© 2016 Shiliang Tian

PROTEIN ENGINEERING USING AZURIN AS THE SCAFFOLD: CAPTURING AND STUDYING NOVEL METAL-SULFENATE AND METAL-NO SPECIES

BY SHILIANG TIAN

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2016

Urbana, Illinois

Doctoral Committee:

Professor Yi Lu, Chair Professor Thomas B. Rauchfuss Professor Wilfred A. van der Donk Assistant Professor Alison R. Fout

Abstract

Metalloproteins account for nearly half of all proteins in nature. Metal ions play important roles in catalyzing numerous important biological processes that necessary to sustain life on the planet, such as photosynthesis, respiration and nitrogen fixation. Much effort has been made to understand the relationship between structures and functions of metalloproteins. Although significant progresses have been made to obtain the knowledge of how metalloproteins work, the ultimate test is to use this knowledge to design new metallproteins that reproduce the structures and functions of native proteins. Protein redesign strategy is one of the most effective approaches in the design and engineering of artificial metalloenzymes. The advantage of a protein redesign strategy is that it can bypass the problem of developing a stable protein fold because many native proteins have remarkable adaptability for changes. The use of small, stable, easy-to- make, and well-characterized blue copper protein azurin as scaffold to design novel metal binding sites has been proven to be a promising way for protein redesign. Not only can its reduction potential be rationally tuned beyond the nature range via secondary coordination sphere engineering, but also the CuA and redox- active nonheme iron sites have been successfully engineered in azurin.

Metal-sulfenate centers are known to play important roles in biology and yet only limited examples are known due to their instability and high reactivity. In Chapter 2, the first copper-sulfenate was characterized in a protein environment, formed at the active site of a cavity mutant of an electron transfer protein, type 1, blue copper azurin. Reaction of hydrogen peroxide with Cu(I)-Met121Gly azurin resulted in a new species with strong visible absorptions at 350 and 452 nm and a relatively low electron paramagnetic resonance gz value of 2.169 in comparison to other normal type 2 copper centers. The presence of a side-on copper-sulfenate species is supported by resonance Raman spectroscopy, electrospray mass spectrometry using isotopically enriched hydrogen peroxide, and density functional theory calculations correlated to the experimental data. In contrast, the reaction with Cu(II)-Met121Gly or Zn(II)-

Met121Gly azurin under the same conditions did not result in Cys oxidation or copper-sulfenate formation.

Structural and computational studies strongly suggest that the secondary coordination sphere non-covalent interactions are critical in stabilizing this highly reactive species, which can further react with oxygen to

ii form a sulfinate and then a sulfonate species, as demonstrated by mass spectrometry. Engineering the electron transfer protein azurin into an active copper enzyme that forms a copper-sulfenate center and demonstrating the importance of non-covalent secondary sphere interactions in stabilizing it constitute important contributions toward the understanding of metal sulfenate species in biological systems.

S-nitrosothiols are known reagents for NO storage and transportation in vivo and regulating factors in many physiological processes. While the S-nitrosylation catalyzed by heme proteins is well known, no direct evidence of S-nitrosylation of Cu(II)-bound cysteine by NO has been reported. In Chapter 3, the blue copper center in WTAz was converted into a red copper center that closely mimics that in nitrosocyanin by rational design of the primary coordination sphere (M121H/H46E) first, and then by tuning its reduction potential via deleting a hydrogen bond in the secondary coordination sphere (F114P). The engineered red copper protein exhibits a significantly longer Cu-S(Cys) bond distance (~2.28 Å), lower reduction potential

(~107 mV at pH 8) and larger hyperfine splitting in the parallel region (~160×10-4 cm-1) as compared to blue copper proteins, and the electronic properties of the engineered protein are comparable to those of the red copper center in nitrosocyanin. Stoichiometric titration of NO to Cu(II)-M121H/H46E/F114PAz yields nearly quantitative S-nitrosylation product with concomitant reduction of the metal center with a second order rate constant of ~ 105 M-1s-1. The resulting S(Cys)-SNO containing protein shows a strong absorbance at 334 nm and a weak absorbance around 540 nm, similar to other reported RSNO species. Reduction of the Cu(II) site to Cu(I) during S-nitrosylation process is confirmed by a loss of over 90% of Cu(II) signal in EPR spectrum and a shift of the Cu K-edge to lower energy in XANES. Further EXAFS fitting reveals that the Cu-S distance increases from 2.18 Å in the reduced engineered red copper protein to 3.98 Å in the

S-nitrosylation species, indicating Cu(I) interacts with N rather than S atom. DFT calculations have identified the most plausible mechanism for S(Cys)-NO formation as the direct radical reaction of NO with

Cu(II)-coordinated S(Cys) enabled by the high covalency of the Cu(II)-S(Cys) bond. Our results provide the first direct evidence of S-nitrosylation of copper-bound cysteine in a protein, and show that such a reaction can modulate solution NO concentration under physiologically relevant conditions to prevent NO inhibition of cytochrome oxidase activity.

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Cupredoxins are known to contain a type 1 copper (T1Cu) center that is coordinately saturated and performs exclusively electron transfer function. Nitrosocyanin has been shown to contain an open-binding site, with water as a ligand, but spectroscopic and X-ray crystallographic studies indicate that it contains a type 2 copper (T2Cu) center. In Chapter 4, a novel cupredoxin isolated from Nitrosopumilus maritimus, called Nmar_1307, was recombinantly expressed in E. coli and characterized with different spectroscopic methods. The protein displays a strong purple color due to strong absorptions around 413 nm (1450 M-1cm-

1) and 558 nm (1770 M-1cm-1) in the UV-vis electronic spectrum and a small hyperfine coupling constant

-4 -1 (A|| < 100x10 cm ) in the EPR spectrum, typical of T1Cu center. X-ray crystal structure at 1.6 Å resolution con-firms that it contains T1Cu center with a Cu atom coordinated by two His and one Cys in a trigonal place and a short Cu(II)-SCys bond (2.25 Å). In contrast to the coordinately saturated T1Cu center observed in other cupredoxins, the Nmar_1307 contains a unique T1Cu center with an open-binding site containing water. Both UV-vis absorption and EPR spectroscopy studies suggest that the Nmar_1307 can oxidase NO to nitrite, whose activity is attributable to the unusually high reduction potential (484±9 mV vs. SHE) of the copper site. These results suggest T1Cu cupredoxins can have a wide range of structural features, including an open-binding site containing water, making this class of proteins even more versatile.

