. Santolini, J., Adak, S., Curran, C. M. & Stuehr, D. J. A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase. J. Biol. Chem. 276, 1233–43 (2001).

39. Massey, V. The chemical and biological versatility of riboflavin. Biochem. Soc. Trans. 28, 283–96 (2000).

40. Powers, H. J. Riboflavin (vitamin B-2) and health. Am. J. Clin. Nutrit. 77, 1352–60 (2003).

41. Dagley, S. Lessons from biodegradation. Ann. Rev. Microbiol. 41, 1–24 (1987).

42. Wojcieszyńska, D., Greń, I., Hupert-Kocurek, K. & Guzik, U. Modulation of FAD-dependent monooxygenase activity from aromatic compounds- degrading Stenotrophomonas maltophilia strain KB2. Acta Biochim. Pol. 58, 421–6 (2011).

43. Weber, S. Light-driven enzymatic catalysis of DNA repair: a review of recent biophysical studies on photolyase. Biochim. Biophys. Acta Bioenergetics 1707, 1–23 (2005).

44. Sancar, A. Photolyase and cryptochrome blue-light photoreceptors. Adv. Protein Chem. 69, 73–100 (2004).

249

References

45. Byrdin, M., Sartor, V., Eker, A. P. M., Vos, M. H., Aubert, C., Brettel, K. & Mathis, P. Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: review and new insights from an “inverse” deuterium isotope effect. Biochim. Biophys. Acta Bioenergetics 1655, 64–70 (2004).

46. Demarsy, E. & Fankhauser, C. Higher plants use LOV to perceive blue light. Curr. Opin. Plant Biol. 12, 69–74 (2009).

47. Kanegae, T., Hayashida, E., Kuramoto, C. & Wada, M. A single chromoprotein with triple chromophores acts as both a phytochrome and a phototropin. Proc. Natl Acad. Sci. USA 103, 17997–8001 (2006).

48. Kimura, M. & Kagawa, T. Phototropin and light-signaling in phototropism. Curr. Opin. Plant Biol. 9, 503–8 (2006).

49. Ghisla, S. & Massey, V. New flavins for old: artificial flavins as active site probes of flavoproteins. Biochem. J. 239, 1–12 (1986).

50. Murataliev, M. B., Feyereisen, R. & Walker, F. A. Electron transfer by diflavin reductases. Biochim. Biophys. Acta 1698, 1–26 (2004).

51. Hammes, G. G. Spectroscopy for the biological sciences. (John Wiley & Sons, Inc., 2005).

52. Masters, B. S., Bilimoria, M. H., Kamin, H. & Gibson, Q. H. The mechanism of 1- and 2-electron transfers catalyzed by reduced triphosphopyridine nucleotide-cytochrome c reductase. J. Biol. Chem. 240, 4081–8 (1965).

53. Iyanagi, T. & Mason, H. S. Properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase. Biochemistry 12, 2297–308 (1973).

54. Roman, L. J., Martásek, P., Miller, R. T., Harris, D. E., De La Garza, M. A., Shea, T. M., Kim, J. J. P. & Masters, B. S. S. The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme

250

References

domains and affect modulation by calmodulin. J. Biol. Chem. 275, 29225–32 (2000).

55. Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S. & Kim, J. J. Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. Proc. Natl Acad. Sci. USA 94, 8411–6 (1997).

56. Gruez, A., Pignol, D., Zeghouf, M., Covès, J., Fontecave, M., Ferrer, J. L. & Fontecilla-Camps, J. C. Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module. J. Mol. Biol. 299, 199–212 (2000).

57. Paine, M. J., Garner, A. P., Powell, D., Sibbald, J., Sales, M., Pratt, N., Smith, T., Tew, D. G. & Wolf, C. R. Cloning and characterization of a novel human dual flavin reductase. J. Biol. Chem. 275, 1471–8 (2000).

58. Watenpaugh, K. D., Sieker, L. C. & Jensen, L. H. The Binding of Riboflavin- 5’ -Phosphate in a Flavoprotein : Flavodoxin at 2.0-A Resolution. Proc. Natl Acad. Sci. USA 70, 3857–3860 (1973).

59. Karplus, P. A., Daniels, M. J. & Herriott, J. R. Atomic structure of ferredoxin- NADP+ reductase: prototype for a structurally novel flavoenzyme family. Science 251, 60–66 (1991).

60. Newton, D. C., Montgomery, H. J. & Guillemette, J. G. The reductase domain of the human inducible nitric oxide synthase is fully active in the absence of bound calmodulin. Arch. Biochem. Biophys. 359, 249–257 (1998).

61. Adak, S., Sharma, M., Meade, A. L. & Stuehr, D. J. A conserved flavin- shielding residue regulates NO synthase electron transfer and nicotinamide coenzyme specificity. Proc. Natl Acad. Sci. USA 99, 13516–21 (2002).

62. Abu-Soud, H. M. & Stuehr, D. J. Nitric oxide synthases reveal a role for calmodulin in controlling electron transfer. Proc. Natl Acad. Sci. USA 90, 10769–72 (1993).

251

References

63. Adak, S., Ghosh, S., Abu-Soud, H. M. & Stuehr, D. J. Role of reductase domain cluster 1 acidic residues in neuronal nitric oxide synthase. J. Biol. Chem. 274, 22313–22320 (1999).

64. Garnaud, P. E., Koetsier, M., Ost, T. W. B. & Daff, S. Redox properties of the isolated flavin mononucleotide- and flavin adenine dinucleotide-binding domains of neuronal nitric oxide synthase. Biochemistry 43, 11035–44 (2004).

65. Noble, M. A., Miles, C. S., Chapman, S. K., Lysek, D. A., MacKay, A. C., Reid, G. A., Hanzlik, R. P. & Munro, A. W. Roles of key active-site residues in flavocytochrome P450 BM3. Biochemical Journal 339, 371–9 (1999).

66. Miller, R. T., Martásek, P., Omura, T. & Masters, B. S. Rapid kinetic studies of electron transfer in the three isoforms of nitric oxide synthase. Biochem. biophys. Res. Commun. 265, 184–8 (1999).

67. Roman, L. J., Martásek, P. & Masters, B. S. S. Intrinsic and extrinsic modulation of nitric oxide synthase activity. Chem Res Toxicol. 102, 1179–90 (2002).

68. Knudsen, G. M., Nishida, C. R., Mooney, S. D. & Ortiz de Montellano, P. R. Nitric-oxide synthase (NOS) reductase domain models suggest a new control element in endothelial NOS that attenuates calmodulin-dependent activity. J. Biol. Chem. 278, 31814–24 (2003).

69. Zhang, J., Martàsek, P., Paschke, R., Shea, T., Siler Masters, B. S. & Kim, J. J. Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase. J. Biol. Chem. 276, 37506–13 (2001).

70. Garcin, E. D., Bruns, C. M., Lloyd, S. J., Hosfield, D. J., Tiso, M., Gachhui, R., Stuehr, D. J., Tainer, J. a & Getzoff, E. D. Structural basis for isozyme- specific regulation of electron transfer in nitric-oxide synthase. J. Biol. Chem. 279, 37918–27 (2004).

252

References

71. Matter, H., Kumar, H. S. A., Fedorov, R., Frey, A., Kotsonis, P., Hartmann, E., Fröhlich, L. G., Reif, A., Pfleiderer, W., Scheurer, P., Ghosh, D. K., Schlichting, I. & Schmidt, H. H. H. W. Structural analysis of isoform-specific inhibitors targeting the tetrahydrobiopterin binding site of human nitric oxide synthases. J. Med. Chem. 48, 4783–92 (2005).

72. Ghosh, D. K. & Stuehr, D. J. Macrophage NO synthase: characterization of isolated oxygenase and reductase domains reveals a head-to-head subunit interaction. Biochemistry 34, 801–807 (1995).

73. McMillan, K. & Masters, B. S. S. Prokaryotic expression of the heme- and flavin-binding domains of rat neuronal nitric oxide synthase as distinct polypeptides: identification of the heme-binding proximal thiolate ligand as cysteine-415. Biochemistry 34, 3686–3693 (1995).

74. Richards, M. K. & Marletta, M. A. Characterization of neuronal nitric oxide synthase and a C415H mutant, purified from a baculovirus overexpression system. Biochemistry 33, 14723–14732 (1994).

75. Marletta, M. A. Nitric oxide synthase: aspects concerning structure and catalysis. Cell 78, 927–30 (1994).

76. Crane, B. R. The structure of nitric oxide synthase oxygenase domain and inhibitor complexes. Science 278, 425–431 (1997).

77. Li, H., Raman, C. S., Glaser, C. B., Blasko, E., Young, T. A., Parkinson, J. F., Whitlow, M. & Poulos, T. L. Crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase. J. Biol. Chem. 274, 21276– 21284 (1999).

78. Feng, C. Mechanism of nitric oxide synthase regulation: electron transfer and interdomain interactions. Coord. Chem. Rev. 256, 393–411 (2012).

79. Stuehr, D. J., Kwon, N. S., Nathan, C. F., Griffiths, O. W., Feldmann, P. L. & Wiseman, J. N omega-hydroxy-L-arginine is an intermediate in the

253

References

biosynthesis of nitric oxide from L-arginine. J. Biol. Chem. 266, 6259–63 (1991).

80. Tayeh, M. A. & Marletta, M. A. Macrophage oxidation of L-arginine to nitric oxide, nitrite, and nitrate. Tetrahydrobiopterin is required as a cofactor. J. Biol. Chem. 264, 19654–19658 (1989).

81. Klatt, P., Schmidt, K., Lehner, D., Glatter, O., Bächinger, H. P. & Mayer, B. Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer. The EMBO J. 14, 3687–95 (1995).

82. Giovanelli, J., Campos, K. L. & Kaufman, S. Tetrahydrobiopterin, a cofactor for rat cerebellar nitric oxide synthase, does not function as a reactant in the oxygenation of arginine. Proc. Natl Acad. Sci. USA 88, 7091–5 (1991).

83. Vásquez-Vivar, J., Kalyanaraman, B., Martásek, P., Hogg, N., Masters, B. S., Karoui, H., Tordo, P. & Pritchard, K. A. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc. Natl Acad. Sci. USA 95, 9220–5 (1998).

84. Vásquez-Vivar, J., Kalyanaraman, B. & Martásek, P. The role of tetrahydrobiopterin in superoxide generation from eNOS: enzymology and physiological implications. Free Rad. Res. 37, 121–127 (2003).

85. Stroes, E., Hijmering, M., Van Zandvoort, M., Wever, R., Rabelink, T. J. & Van Faassen, E. E. Origin of superoxide production by endothelial nitric oxide synthase. FEBS Lett. 438, 161–4 (1998).

86. Schmidt, T. S. & Alp, N. J. Mechanisms for the role of tetrahydrobiopterin in endothelial function and vascular disease. Clin. Sci. 113, 47–63 (2007).

87. Crane, B. R., Arvai, A. S., Ghosh, S., Getzoff, E. D., Stuehr, D. J. & Tainer, J. A. Structures of the N(omega)-hydroxy-L-arginine complex of inducible nitric oxide synthase oxygenase dimer with active and inactive pterins. Biochemistry 39, 4608–21 (2000).

254

References

88. Gorren, A. & Mayer, B. Tetrahydrobiopterin in nitric oxide synthesis: a novel biological role for pteridines. Curr Drug Metab. 3, 133–157 (2002).

89. Babu, Y. S., Bugg, C. E. & Cook, W. J. Structure of calmodulin refined at 2.2 A resolution. J. Mol. Biol. 204, 191–204 (1988).