7 8 Mononuclear nitrosyl iron complexes – mononitrosyl iron {FeNO} , dinitrosyl iron {Fe(NO)2}

9 and {Fe(NO)2} – are key intermediates in the nitrosylation of nonheme iron proteins in biology. It has been difficult to capture all three species in identical host complexes for both native enzymes and synthetic analogs, which is necessary to elucidate changes to the structure of the iron center during nitrosylation and the factors that control their chemical reactivities. More importantly, to the best of our knowledge, the

8 {Fe(NO)2} species which is proposed as an intermediate in cis-FeB mechanism of nitric oxide reductases

(NORs) has never been unambiguously characterized and thus the one-electron reduction process from

8 9 {Fe(NO)2} to {Fe(NO)2} is not fully understood. In Chapter 5, a stepwise nitrosylation process of an engineered non heme iron site in blue copper azurin was captured and characterized. All {FeNO}7,

8 9 {Fe(NO)2} and {Fe(NO)2} species are successfully isolated by controlling the amount of nitric oxide

8 9 added and reaction time. The {Fe(NO)2} species can be reduced to {Fe(NO)2} with either dithionite or

7 8 9 excess NO. Structural information of the {FeNO} , {Fe(NO)2} and {Fe(NO)2} species are gained by combining the results of electron nuclear double resonance (ENDOR), hyperfine sub-level correlation

(HYSCORE) and nuclear resonance vibrational spectroscopy (NRVS) techniques. The entire pathway of

7 8 engineered non heme iron protein nitrosylation process from reduced form to {FeNO} , then to {Fe(NO)2}

9 and {Fe(NO)2} is discovered. These results not only enhance our understanding of the pathological and physiological roles of nitric oxide in non heme iron proteins’ regulation but also shed light on the mechanism of NORs.

Compared to other virus presented in drinking water, adenoviruses show high resistance to monochloramine and low pressure ultraviolet light but significantly disinfected by free chlorine. A systematic understanding of adenovirus inactivation by free chlorine would potentially direct the development and optimization of water treatments and strengthen our understanding of virus inactivation mechanism. Previous study suggests that genome damage and loss of the interaction between fiber and

CAR receptors are not the cause of disinfection but the disruption of penton or hexon protein structure may play an important role in adenovirus inactivation by free chlorine. In Chapter 6, adenovirus penton and hexon with 6×His-tag at C-terminal was first time expressed in E. coli. This novel method enable us to produce all three major capsid proteins of human adenovirus serotype 2 in a fast and high quality way.

Around 90% sequence coverages are achieved after trypsin digestion – MS/MS studies. This approach allowed us to detect modifications at amino acid sidechains and provide insight into the mechanism of Ad2 inactivation by free chlorine.

In summary, this thesis details in capture and characterization of novel copper-sulfenate, copper-

SNO and dinitrosyl iron species in engineered azurin with different spectroscopic methods and DFT calculation.

To my beloved wife Jing, who comforts my pain, cheers me up and shares my happiness,

and my cute son Ryan, whose smile always brightens my day.

Acknowledgements

There are many people without whom I would never have been able to finish my dissertation. First and foremost I would like to express my deepest gratitude to my advisor Dr. Yi Lu for his guidance, caring, patience, teaching and support throughout the years. I also wish to thank my committee members Dr. Tom

Rauchfuss, Dr. Wilfred van der Donk and Dr. Alison Fout for their helpful discussions, guiding my research, providing me with an excellent atmosphere for doing research during the past several years.

Thank you also to all of my coworkers in the Lu lab, both current and past, who have been very supportive over the years. In particular I would like to thank Dr. Nathan Sieracki for training and mentoring me during my early years. I would also like to thank the postdocs, graduate students and undergraduates who I have worked with directly on different projects including: Dr. Hongchao Guo, Dr. Yingwu Lin, Dr.

Hui Wei, Dr. Nick Marshall, Dr. Yang Yu, Dr. Parisa Hosseinzadeh, Chang Cui, Kevin Harnden, Kevin

Hwang and Chu Zheng. I would also like to thank Dr. Kyle Miner, Dr. Tiffany Wilson, Dr. Arnab

Mukherjee, Dr. Saumen Chakraborty, Dr. Zhou Dai, Igor Petrik, Bryant Kearl, Ambika Bhagi, Julian Reeds and Evan Mirts for their useful discussion and support throughout the years.

I would also like to thank my collaborators outside the group. Specially thank Dr. Ryan Hadt, Dr.

Ryan Cowley and Dr. Ed Solomon from Stanford University, who performed DFT calculation on both copper-sulfenate and copper-SNO projects, providing insightful explanation of the experimental results. I would like to thank Dr. Ninian Blackburn from Oregon Health and Science University, Dr. Erik Farquhar from Brookhaven National Lab for their help in X-ray absorption spectroscopy experiment. I would like to thank Dr. Yisong Guo from Carnegie Mellow University and Dr. Chuck Schulz from Knox College for their help in collecting Mössbauer data on dinitrosyl iron project. I would like to thank Dr. Tim Sage and

Nicole Branagan from Northeastern University for their help in NRVS data analysis. I am grateful to Dr.

Mark Nilges for his help from EPR instrument setup, data collection, EPR simulations to data interpretation.

Thanks to Dr. Peter Yau and Dr. Brian Imai at Roy J. Carver Biotechnology Center for their analysis of submitted HPLC/MS/MS samples and results discussion.

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I would also like to thank my mom, who was always supporting me and encouraging me with her best wishes.

Finally, I would like to thank my wife, Jing Liu. She was always there cheering me up and stood by me through the good times and bad.