90. Chattopadhyaya, R., Meador, W. E., Means, A. R. & Quiocho, F. A. Calmodulin structure refined at 1.7 A resolution. J. Mol. Biol. 228, 1177–92 (1992).

91. Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B. & Bax, A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 256, 632–8 (1992).

92. Meador, W. E., Means, A. R. & Quiocho, F. A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science 257, 1251–5 (1992).

93. Yuan, T., Vogel, H. J., Sutherland, C. & Walsh, M. P. Characterization of the Ca2+ -dependent and -independent interactions between calmodulin and its binding domain of inducible nitric oxide synthase. FEBS Lett. 431, 210–4 (1998).

94. Matsubara, M., Hayashi, N., Titani, K. & Taniguchi, H. Circular dichroism and 1 H NMR studies on the structures of peptides derived from the calmodulin-binding domains of inducible and endothelial nitric-oxide synthase in solution and in complex with calmodulin. J. Biol. Chem. 272, 23050–23056 (1997).

95. Cho, H. J., Xie, Q. W., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D. & Nathan, C. Calmodulin is a subunit of nitric oxide synthase from macrophages. J. Exp. Med. 176, 599–604 (1992).

96. Murrin, F. & Staveley, B. www.mun.ca. Pearson Education Inc. 9, (2009).

255

References

97. Feng, C., Tollin, G., Hazzard, J. T., Nahm, N. J., Guillemette, J. G., Salerno, J. C. & Ghosh, D. K. Direct measurement by laser flash photolysis of intraprotein electron transfer in a rat neuronal nitric oxide synthase. J. Am. Chem. Soc. 129, 5621–5629 (2007).

98. Feng, C., Taiakina, V., Ghosh, D. K., Guillemette, J. G. & Tollin, G. Intraprotein electron transfer between the FMN and heme domains in endothelial nitric oxide synthase holoenzyme. Biochim. Biophys. Acta 1814, 1997–2002 (2011).

99. Feng, C., Tollin, G., Holliday, M. A., Thomas, C., Salerno, J. C., Enemark, J. H. & Ghosh, D. K. Intraprotein electron transfer in a two-domain construct of neuronal nitric oxide synthase: the output state in nitric oxide formation. Biochemistry 45, 6354–62 (2006).

100. Newman, E., Spratt, D. E., Mosher, J., Cheyne, B., Montgomery, H. J., Wilson, D. L., Weinberg, J. B., Smith, S. M. E., Salerno, J. C., Ghosh, D. K. & Guillemette, J. G. Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras. J. Biol. Chem. 279, 33547–57 (2004).

101. Gachhui, R., Presta, A., Bentley, D. F., Abu-Soud, H. M., Mcarthur, R., Brudvig, G., Ghosh, D. K. & Stuehr, D. J. Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. J. Biol. Chem. 271, 20594–20602 (1996).

102. Wada, F., Shibata, H., Goto, M. & Sakamoto, Y. Participation of the microsomal electron transport system involving cytochrome p450 in oxidation of fatty acids. Biochim. Biophys. Acta 162, 518–524 (1968).

103. Glazer, R. I., Schenkman, J. B. & Sartorelli, A. C. Immunochemical studies on the role of reduced nicotinamide adenine dinucleotide phosphate- cytochrome c (P-450) reductase in drug oxidation. Mol Pharmacol 7, 683–688 (1971).

104. Masters, B. S. S., Nelson, E. B., Ziegler, D. M., Baron, J., Raj, P. P. & Isaacson, E. L. Immunochemical studies utilizing antibody to NADPH-

256

References

cytochrome c reductase as a specific inhibitor of microsomal electron transport. Chem. Biol. Interact. 3, 296–299 (1971).

105. Estabrook, R. W., Franklin, M. R., Cohen, B., Shigamatzu, A. & Hildebrandt, A. G. Biochemical and genetic factors influencing drug metabolism. Influence of hepatic microsomal mixed function oxidation reactions on cellular metabolic control. Metabolism: Clin. Exp. 20, 187–99 (1971).

106. Shephard, E. A., Phillips, I. R., Bayney, R. M., Pike, S. F. & Rabin, B. R. Quantification of NADPH: cytochrome P-450 reductase in liver microsomes by a specific radioimmunoassay technique. Biochemical Journal 211, 333–40 (1983).

107. Feyereisen, R., Koener, J. F., Cariño, F. A. & Daggett, A. S. Biochemistry and molecular biology of insect cytochrome P450. Mol. Insect Sci. 262, 263–272 (1990).

108. Porter, T. D. & Kasper, C. B. Coding nucleotide sequence of rat NADPH- cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains. Proc. Natl Acad. Sci. USA 82, 973–7 (1985).

109. Iyanagi, T. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain. Biochem. biophys. Res. Commun. 338, 520–8 (2005).

110. Lu, A. Y. H., Junk, K. W. & Coon, M. J. Resolution of the cytochrome P450- containing hydroxylation system of lver microsomes into three components. J. Biol. Chem. 244, 3714–3721 (1969).

111. Coon, M. J., Strobel, H. W. & Boyer, R. F. On the mechanism of hydroxylation reactions catalyzed by cytochrome P450. Drug Metab. Dispos. 1, 92–97 (1973).

112. Enoch, H. G. & Strittmatter, P. Formation and properties of 1000-A-diameter, single-bilayer phospholipid vesicles. Proc. Natl Acad. Sci. USA 76, 145–149 (1979).

257

References

113. Schacter, B. A., Nelson, E. B., Marver, H. S. & Masters, B. S. Immunochemical evidence for an association of heme oxygenase with the microsomal electron transport system. J. Biol. Chem. 247, 3601–7 (1972).

114. Ono, T., Ozasa, S., Hasegawa, F. & Imai, Y. Involvement of NADPH- cytochrome c reductase in the rat liver squalene epoxidase system. Biochim. Biophys. Acta 486, 401–407 (1977).

115. Guengerich, F. P. Cytochrome p450 and chemical toxicology. Chem Res Toxicol. 21, 70–83 (2008).

116. Hannemann, F., Bichet, A., Ewen, K. M. & Bernhardt, R. Cytochrome P450 systems--biological variations of electron transport chains. Biochim. Biophys. Acta 1770, 330–44 (2007).

117. Berka, K., Hendrychová, T., Anzenbacher, P. & Otyepka, M. Membrane position of ibuprofen agrees with suggested access path entrance to cytochrome P450 2C9 active site. J. Phys. Chem. 115, 11248–55 (2011).

118. Whitehouse, C. J. C., Bell, S. G. & Wong, L.-L. P450(BM3) (CYP102A1): connecting the dots. Chemical Society reviews 41, 1218–60 (2012).

119. Munro, A. W., Leys, D. G., McLean, K. J., Marshall, K. R., Ost, T. W. B., Daff, S., Miles, C. S., Chapman, S. K., Lysek, D. a, Moser, C. C., Page, C. C. & Dutton, P. L. P450 BM3: the very model of a modern flavocytochrome. Trends Biol. Sci. 27, 250–7 (2002).

120. Roitel, O., Scrutton, N. S. & Munro, A. W. Electron transfer in flavocytochrome P450 BM3: kinetics of flavin reduction and oxidation, the role of cysteine 999, and relationships with mammalian cytochrome P450 reductase. Biochemistry 42, 10809–21 (2003).

121. Di Nardo, G. & Gilardi, G. Optimization of the bacterial cytochrome P450 BM3 system for the production of human drug metabolites. Int. J. Mol. Sci. 13, 15901–15924 (2012).

258

References

122. Narhi, L. O. & Fulco, A. J. Characterization of a catalytically self-sufficient 119,000-Da cytochrome P-450 monooxygenase induced by barbiturates Bacillus megaterium. J. Biol. Chem. 261, 7160–7169 (1986).

123. Narhi, L. O. & Fulco, A. J. Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium. J. Biol. Chem. 262, 6683–90 (1987).

124. Narhi, L. O., Kim, B. H., Stevenson, P. M. & Fulco, A. J. Partial characterisation of a barbiturane-induces cytochrome P450-dependent. Biochem. biophys. Res. Commun. 116, 851–858 (1983).

125. Munro, A. W., Daff, S., Coggins, J. R., Lindsay, J. G. & Chapman, S. K. Probing electron transfer in flavocytochrome P-450 BM3 and its component domains. FEBS Journal 239, 403–9 (1996).

126. Capeillere-Blandin, C. Simulation studies of the electron-transfer reactions among the prosthetic groups. Eur J Biochem. 56, 91–101 (1975).

127. Pompon, D. Flavocytochrome b2 from Baker’s Yeast. Eur J Biochem. 106, 151–159 (1980).

128. Daff, S., Sharp, R. E., Short, D. M., Bell, C., White, P., Manson, F. D., Reid, G. A. & Chapman, S. K. Interaction of cytochrome c with flavocytochrome b2. Biochemistry 35, 6351–7 (1996).

129. Chapman, S. K., Welsh, F., Moysey, R., Mowat, C. G., Doherty, M. K., Turner, K. L., Munro, A. W. & Reid, G. A. Flavocytochromes: transceivers and relays in biological electron transfer. Biochem. Soc. Trans. 27, 185–9 (1999).

130. Tegoni, M., Silvestrini, M. C., Guigliarelli, B., Asso, M., Brunori, M. P. & Bertrand, P. Temperature-jump and potentiometric studies on recombinant wild type and Y143F and Y254F mutants of Saccharomyces cerevisiae

259

References

flavocytochrome b2: role of the driving force in intramolecular electron transfer kinetics. Biochemistry 37, 12761–71 (1998).

131. Henriksson, G., Johansson, G. & Pettersson, G. A critical review of cellobiose dehydrogenases. J. Biotechnol. 78, 93–113 (2000).

132. Igarashi, K., Momohara, I., Nishino, T. & Samejima, M. Kinetics of interdomain electron transfer in flavocytochrome cellobiose dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Biochem. J. 365, 521–6 (2002).

133. Gardner, P. R., Gardner, A. M., Martin, L. A. & Salzman, A. L. Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc. Natl Acad. Sci. USA 95, 10378–83 (1998).

134. Gardner, P. R., Gardner, A. M., Martin, L. A., Dou, Y., Li, T., Olson, J. S., Zhu, H. & Riggs, A. F. Nitric-oxide dioxygenase activity and function of flavohemoglobins. sensitivity to nitric oxide and carbon monoxide inhibition. J. Biol. Chem. 275, 31581–7 (2000).

135. Koppenol, W. H., Moreno, J. J., Pryor, W. A., Ischiropoulos, H. & Beckman, J. S. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem Res Toxicol. 5, 834–842 (1992).

136. Hausladen, A., Gow, A. & Stamler, J. S. Flavohemoglobin denitrosylase catalyzes the reaction of a nitroxyl equivalent with molecular oxygen. Proc. Natl Acad. Sci. USA 98, 10108–12 (2001).

137. Mills, C. E., Sedelnikova, S., Søballe, B., Hughes, M. N. & Poole, R. K. Haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide. Biochemical Journal 213, 207–213 (2001).

138. Forrester, M. T. & Foster, M. W. Protection from nitrosative stress: a central role for microbial flavohemoglobin. Free Radical Biol. Med. 52, 1620–33 (2012).

260

References

139. Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H. H., Rommens, J. M., Scherer, S. W., Rosenblatt, D. S. & Gravel, R. a. Cloning and mapping of a cDNA for methionine synthase reductase, a flavoprotein defective in patients with homocystinuria. Proc. Natl Acad. Sci. USA 95, 3059–64 (1998).