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Table of Contents List of Figures ...... xiii List of Tables ...... xvii Chapter 1 Engineering Novel Metal-Binding Sites Using Pseudomonas aeruginosa Azurin as the Scaffold ...... 1 1.1 Rational protein design: a valuable tool for understanding how metalloproteins work and achieving novel catalytic biochemical transformation ...... 1 1.1.1 De novo design: metalloproteins design from scratch ...... 1 1.1.2 Protein redesign: metalloproteins design through strategic modification of native protein scaffolds ...... 4 1.2 Engineering novel metal sites using Pseudomonas aeruginosa azurin as the scaffold...... 7 1.2.1 Rationally tuning the reduction potential of blue copper protein azurin beyond the natural range ...... 8 1.2.2 Design mononuclear Cu-binding site with novel electronic structures ...... 10

1.2.3 Design binuclear CuA site in azurin ...... 11 1.2.4 Design a redox-active mononuclear nonheme iron binding site in azurin ...... 13 1.3 Summary ...... 14 1.4 References ...... 14

Chapter 2 A Novel Copper-Sulfenate Complex from Oxidation of a Cavity Mutant of Pseudomonas aeruginosa Azurin ...... 21 2.1 Introduction ...... 21 2.2 Materials and methods ...... 23 2.2.1 Site-directed mutagenesis and protein purification ...... 23 2.2.2 Spectroscopic measurements ...... 24 2.2.3 Mass spectrometry measurements ...... 24 2.2.4 Preparation of Cu(II)-M121G azurin ...... 24 2.2.5 Crystallography of Cu(II)-M121G azurin ...... 25 2.2.6 Preparation of Cu(I)-M121G azurin ...... 25 2.2.7 Trypsin digest and HPLC MS/MS analysis ...... 25 2.2.8 DFT Calculations ...... 26 2.3 Results and discussion ...... 26 2.3.1 UV-vis and crystallographic characterization of Cu(II)-M121G azurin ...... 26 2.3.2 EPR investigation of the air-sensitive species ...... 33 2.3.3 Resonance Raman of the air-sensitive species ...... 34 2.3.4 Capture of sulfenic acid with the selective labeling agent, dimedone ...... 37

2.3.5 Isotopic enrichment study of Cys-SO3H formation ...... 39 2.3.6 DFT calculations ...... 40 2.4 Summary ...... 49 2.5 References ...... 49

Chapter 3 Reversible S-Nitrosylation in an Engineered Azurin and its Application as NO regulator under Physiological Conditions ...... 54 3.1 Introduction ...... 54 3.2 Materials and methods ...... 55 3.2.1 Site-directed mutagenesis and protein purification ...... 55 3.2.2 UV-vis spectroscopy measurements ...... 56 3.2.3 Mass spectrometry measurements ...... 56 3.2.4 Electron paramagnetic resonance (EPR) spectroscopy measurements ...... 56 3.2.5 Electrochemical measurements ...... 57 3.2.6 Collection and analysis of XAS data ...... 57 3.2.7 Reconstitution of M121H/H46EAz and M121H/H46E/F114PAz with Cu(II) ...... 58 3.2.8 Crystallography of Cu(II)-M121H/H46EAz ...... 59 3.2.9 Preparation of Cu(I)-M121H/H46E/F114PAz ...... 59 3.2.10 Reaction of Cu(II)-M121H/H46E/F114PAz with nitric oxide (generated from DEA NONOate) ...... 60 3.2.11 Reaction of Cu(II)-M121H/H46E/F114PAz with nitric oxide (prepared from nitric oxide saturated buffer) with stopped-flow ...... 60

3.2.12 Effect of Cu(II)-M121H/H46EAz on NO-inhibition of E. coli cytochrome bo3 oxidase ...... 61 3.2.13 Electronic structure calculations ...... 61 3.3 Results ...... 62 3.3.1 Design, spectroscopic, crystallographic and redox studies of an engineered red copper protein in azurin (Cu(II)-M121H/H46EAz) ...... 62 3.3.2 The design, spectroscopic, and redox studies of Cu(II)-M121H/H46E/F114PAz ...... 67 3.3.3 S-nitrosylation in the engineered red copper protein ...... 69 3.3.4 X-ray absorption spectroscopic studies of S-nitrosylation ...... 75 3.3.5 DFT calculations and mechanism of nitrosylation ...... 78 3.3.6 Using the engineered red copper protein to regulate NO concentration under physiological conditions by S-nitrosylation ...... 84 3.4 Discussion ...... 86 3.5 Summary ...... 87 3.6 References ...... 88

Chapter 4 A Novel Purple Cupredoxin from Nitrosopumilus maritimus Containing a Mononuclear Type 1 Copper Center with an Open Binding Site ...... 95 4.1 Introduction ...... 95 4.2 Materials and methods ...... 96 4.2.1 Cell culture and expression of Nmar_1307 ...... 96 4.2.2 Osmotic shock procedure for periplasmically expressed Nmar_1307 ...... 97 4.2.3 Column purification of Nmar_1307 and reconstitution with copper ...... 97 4.2.4 Electrochemical characterization of Cu(II)-Nmar_1307 ...... 98 4.2.5 Spectroscopic characterization of Cu(II)-Nmar_1307 ...... 98 4.2.6 Crystallization of Cu(II)-Nmar_1307 ...... 99 4.2.7 Activity tests ...... 99 4.2.8 Griess assay ...... 99 4.3 Results and discussion ...... 100 4.3.1 Recombinant expression of apo-Nmar_1307 in E. coli ...... 100 4.3.2 Preparation and UV-vis, EPR characterization of Cu(II)-NMar_1307 ...... 102 4.3.3 X-ray crystallography study of Cu(II)-Nmar_1307 ...... 103 4.3.4 Redox property of Cu(II)-Nmar_1307 ...... 106 4.3.5 Potential functions of Cu(II)-Nmar_1307...... 107 4.4 Summary ...... 111 4.5 References ...... 111