140. Olteanu, H. & Banerjee, R. Human methionine synthase reductase, a soluble P-450 reductase-like dual flavoprotein, is sufficient for NADPH-dependent methionine synthase activation. J. Biol. Chem. 276, 35558–63 (2001).

141. Deng, L., Elmore, C. L., Lawrance, A. K., Matthews, R. G. & Rozen, R. Methionine synthase reductase deficiency results in adverse reproductive outcomes and congenital heart defects in mice. Mol. Genet. Metab. 94, 336– 42 (2008).

142. Ludwig, M. L. & Matthews, R. G. Structure-based perspectives on B12- dependent enzymes. Annu. Rev. Biochem. 66, 269–313 (1997).

143. Watkins, D. & Rosenblatt, D. S. Functional methionine synthase deficiency (cblE and cblG): clinical and biochemical heterogeneity. Am J Med Genet. 34, 427–434 (1989).

144. Harding, C. O., Arnold, G., Barness, L. A., Wolff, J. A. & Rosenblatt, D. S. Functional methionine synthase deficiency due to cblG disorder: a report of two patients and a review. Am J Med Genet. 71, 384–90 (1997).

145. Refsum, H., Ueland, P. M., Nygard, O. & Vollset, S. E. Homocysteine and cardiovascular disease. Annu. Rev. Medicine 49, 31–62 (1998).

146. Mills, J. L., Lee, Y. J., Conley, M. R., Kirke, P. N., McPartlin, J. M., Weir, D. G. & Scott, J. M. Homocysteine metabolism in pregnancies complicated by neural-tube defects. The Lancet 345, 149–151 (1995).

147. Clarke, R., Smith, A. D., Jobst, K. A., Refsum, H., Sutton, L. & Ueland, P. M. Folate, vitamin B12, and serum total homocysteine levels in confirmed Alzheimer disease. Arch. Neurol. 55, 1449–55 (1998).

261

References

148. Ostrowski, J., Barbertllii, M. J., Ruegerqll, D. C., Miller, B. E., Siegel, L. M. & Vi, N. M. K. Characterization of the flavoprotein moieties of NADPH- sulfite reductase from Salmonella typhimurium and Escherichia coli. J. Biol. Chem. 264, 15796–15808 (1989).

149. Siegel, L. M. & Murphy, J. Reduced nicotinamide adenine reductase of Enterobacteria dinucleotide. J. Biol. Chem. 248, (1973).

150. Siegel, L. M. & Davis, P. S. Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of Enterobacteria. J. Biol. Chem. 249, 1587–1598 (1974).

151. Porter, T. D. An unusual yet strongly conserved flavoprotein reductase in bacteria and mammals. Trends Biol. Sci. 16, 154–158 (1991).

152. Eschenbrenner, M., Covès, J. & Fontecave, M. NADPH-sulfite reductase flavoprotein from Escherichia coli: contribution to the flavin content and subunit interaction. FEBS Lett. 374, 82–4 (1995).

153. Eschenbrenner, M., Covès, J. & Fontecave, M. The flavin Rreductase activity of the flavoprotein component of sulfite reductase from Escherichia coli. A new model for the protein structure. J. Biol. Chem. 270, 20550–20555 (1995).

154. Crane, B. R., Siegel, L. M. & Getzoff, E. D. Sulfite reductase structure at 1. 6 A : evolution and catalysis for reduction of inorganic anions. Science 270, 59– 67 (1994).

155. Pinto, R., Harrison, J. S., Hsu, T., Jacobs, W. R. & Leyh, T. S. Sulfite reduction in mycobacteria. J. Bacteriol. 189, 6714–22 (2007).

156. Parey, K., Warkentin, E., Kroneck, P. M. H. & Ermler, U. Reaction cycle of the dissimilatory sulfite reductase from Archaeoglobus fulgidus. Biochemistry 49, 8912–21 (2010).

157. Yoshimoto, A. & Sato, R. Y. O. Studies on yeast sulphite reductase. Biochim. Biophys. Acta 153, 555–575 (1967).

262

References

158. Siegel, L. M., Davis, P. S. & Kamin, H. Reduced nicotinamide adenine Dinucleotide Sulfite Reductase of Enterobacteria. J. Biol. Chem. 249, 1572– 1586 (1974).

159. Coucouvanis, E. & Martin, G. R. Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83, 279–287 (1995).

160. Vermilion, J. L. & Coon, M. J. Purified liver microsomal reductase NADPH- cytochrome P-450. J. Biol. Chem. 253, 2694–2704 (1978).

161. Shen, A. L., Porter, T. D., Wilson, E. & Kasperll, C. B. Structural analysis of the FMN binding domain of NADPH-cytochrome P450 oxidoreductase by site-directed mutagenesis. J. Biol. Chem. 164, 7584–7589 (1989).

162. Shen, A. L. & Kasper, C. B. Role of acidic residues in the interaction of NADPH-cytochrome P450 oxidoreductase with cytochrome P450 and cytochrome c. J. Biol. Chem. 270, 27475–80 (1995).

163. Olteanu, H. & Banerjee, R. Redundancy in the pathway for redox regulation of mammalian methionine synthase: reductive activation by the dual flavoprotein, novel reductase 1. J. Biol. Chem. 278, 38310–4 (2003).

164. Omer, N., Rohilla, A., Rohilla, S. & Kushnoor, A. Review article nitric oxide : role in human biology. Int. J. Pharma. Sci. Drug Res. 4, 105–109 (2012).

165. Palmer, R. J. M. & Moncada, S. A novel citrulline-forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem. biophys. Res. Commun. 158, 348–352 (1989).

166. Malinski, T. & Taha, Z. Nitric oxide release from a single cell measured in situ by a porphyrinic-based microsensor. Nature 358, 676–678 (1992).

167. Malinski, T., Radomski, M. W., Taha, Z. & Moncada, S. Direct electrochemical measurement of nitric oxide released from human platelets. Biochem. biophys. Res. Commun. 194, 960–965 (1993).

263

References

168. Anggard, E. Nitric oxide : mediator , murderer , and medicine. The Lancet 343, 1199–1206 (1994).

169. Antosova, M., Plevkova, J., Strapkova, A. & Buday, T. Nitric oxide— important messenger in human body. Open. J. Mol. Integr. Physiol. 02, 98– 106 (2012).

170. Malinski, T. The vital role of nitric oxide. The Oakland Journal 1, 47–57 (2000).

171. Jerca, L., Jerca, O., Mancaş, G., Constantinescu, I. & Lupuşoru, R. Mechanism of action and biochemical effects of nitroc oxide. Am. J. Prev. Med. 10, 35–45 (2002).

172. Bredt, D. S. Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Rad. Res. 31, 577–96 (1999).

173. Bredt, D. S. Nitric oxide signaling specificity - the heart of the problem. J. Cell Science 116, 9–15 (2003).

174. Seddon, M., Shah, A. M. & Casadei, B. Cardiomyocytes as effectors of nitric oxide signalling. Cardiovasc. Res. 75, 315–26 (2007).

175. Bon, C. L. M. & Garthwaite, J. On the role of nitric oxide in hippocampal

long-term potentiation. J. Neurosci. 23, 1941–8 (2003).

176. Paul, V. & Ekambaram, P. Involvement of nitric oxide in learning & memory processes. Indian J. Med. Res. 133, 471–8 (2011).

177. Arancio, O., Lev-Ram, V., Tsien, R. Y., Kandel, E. R. & Hawkins, R. D. Nitric oxide acts as a retrograde messenger during long-term potentiation in cultured hippocampal neurons. J. Physiology (Paris) 90, 321–2 (1996).

178. Alger, B. E. Retrograde signaling in the regulation of synaptic transmission: focus on endocannabinoids. Prog. Neurobiol. 68, 247–86 (2002).

264

References

179. Malenka, R. C. & Bear, M. F. LTP and LTD: an embarrassment of riches. Neuron 44, 5–21 (2004).

180. Huk, I., Nanobashvili, J., Neumayer, C., Punz, A., Mueller, M., Afkhampour, K., Mittlboeck, M., Losert, U., Polterauer, P., Roth, E., Patton, S. & Malinski, T. L-Arginine treatment alters the kinetics of nitric oxide and superoxide release and reduces ischemia/reperfusion injury in skeletal muscle. Circulation 96, 667–675 (1997).

181. Momohara, Y., Sakamoto, S., Obayashi, S., Aso, T., Goto, M. & Azuma, H. Roles of endogenous nitric oxide synthase inhibitors and endothelin-1 for regulating myometrial contractions during gestation in the rat. Mol. Hum. Reprod. 10, 505–12 (2004).

182. Sugino, N., Takiguchi, S., Ono, M., Tamura, H., Shimamura, K., Nakamura, Y., Tsuruta, R., Sadamitsu, D., Ueda, T., Maekawa, T. & Kato, H. Nitric oxide concentrations in the follicular fluid and apoptosis of granulosa cells in human follicles. Human reproduction 11, 2484–7 (1996).

183. Davidoff, M. S., Middendorff, R., Mayer, B., DeVente, J., Koesling, D. & Holstein, A. F. Nitric oxide/cGMP pathway components in the Leydig cells of the human testis. Cell and Tissue Res. 287, 161–170 LA – English (1996).

184. Hagen, W. R. Biomolecular EPR spectroscopy. (CRC Press, 2009).

185. Jonas, M. Concepts and methods of ESR dating. Radiat. Meas. 27, 943–973 (1997).

186. Patel, K. N., Patel, J. K., Rajput, G. C. & Rajgor, N. B. Derivative spectrometry method for chemical analysis. Der Pharmacia Lettre 2, 139–150 (2010).

187. Hagen, W. R. High-frequency EPR of transition ion complexes and metalloproteins. Coord. Chem. Rev. 190-192, 209–229 (1999).

265

References

188. Schweiger, A. & Jeschke, G. Principles of pulsed electron paramagnetic resonance. (Oxford University Press, 2001).

189. Jeschke, G. & Polyhach, Y. Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance. Physical chemistry chemical physics: PCCP 9, 1895–910 (2007).

190. Puddephat, M. http://www.mikepuddephat.com. (2010). at

191. Del Barco, E. http://physics.ucf.edu. at

192. Roessler, M. M., King, M. S., Robinson, A. J., Armstrong, F. A., Harmer, J. & Hirst, J. Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron-electron resonance. Proc. Natl Acad. Sci. USA 107, 1930–5 (2010).

193. Dunford, A. J., Marshall, K. R., Munro, A. W. & Scrutton, N. S. Thermodynamic and kinetic analysis of the isolated FAD domain of rat neuronal nitric oxide synthase altered in the region of the FAD shielding residue Phe1395. FEBS Journal 271, 2548–60 (2004).

194. Knight, K. & Scrutton, N. S. Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase. Biochem. J. 367, 19–30 (2002).

195. Adak, S., Aulak, K. S. & Stuehr, D. J. Chimeras of nitric-oxide synthase types I and III establish fundamental correlates between heme reduction, heme-NO complex formation, and catalytic activity. J. Biol. Chem. 276, 23246–52 (2001).

266

References

196. Aliverti, A., Curti, B. & Vanoni, M. A. in Methods in Molecular Biology: Flavoprotein Protocols (Chapman, S. K. . & Reid, G. A.) 9–23 (Humana Press Inc., 1999).

197. Horecker, B. L. & Kornberg, A. The extinction coefficients of the reduced band of pyridine nucleotides. J. Biol. Chem. 175, 385–390 (1948).

198. Masters, B. S. S., Williams, C. H. & Kamin, H. The preparation and properties of microsomal TPNH-cytochrome c reduetase from pig liver. Method. Enzymol. 10, 565–573 (1967).