7 8 9 Chapter 5 From {FeNO} to {Fe(NO)2} to {Fe(NO)2} : Stepwise Nitrosylation of the Biological Nonheme Iron Proteins Observed in an Engineered Mutant of Pseudomonas aeruginosa Azurin ...... 115 5.1 Introduction ...... 115 5.2 Materials and methods ...... 117 5.2.1 Materials and chemicals ...... 117 5.2.2 Site-directed mutagenesis and protein purification...... 117 5.2.3 UV-vis spectroscopy measurements...... 117 5.2.4 Mass spectrometry measurements...... 118 5.2.5 Electron paramagnetic resonance (EPR) spectroscopy measurements...... 118 5.2.6 Fe(II) titration...... 118 5.2.7 Determination of extinction coefficient of Fe(II)-M121H/H46EAz...... 119 5.2.8 Fe(II) incorporation...... 119 5.2.9 Mössbauer spectroscopy...... 119 5.3 Results ...... 120

5.3.1 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by UV-vis spectrum ...... 120 5.3.2 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by cwEPR spectrum ...... 123 9 5.3.3 The characterization of {Fe(NO)2} species using ENDOR and HYSCORE ...... 127 5.3.4 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by Mössbauer spectrum ...... 131 5.3.5 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by NRVS ...... 133 5.3.6 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by XAS ...... 134 5.4 Summary ...... 137 5.5 References ...... 138

Chapter 6 Mechanistic Studies into Adenovirus Serotype 2 Inactivation by Free Chlorine via Expression of Major Capsid Proteins in E.coli ...... 144 6.1 Introduction ...... 144 6.2 Materials and methods ...... 145 6.2.1 Construction of plasmid for the synthesis of adenovirus fiber, penton and hexon proteins .... 145 6.2.2 Expression of the adenovirus fiber, penton and hexon proteins ...... 146 6.2.3 Purification of the adenovirus fiber, penton and hexon proteins ...... 146 6.2.4 Transmission electron microscopy (TEM) investigation ...... 147 6.2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot . 147 6.2.6 Inactivate major capsid proteins with free chlorine ...... 148 6.2.7 Trypsin digest and HPLC MS/MS analysis ...... 148 6.3 Results ...... 149 6.3.1 Adenovirus fiber, penton and hexon expression in E. coli ...... 149 6.3.2 Mechanism investigation of adenovirus inactivation by free chlorine ...... 155 6.4 Discussion ...... 157 6.5 Summary ...... 160 6.6 References ...... 160

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List of Figures Figure 1.1 Important structural features for the original and redesigned DFsc proteins ...... 3 Figure 1.2 Ribbon diagrams of the Hg, Zn-binding α-helix buddle structure...... 4

Figure 1.3 FeBMb, designed prior to structure elucidation of NOR, is structurally similar to NOR and displays NOR activity ...... 5 Figure 1.4 A designed oxdiase achiving the catalytic rate of a native enzyme ...... 6 Figure 1.5 Rational tuning of the reduction potential of azurin ...... 8 Figure 1.6 The Cu(II) binding sites of C112D in Type zero copper proteins ...... 10

Figure 1.7 CuA in biosynthetic model in P. aeruginosa azurin ...... 12 Figure 1.8 Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron protein ... 13 Figure 2.1 List of different products during cysteine oxidation ...... 21 Figure 2.2 Cysteine sulfenic acid Cys13-SOH in SarZ...... 22 Figure 2.3 The active site of nitrile hydratase contains either Fe or Co ...... 23 Figure 2.4 X-ray crystal structures showing the active sites of wild type azurin and M121G azurin ...... 27 Figure 2.5 Time course of UV-vis and EPR spectra upon formation of the air sensitive species and its decomposition upon air expose ...... 29

Figure 2.6 UV-vis spectrum upon addition of 5 eq. H2O2 to different protein solutions ...... 31

Figure 2.7 Electrospray mass spectrum upon addition of 5 eq. H2O2 to different protein solutions ...... 32 Figure 2.8 EPR study and simulation of the air sensitive species ...... 33 Figure 2.9 Resonance Raman spectroscopy of the air sensitive species ...... 35 Figure 2.10 Mass spectra of the air sensitive species...... 38

Figure 2.11 MS/MS data of NH2-LKEGEQYMFF(C-dimedone)TFPGHSALGK-COOH formed from trypsin digestion of dimedone treated reaction solution ...... 39 Figure 2.12 Calculated and experimental isotopic distributions for product

18 EGEQYMFF(C-SO3H)TFPGHSALGK tryptic peptides with progressive O labeling ...... 40

Figure 2.13 Small DFT structures for M121G-H2O and models for the air-sensitive species ...... 41 Figure 2.14 Time dependent DFT calculated absorption spectra. TDDFT calculationed absorption spectra for potential air-sensitive species ...... 43

Figure 2.15 Large DFT structures for M121G-H2O (L-M121G-H2O) and the air-sensitive species ...... 45 Figure 2.16 Comparison of the ground state wave functions ...... 48 Figure 3.1 UV-vis spectroscopic study of Cu(II)-WTAz reacting with NO ...... 62

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Figure 3.2 The Cu(II) binding sites of WTAz, nitrosocyanin and M121H/H46EAz ...... 63

Figure 3.3 Titration of apo-M121H/H46EAz with CuSO4 ...... 64 Figure 3.4 Measurement of the extinction coefficient of Cu(II)-M121H/H46EAz ...... 65

Figure 3.5 Titration of apo-M121H/H46E/F114PAz with CuSO4 ...... 67 Figure 3.6 Measurement of the extinction coefficient of Cu(II)-M121H/H46E/F114PAz ...... 68 Figure 3.7 Cyclic Voltammetry of Cu(II)-M121H/H46E/F114PAz ...... 68 Figure 3.8 EPR spectrum of Cu(II)-M121H/H46E/F114PAz...... 69 Figure 3.9 UV-Vis absorption, mass spectrometric and EPR spectroscopic characterization of S-nitrosylation in engineered red copper protein ...... 71 Figure 3.10 High-resolution mass spectrometric study of the S-nitrosylation reaction in engineered azurin ...... 72 Figure 3.11 Stopped-flow kinetics of Cu(II)-M121H/H46E/F114P directly reacting with nitric oxide in 50 mM MES buffer pH6.0 ...... 73 Figure 3.12 The reversibility of Cu(I)-SNO species formation towards air exposure ...... 74 Figure 3.13 The reversibility of Cu(I)-SNO species formation towards vacuum ...... 74