199. Van Gelder, B. F. & Slater, E. C. The extinction coefficient of cytochrome c. Biochim. Biophys. Acta 58, 593–595 (1962).

200. Onufriev, M. V & Gulyaeva, N. V. Recording of NADPH oxidation as a means of estimating NO synthase activity. B. Exp. Biol. Med. 120, 792–795

201. Wang, R. Y. in Applications of Physical Methods to Inorganic and Bioinorganic Chemistry (Scott, R. A. W. & Lukehart, C. M.) (John Wiley & Sons, Ltd, 2007).

202. Salter, M. & Knowles, R. G. in Methods in Molecular Biology: Nitric Oxide Protocols (Titheradge, M. A.) 61–65 (Springer-Verlag, 1998).

203. Doyle, M. P. & Hoekstra, J. W. Oxidation of nitrogen oxides by bound dioxygen in hemoproteins. J. Inorg. Chem. 14, 351–358 (1981).

204. Adak, S., Wang, Q. & Stuehr, D. J. Arginine conversion to nitroxide by tetrahydrobiopterin-free neuronal nitric-oxide synthase. Implications for mechanism. J. Biol. Chem. 275, 33554–61 (2000).

205. Dutton, P. L. Redox potentiometry: determination of midpoint potentials of oxidation-reduction components of biological electron-transfer systems. Method. Enzymol. 54, 411–435 (1978).

267

References

206. Dunford, A. J., Rigby, S. E. J., Hay, S., Munro, A. W. & Scrutton, N. S. Conformational and thermodynamic control of electron transfer in neuronal nitric oxide synthase. Biochemistry 46, 5018–5029 (2007).

207. Murataliev, M. B., Klein, M., Fulco, A. & Feyereisen, R. Functional interactions in cytochrome P450BM3: flavin semiquinone intermediates, role of NADP(H), and mechanism of electron transfer by the flavoprotein domain. Biochemistry 36, 8401–12 (1997).

208. Hay, S., Brenner, S., Khara, B., Quinn, A. M., Rigby, S. E. J. & Scrutton, N. S. Nature of the energy landscape for gated electron transfer in a dynamic redox protein. J. Am. Chem. Soc. 132, 9738–45 (2010).

209. Weber, S., Mo, K., Richter, G. & Kay, C. W. M. The electronic structure of the flavin cofactor in DNA photolyase. J. Am. Chem. Soc. 123, 3790–3798 (2001).

210. Schleicher, E. & Weber, S. Radicals in flavoproteins. Top Curr Chem 321, 41–66 (2012).

211. Kay, C. W. M., Elsässer, C., Bittl, R., Farrell, S. R. & Thorpe, C. Determination of the distance between the two neutral flavin radicals in augmenter of liver regeneration by pulsed ELDOR. J. Am. Chem. Soc. 128, 76–7 (2006).

212. Tiso, M., Konas, D. W., Panda, K., Garcin, E. D., Sharma, M., Getzoff, E. D. & Stuehr, D. J. C-terminal tail residue Arg1400 enables NADPH to regulate electron transfer in neuronal nitric-oxide synthase. J. Biol. Chem. 280, 39208– 19 (2005).

213. Konas, D. W., Zhu, K., Sharma, M., Aulak, K. S., Brudvig, G. W. & Stuehr, D. J. The FAD-shielding residue Phe1395 regulates neuronal nitric-oxide synthase catalysis by controlling NADP+ affinity and a conformational equilibrium within the flavoprotein domain. J. Biol. Chem. 279, 35412–25 (2004).

268

References

214. Gutierrez, A., Paine, M., Wolf, C. R., Scrutton, N. S. & Roberts, G. C. K. Relaxation kinetics of cytochrome P450 reductase: internal electron transfer is limited by conformational change and regulated by coenzyme binding. Biochemistry 41, 4626–37 (2002).

215. Gutierrez, A., Munro, A. W., Grunau, A., Wolf, C. R., Scrutton, N. S. & Roberts, G. C. K. Interflavin electron transfer in human cytochrome P450 reductase is enhanced by coenzyme binding. Relaxation kinetic studies with coenzyme analogues. Eur. J. Biochem. 270, 2612–2621 (2003).

216. Hamdane, D., Xia, C., Im, S. C., Zhang, H., Kim, J. J. P. & Waskell, L. Structure and function of an NADPH-cytochrome P450 oxidoreductase in an open conformation capable of reducing cytochrome P450. J. Biol. Chem. 284, 11374–84 (2009).

217. Förstermann, U. & Sessa, W. C. Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829–37, 837a–837d (2012).

218. Abu-Soud, H. M., Yoho, L. L. & Stuehr, D. J. Calmodulin controls neuronal nitric-oxide synthase by a dual mechanism. J. Biol. Chem. 269, 32047–32050 (1994).

219. Masters, B. S. S. Nitric oxide synthases: why so complex? Annu. Rev. Nutr. 14, 131–45 (1994).

220. Matsuda, H. & Iyanagi, T. Calmodulin activates intramolecular electron transfer between the two flavins of neuronal nitric oxide synthase flavin domain. Biochim. Biophys. Acta 1473, 345–55 (1999).

221. Roman, L. J. & Masters, B. S. S. Electron transfer by neuronal nitric-oxide synthase is regulated by concerted interaction of calmodulin and two intrinsic regulatory elements. J. Biol. Chem. 281, 23111–8 (2006).

222. Gao, Y. T., Smith, S. M. E., Weinberg, J. B., Montgomery, H. J., Newman, E., Guillemette, J. G., Ghosh, D. K., Roman, L. J., Martasek, P. & Salerno, J.

269

References

C. Thermodynamics of oxidation-reduction reactions in mammalian nitric- oxide synthase isoforms. J. Biol. Chem. 279, 18759–66 (2004).