Figure 3.14 ESI-MS and EPR investigation of S-nitrosylated protein with O2 ...... 75 Figure 3.15 XAS investigation of S-nitrosylation process in engineered red copper protein ...... 76 Figure 3.16 The Fourier transform and fitting of EXAFS data...... 77 Figure 3.17 Ground state wavefunctions of optimized Cu(II) M121H/H46E azurin ...... 79 Figure 3.18 Electrophilic and radical pathways for (Cys)S-NO bond formation and possible isomers of the Cu(I)/(Cys)S-NO product ...... 80 Figure 3.19 Frontier molecular orbitals of the {CuNO}10 complex ...... 81

Figure 3.20 Frontier α molecular orbitals at several points along the direct SCys-NO reaction coordinate ...... 81

Figure 3.21 Frontier β molecular orbitals at several points along the direct SCys-NO reaction coordinate ...... 82

2 Figure 3.22 Spin polarization (~~) and spin density plots along the SCys-NO radical coupling reaction coordinate ...... 83~~

~~Figure 3.23 NO inhibition of E. coli cytochrome bo3 oxidase ...... 84~~

~~Figure 3.24 Effect of Cu(II)-M121H/H46E/F114PAz on NO inhibition of E. coli cytochrome bo3 oxidase...... 85~~

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Figure 3.25 Negative control of Cu(II)-M121H/H46E/F114PAz on NO inhibition of E. coli cytochrome bo3 oxidase ...... 86 Figure 4.1 Structure-based sequence alignment of Nmar_1307 and a number of other cupredoxins proteins ...... 101 Figure 4.2 Purification of Nmar_1307 via Q-FF and SEC columns ...... 101 Figure 4.3 ESI-MS spectra of Cu(II)-Nmar_1307 ...... 102 Figure 4.4 UV-vis and EPR spectra of Cu(II)-Nmar_1307 in 50 mM Tris buffer pH 8.0...... 103 Figure 4.5 Crystal Structure of Cu(II)-Nmar_1307 ...... 105 Figure 4.6 Cyclic voltammogram of Cu(II)-Nmar_1307 at 100 mM Tris pH8.0 supplemented with 100 mM NaCl ...... 107 Figure 4.7 Kinetic UV-vis and EPR spectra of Cu(II)-Nmar_1307 reacting with NO at pH8.0...... 108

- Figure 4.8 NO2 detection by Griess assay after Cu(II)-Nmar1307 reacting with NO...... 109 Figure 4.9 The NO oxidation activity is highly pH-dependent ...... 110 Figure 4.10 Kinetic UV-vis spectra and absorbance traces monitored at 412 and 560 nm of Cu(II)-Nmar_1307 reacting with NO at pH6.0...... 110

Figure 5.1 Schematic representation of the trans, cis-heme b3, and cis-FeB mechanisms ...... 116 Figure 5.2 Fe(II) titration and extinction coefficient of Fe(II)-M121H/H46E ...... 121 Figure 5.3 UV-vis spectrum characterization of dinitrosyl iron complexes formation in engineered non heme iron site in Az ...... 122 Figure 5.4 UV-vis spectrum characterization of dinitrosyl iron complexes formation in engineered non heme iron site in Az ...... 122 Figure 5.5 UV-vis spectra characterization of oxidizing dinitrosyl iron complexes with

~~Na2IrCl6 in engineered non heme iron site in Az ...... 123 Figure 5.6 cw-EPR investigation of dinitrosyl iron species formed in engineered non heme iron Az ..... 125~~

~~9 Figure 5.7 EPR spectra study of the {Fe(NO)2} species formation ...... 126~~

~~9 Figure 5.8 The EPR spectra of the {Fe(NO)2} under different temperature...... 126~~

9 Figure 5.9 EPR power saturation study of the {Fe(NO)2} species ...... 127 Figure 5.10 Pulsed EPR investigation of dinitrosyl iron species formed in engineered non heme iron Az ...... 128 Figure 5.11 N-ENDOR investigation of dinitrosyl iron species formed in engineered non heme iron Az ...... 129

Figure 5.12 HYSCORE investigation of dinitrosyl iron species formed in engineered non heme iron Az ...... 130 Figure 5.13 Zero-field Mӧssbauer spectra of 57Fe enriched non heme iron Az samples ...... 132 Figure 5.14 Nuclear resonance vibrational spectroscopy (NRVS) investigation of dinitrosyl iron species formed in engineered non heme iron Az ...... 134 Figure 5.15 XAS investigation of dinitrosyl iron species formed in engineered non heme iron Az ...... 135 Figure 5.16 EXAFS investigation of dinitrosylation in an engineered nonheme iron protein ...... 136 Figure 5.17 Schematic representation of the stepwise nitrosylation in an engineered nonheme iron protein ...... 137 Figure 6.1 SDS-PAGE gel investigation the overexpression of Ad2 fiber protein in different media. .... 150 Figure 6.2 SDS-PAGE gel investigation the overexpression of Ad2 penton and hexon under different concentrations of IPTG and induction time...... 150 Figure 6.3 The western blot of Ad2 fiber expressed in E. coli ...... 151 Figure 6.4 The western blot of Ad2 hexon expressed in E. coli ...... 151 Figure 6.5 Purification of Ad2 fiber, penton and hexon proteins expressed in E. coli ...... 152 Figure 6.6 The TEM images of recombinant Ad2 hexon protein expressed in E. coli ...... 154 Figure 6.7 Visualizations of adenovirus fiber interacting with CAR receptor ...... 158

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~~List of Tables Table 2.1 Diffraction and refinement data for Cu(II)-M121G azurin crystal structure 28 Table 2.2 Summary of EPR spectral parameters and percentages for select complexes 34~~

~~Table 2.3 Resonance Raman vibrational frequencies and isotope obtained using λex = 457.9 nm~~

16 18 for Cu(I) + H2 O2 and H2 O2 36 Table 2.4 Comparison between experimental bond distances and DFT structures for small M121G models and possible structures for the air-sensitive species 41 Table 2.5 DFT vibrational frequencies for small M121G models and potential structures of the air- sensitive species. 43 Table 2.6 DFT vibrational frequencies for large M121G models and potential structures of the air- sensitive species 46 Table 2.7 DFT structures for large M121G models and potential structures of the air-sensitive species upon removal of specific second sphere interactions 47 Table 3.1 Diffraction and refinement data for Cu(II)-M121H/H46EAz crystal structure 66 Table 3.2 Spectroscopic and EPR spin parameters of nitrosocyanin, Cu(II)-WTAz and Cu(II)- M121H/H46E/F114PAz 69 Table 3.3 EXAFS fitting parameters 78 Table 4.1 EPR simulation parameters of Cu(II)-Nmar_1307 103 Table 4.2 Data collection and refinement statistics of Cu(II)-Nmar_1307 crystal 105 Table 4.3 Relevant bond length near the Cu binding site of Cu(II)-Nmar_1307 106 Table 5.1 The simulation parameters for the 14N-ENDOR fittings 130 Table 5.2 EPR and Mössbauer parameters for all the species at 30 K 133 Table 5.3 EXAFS fitting parameters 137 Table 6.1 The sequence coverage of trypsin digestion – MS/MS studies of recombinant AD2 fiber, penton and hexon expressed in E.coli 156 Table 6.2 Modified amino acid sidechains by MS/MS studies 157

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~~Chapter 1 Engineering Novel Metal-Binding Sites Using Pseudomonas aeruginosa Azurin as the~~

~~Scaffold~~

~~1.1 Rational protein design: a valuable tool for understanding how metalloproteins work and achieving novel catalytic biochemical transformation~~

Metalloproteins account for nearly half of all proteins in nature.1 Metal ions play important roles in catalyzing numerous important biological processes that necessary to sustain life on the planet, such as photosynthesis,2 respiration3 and nitrogen fixation.4 Much effort has been made to understand the relationship between structures and functions of metalloproteins. Although significant progresses have been made to obtain the knowledge of how metalloproteins work, the ultimate test is to use this knowledge to design new metallproteins that reproduce the structures and functions of native proteins.5-11 Metalloprotein design not only duplicates biochemical and biophysical studies of native metalloproteins but also elucidates structure features that may remain hidden in those studies. Whereas biochemical and biophysical studies mostly reveal individual features that result in a loss of function, design requires the incorporation of all the structural features needed to attain a function. Equipped with insights from design processes, metalloprotein design has advanced to a stage where it is possible to create structures never seen in nature, or functions not found in nature while exhibiting desired properties. Metalloprotein design mainly focuses on creating novel metal-binding sites in either de novo-designed or native protein scaffolds.

~~1.1.1 De novo design: metalloproteins design from scratch~~

The purest and most challenging form of metalloprotein design is de novo design. De novo design of a metallprotein involves constructing a polypeptide sequence that is not directly related to any natural protein and that folds precisely into a defined three-dimensional structure that binds a metal ion.12 Most work in this area has focused on introducing metal-binding sites into designed α-helical bundles , which are

1 among the first de novo-designed proteins.12 These α-helical bundles are a common scaffold for a number of heme proteins in nature so that heme group was among one of the first metal centers incorporated into de novo α-helical bundles,13 from mono-heme14 to multi-heme binding,15 mimicking long-range electron transfer proteins. These proteins were rationally designed by direct comparison with native proteins, such as cytochromes. Besides electron-transfer function,16,17 variety of functions such as hydroxylase,18 peroxidase19 and oxygenase20 have also been achieved via de novo protein design. An exciting development in this field is the design of a di-heme-containing four helix bundle with oxygen transport properties, whose

~~21 O2 affinities and exchange timescales match those of natural globins.~~

In addition to heme, other metal ions/cofactors have been engineered into helical bundles by introducing metal-binding ligands at specific locations to mimic those in native proteins. A wide range of dinuclear metalloproteins in α-helical bundles have been reported to bind two Zn(II), Co(II) or Fe(II), showing structure analogous to the di-iron site of ribonucleotide reductase and ferritin.22 Motivated by the natural occurrence of Cys-rich coordination sites in proteins, the introduction of Cys ligands led to the development of α-helical coiled-coil metalloproteins capable of binding Cd(II), Hg(II) and As(III).23-25 One interesting property of these de novo-designed proteins is that they can stabilize unusual metal-coordination states, for example three-coordinate Hg(II) stabilized by a trihelical bundle, in preference to more normal coordination states, such as the bis-coordination often preferred by Hg(II).26,27 The exchange kinetics of

~~Cd(II) binding in a variety of de novo designed three-stranded coiled-coil peptides was investigated using~~

~~113Cd NMR spectroscopy.28 The results indicate that Cd(II) interfaces between surface ion pairs destabilize the helical structure and perturb the primary Cd(II) binding site.~~

Figure 1.1 Important structural features for the original and redesigned DFsc proteins. (A) Surface models of DFsc (top) and G4DFsc (bottom) based on the initial DFsc computational design. The four Ala to Gly substitutions (shown in white) significantly open the substrate access channel. (B) Illustration of the structure of 3His-G2DFsc variant (PDB 2LFD) that highlights the added active-site His residue (H100) and supporting mutations (I37N and L81H). The helix closest to the viewer is shown as transparent to allow a view of the ligands. The structure shown is for a variant with two Ala and two Gly residues along the substrate access channel, which proved more stable during the extended data-collection times required for the structure determination. This variant still exhibited N-oxygenase activity, but to a lesser extent than 3His-G4DFsc.29

The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function. One recent example of this is the design of an O2-dependent phenol oxidase, which catalyzes the two-electron oxidation of 4-aminophenol to corresponding quinone monoamine.30,31 More recently, the originally created to catalyze the O2-dependent phenol oxidase was reprogrammed to catalyze the selective

~~N-hydroxylation of arylamines by remodeling the substrate access cavity and introducing a critical third~~

His ligand to the metal-binding site (Figure 1.1).29 Additional second- and third-shell modifications were required to stabilize the His ligand in the core of the protein. These structural changes resulted in at least a

106-fold increase in the relative rate between the arylamine N-hydroxylation and hydroquinone oxidation reactions. The other representative example is the design of an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions—a Zn(II) ion (Figure 1.2), which is important for catalytic activity, and a Hg(II) ion, which provides structural stability.32 Although it catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency of 100 fold less than that of human carbonic anhydrase II, it is at least 550-fold better than comparable synthetic complexes. The kinetic and structural

~~3 analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.~~

Figure 1.2 Ribbon diagrams of the Hg, Zn-binding α-helix buddle structure. (A) One of two trimers found in the asymmetric unit of the crystal structure. (B) Top-down view of the structural trigonal thiolate site, Hg(II)S3.(C) Side view of the tetrahedral catalytic site, Zn(II)N3O.