223. Jeschke, G. DEER distance measurements on proteins. Annu. Rev. Phys. Chem. 63, 419–46 (2012).

224. Pannier, M., Veit, S., Godt, A., Jeschke, G. & Spiess, H. W. Dead-time free measurement of dipole-dipole interactions between electron spins. J. Magn. Reson. 142, 331–40 (2000).

225. Schiemann, O. & Prisner, T. F. Long-range distance determinations in biomacromolecules by EPR spectroscopy. Quart. Rev. Biophys. 40, 1–53 (2007).

226. Roman, L. J., Sheta, E. A., Martasek, P., Gross, S. S., Liu, Q. & Masters, B. S. High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli. Proc. Natl Acad. Sci. USA 92, 8428–32 (1995).

227. Adak, S., Santolini, J., Tikunova, S., Wang, Q., Johnson, J. D. & Stuehr, D. J. Neuronal nitric-oxide synthase mutant (Ser-1412 --> Asp) demonstrates surprising connections between heme reduction, NO complex formation, and catalysis. J. Biol. Chem. 276, 1244–52 (2001).

228. Panda, K., Adak, S., Aulak, K. S., Santolini, J., McDonald, J. F. & Stuehr, D. J. Distinct influence of N-terminal elements on neuronal nitric-oxide synthase structure and catalysis. J. Biol. Chem. 278, 37122–31 (2003).

229. Stuehr, D. J., Wei, C. C., Wang, Z. & Hille, R. Exploring the redox reactions between heme and tetrahydrobiopterin in the nitric oxide synthases. J. R. Soc. Chem. Dalton transactions 3427–35 (2005). doi:10.1039/b506355h

230. Astashkin, A. V, Elmore, B. O., Fan, W., Guillemette, J. G. & Feng, C. Surface charges and regulation of FMN to heme electron transfer in nitric- oxide synthase. J. Am. Chem. Soc. 132, 12059–12067 (2010).

270

References

231. Ghosh, D. K., Holliday, M. A., Thomas, C., Weinberg, J. B., Smith, S. M. E. & Salerno, J. C. Nitric-oxide synthase output state. Design and properties of nitric-oxide synthase oxygenase/FMN domain constructs. J. Biol. Chem. 281, 14173–83 (2006).

232. Lange, R., Bec, N., Anzenbacher, P., Munro, A. W., Gorren, A. C. F. & Mayer, B. Use of high pressure to study elementary steps in P450 and nitric oxide synthase. J. Inorg. Biochem. 87, 191–195 (2001).

233. Marchal, S., Gorren, A. C. F., Andersson, K. K. & Lange, R. Hunting oxygen complexes of nitric oxide synthase at low temperature and high pressure. Biochem. biophys. Res. Commun. 338, 529–35 (2005).

234. Balny, C. Pressure effects on weak interactions in biological systems. J. Phys.: Condens. Matter 16, S1245–S1253 (2004).

235. Bailey, J. A., James, C. A. & Woodruff, W. H. Flow-flash kinetics of O2 binding to cytochrome c oxidase at elevated [O2]: observations using high pressure stopped flow for gaseous reactants. Biochem. biophys. Res. Commun. 220, 1055–60 (1996).

236. Jung, C. Cytochrome P-450-CO and substrates: lessons from ligand binding under high pressure. Biochim. Biophys. Acta 1595, 309–28 (2002).

237. Gross, M. & Jaenicke, R. Proteins under pressure. The influence of high hydrostatic pressure on structure, function and assembly of proteins and protein complexes. Eur. J. Biochem. 221, 617–630 (1994).

238. Gow, A. J., Luchsinger, B. P., Pawloski, J. R., Singel, D. J. & Stamler, J. S. The oxyhemoglobin reaction of nitric oxide. Proc. Natl Acad. Sci. USA 96, 9027–32 (1999).

239. Gorren, A. C. F., Marchal, S., Sørlie, M., Andersson, K. K., Lange, R. & Mayer, B. High-pressure studies of the reaction mechanism of nitric-oxide synthase. Biochim. Biophys. Acta 1764, 578–85 (2006).

271

References

240. Marchal, S., Girvan, H. M., Gorren, A. C. F., Mayer, B., Munro, A. W., Balny, C. & Lange, R. Formation of transient oxygen complexes of cytochrome p450 BM3 and nitric oxide synthase under high pressure. Biophys. J. 85, 3303–9 (2003).

241. Abu-soud, H. M., Ichimori, K., Presta, A. & Stuehr, D. J. Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric- oxide synthase. J. Biol. Chem. 275, 17349–17357 (2000).

242. Feng, C., Thomas, C., Holliday, M. A., Tollin, G., Salerno, J. C., Ghosh, D. K., Enemark, J. H., Carolina, N. & York, N. Direct measurement by laser flash photolysis of intramolecular electron transfer in a two-domain construct of murine inducible nitric oxide synthase biochemical characterization of the murine iNOS con- deazariboflavin semiquinone. J. Am. Chem. Soc. 11, 3808– 3811 (2006).

243. Abu-Soud, H. M., Feldman, P. L., Clark, P. & Stuehr, D. J. Electron transfer in the nitric-oxide synthases. J. Biol. Chem. 269, 32318–32326 (1994).

244. Abu-Soud, H. M., Wang, J., Rousseau, D. L., Fukuto, J. M., Ignarro, L. J. & Stuehr, D. J. Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis. J. Biol. Chem. 270, 22997– 23006 (1995).

245. Haque, M. M., Tejero, J., Bayachou, M., Wang, Z. Q., Fadlalla, M. & Stuehr, D. J. Thermodynamic characterization of five key kinetic parameters that define neuronal nitric oxide synthase catalysis. FEBS Journal 280, 4439–53 (2013).

246. Li, H., Jamal, J., Chreifi, G., Venkatesh, V., Abou-Ziab, H. & Poulos, T. L. Dissecting the kinetics of the NADP(+)-FADH2 charge transfer complex and flavin semiquinones in neuronal nitric oxide synthase. J. Inorg. Biochem. 124, 1–10 (2013).

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