~~1.1.2 Protein redesign: metalloproteins design through strategic modification of native protein scaffolds~~

A recent survey of >38,000 protein crystal structures in the Protein Data Bank revealed that all of these proteins belong to only ~1,200 different scaffolds and many folds, such as Greek key β barrel, are used by hundreds of proteins with different activities. The observation that Nature achieves almost unlimited functional diversity using a limited number of scaffolds suggests that, instead of designing a new scaffold for every new function, it is possible to use naturally evolved proteins as scaffolds to design and engineer various new structures and functions.10 The ultimate goal of protein redesign is to “teach old proteins new tricks”, for example, new activities, inverted stereoselectivity, or new substrate specificity

4 into existing proteins.33,34 This approach relies on the structural differences between template and target proteins and it is powerful in elucidating important structural features for the functions. The advantage of a protein redesign strategy is that it can bypass the problem of developing a stable protein fold because many native proteins have remarkable adaptability for changes. Protein redesign strategy has proven to be one of the most effective approaches in the design and engineering of artificial metalloenzymes.

Engineering a Zn(II)-binding site around the dimerization interface known from the X-ray structure of green fluorescent protein (GFP) is applied to the cyan and the yellow spectral variant of GFP to stabilize the heterodimeric form of these molecules and thereby increase FRET signaling.35,36 The Zn(II)-binding site was designed in a coiled-coil buddle which was inserted into a circularly permutated GFP (cpGFP), where the fluorescent emission from cpGPF was induced by Zn(II) coordination to the coiled-coil.37 The

~~2+ metal-binding site of NikR, a DNA-binding protein, has been redesigned to bind UO2 rather than its native~~

~~2+ 2+ 38 Ni cofactor, resulting in an artificial protein that binds DNA only in the presence of UO2 .~~

~~Figure 1.3 FeBMb, designed prior to structure elucidation of NOR, is structurally similar to NOR and displays NOR activity. Incorporation of I107E mutation doubles nitric oxide reduction activity.10~~

A great example of protein redesign is the design of a non-heme FeB site in the distal heme pocket of myoglobin (FeBMb) to mimic the heme-non-heme di-iron catalytic center of bacterial nitric oxide reductase (NOR). In the absence of a structure of NOR, due to inherent difficulties of crystallizing large

5 membrane proteins, the design was guided by the known structure of heme-copper oxidase (HCO) and its sequence homology with NOR. A glutamate and two histidines known to be conserved in the NOR FeB site were introduced into the distal site of Mb, resulting in the FeBMb, which binds Fe(II) and promotes NOR activity.39 Encouraged by this success and based on the hypothesis that at least one more conserved Glu may play a role in NOR, an additional Glu, I107E, was introduced into FeBMb, as a potential hydrogen- bonding donor to the NO substrate (Figure 1.3). Such a design of the non-covalent interaction increased the

40 NOR activity by 100%. Additionally, investigation of the effect of different metals in I107E FeBMb revealed the critical role of the metal in the FeB site in weakening the heme Fe–His bond and raising the heme reduction potential by up to 70 mV. An alternative model with one of the His ligands of FeBMb replaced with a Glu, mimicking the proposed 2-His-1-Asp active site of gNORs, was also investigated and

~~41 found to successfully reduce NO to N2O with Fe or Cu in the non-heme metal site.~~

~~Figure 1.4 A designed oxdiase achiving the catalytic rate of a native enzyme.42~~

The other example is to transform myoglobin (Mb) into an oxidase, where the conserved structural elements of heme-copper oxidases were introduced into the distal pocket of Mb, such as one tyrosine and two histidines. One such variant, L29H/F43H/G65Y Mb (called G65Y-CuBMb), was able to reduce O2 to water with a catalytic oxygen reduction rate of 0.30 s−1 and over 1000 turnovers using ascorbate as a reductant and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) as a redox mediator.43 While the above results demonstrate that it is possible to rationally design a functional enzyme with a high number of turnovers, the catalytic rate is still far below that of native enzymes. Later, to enhance the electron transfer

~~6 rate, D44K/D60K/E85K variant was designed through engineering of more favorable electrostatic~~

~~-1 interactions and exhibited the O2 reductase activity with an O2 reduction rate of 52 S , comparable to that~~

~~-1 42 of a native cytochrome cbb3 oxidase (50 S ) (Figure 1.4).~~

~~1.2 Engineering novel metal sites using Pseudomonas aeruginosa azurin as the scaffold~~

The use of small, stable, easy-to-make, and well-characterized proteins as scaffold to mimic the functions of large, multi-domain, membrane proteins has been proven to be an effective way of protein redesign. Pseudomonas aeruginosa azurin, also called blue copper azurin, is in Greek key β barrel scaffold, which is used by hundreds of proteins with different activities. This periplasmic protein only has 128 amino acids and can be overexpressed in E. coli with high yield. Engineering azurin and tuning its function from electron transfer to an enzyme have been demonstrated. Several representative examples are summarized below.

~~1.2.1 Rationally tuning the reduction potential of blue copper protein azurin beyond the natural range~~

Figure 1.5 Rational tuning of the reduction potential of azurin. (A) X-ray structure of native azurin (PDB 4AZU). (B) X-ray structure of N47S/M121L azurin. (C) X-ray structure of N47S/F114N azurin. (D) X-ray structure of F114P/M121Q azurin. Copper is shown in green, carbon in cyan, nitrogen in blue, oxygen in red and sulphur in yellow. Hydrogen-bonding interactions are shown by dashed red lines. (E) Plot of Eo versus log P for the Az mutants from this study (here P is the partition coefficient of the side chain of the residue between octanol and water; that is, it is a measure of hydrophobicity). (F) Plot showing 44 the Eo value for each azurin variant.

Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer in photosynthesis and respiration to catalysis in industrial and fuel cell research. A long- standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or electron-transfer properties. Marshall et al. reported tuning the reduction potential of the cupredoxin azurin over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins.44 Such redox tuning is achieved by redesigning two important

8 secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding (Figure 1.5). For instance, N47S affects the rigidity of the copper binding site and, probably, the direct hydrogen bonds between the protein backbone and Cys 112 (Figure 1.5B). F114N introduces a hydrogen-bond donor at position 114 perturbs hydrogen-bonding near the copper binding site, possibly disrupting donor–acceptor interactions to His 117, or ionic interactions between the copper and the carbonyl oxygen of Gly 45 (Figure

~~1.5C). F114P deletes a direct hydrogen bond to Cys 112 resulting in a lower redox potential (Figure 1.5D).~~

It also demonstrates that the effects of individual structural features are additive and that redox potential tuning of azurin is now predictable across the full range of cupredoxin potentials (Figure 1.5E, F). This research not only advances our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allows us to design redox-active proteins with tunable redox potentials for industrial and biological applications.

~~1.2.2 Design mononuclear Cu-binding site with novel electronic structures~~

Figure 1.6 The Cu(II) binding sites of C112D in Type zero copper proteins (a, 1.9 Å, PDBID: 3FQY), C112D/M121L (b, 2.1 Å, PDBID: 3FPY), C112D/M121F (c, 1.9 Å, PDBID: 3FQ2), and C112D/M121I (d, 1.9 Å, PDBID: 3FQ1) azurins are displayed with Cu–heteroatom bond distances indicated in Å. Oxygen atoms are red; nitrogen atoms are blue.45

Many proteins contain copper in a range of coordination environments, where it has various biological roles, such as transferring electrons or activating dioxygen. These copper sites can be classified by their function or spectroscopic properties. Those with a single copper atom are either type 1, with an intense absorption band near 600 nm, or type 2, with weak absorption in the visible region. Lancaster et al. have built a novel copper(II) binding site within structurally modified Pseudomonas aeruginosa azurin that does not resemble either existing type, which they therefore call ‘type zero’.45 X-ray crystallographic analysis shows that these sites adopt distorted tetrahedral geometries, with an unusually short Cu–O (G45 carbonyl) bond (Figure 1.6). Relatively weak absorption near 800 nm and narrow parallel hyperfine

~~10 splittings in electron paramagnetic resonance spectra are the spectroscopic signatures of type zero copper.~~

~~Cyclic voltammetric experiments demonstrate that the electron transfer reactivities of type-zero azurins are enhanced relative to that of the corresponding type 2 (C112D) protein.~~

~~1.2.3 Design binuclear CuA site in azurin~~

~~CuA is a binuclear copper center bridged by two cysteine ligands to form a Cu2S2 “diamond-core”~~

46-48 49,50 structure which has only been found in cytochrome c oxidase (CcO) , nitrous oxide reductase (N2OR) , and the oxidase from Sulfolobus acidocaldarius (SoxH)51 in biology to date. The one of the most important features in CuA sites is that the two copper ions form a direct metal-metal bond with each other. Therefore, the unpaired electron is delocalized between two copper ions and the resting state of CuA center is a

Cu(+1.5)-Cu(+1.5) rather than Cu(+2)-Cu(+1). The UV-vis absorption spectrum of CuA shows two intense absorbance at ~480 nm and 530 nm which arises from S(Cys) → Cu charge transfer bands in the visible region and also a broad band at 760 - 800 nm which arises from Cu(+1.5)-Cu(+1.5) intervalence charge transfer.52-55 The reduced Cu(I)-Cu(I) form is colorless because of the d10 electronic configuration at each copper center. The more oxidized Cu(II)-Cu(II) state has not been observed to date. Therefore, CuA site acts as one-electron transfer center.56

Figure 1.7 CuA in biosynthetic model in P. aeruginosa azurin. (A) Crystal structure of biosynthetic model of CuA site in azurin (PDB: 1CC3). (B) The comparison of UV-vis spectra between soluble CuA domain in cytochrome c oxidase (green line), wild type azurin (blue line) and biosynthetic CuA model in azurin (purple line), 4-line splitting vs. 7-line splitting.57

~~Hay et al. constructed a purple copper protein from a recombinant blue copper protein, P. aeruginosa azurin, by replacing the loop containing the three ligands to the blue copper center with the~~

58 corresponding loop of the CuA site in CcO from P. dentrificans (Figure 1.7A). The UV-vis and EPR spectra of this protein analog were remarkably similar to those of native CuA sites in CcO from P. dentrificans. The UV-vis absorption spectrum of CuAAz features two S(Cys)→Cu CT bands at 485 (ε ~3700

~~-1 -1 -1 -1 52,59 M cm ) and 530 nm (ε ~ 3400 M cm ) , compared to 480-485 nm and 530-540 nm for native CuA~~

60 -1 -1 centers (Figure 1.7B). CuAAz also featured a broad band centered at 760-800 nm (ε ~ 2000 M cm ), typical of the Cu-Cu ψ→ψ* transition, suggesting that CuAAz had reproduced the Cu-Cu bond.

~~Additionally, the EPR spectrum of CuAAz displayed a 7-line hyperfine splitting pattern, demonstrating that~~

~~58,59 this biosynthetic model duplicated the mixed-valence ground state of native CuA centers (Figure 1.7C).~~

~~EXAFS, CD, MCD and resonance Raman analyses of the CuA in azurin also suggested a high level of~~

~~52,58,59,61,62 electronic and structural identity with CuA centers from CcO. X-ray crystal structure of CuAAz~~

~~12 showed a very similar arrangement of ligands about the copper ions, and a Cu-Cu distance that was even~~

63 slightly shorter than the native CuA center in CcO, confirming the presence of a Cu-Cu bond. CuAAz’s small size and relative ease of expression and purification make this biosynthetic model highly amenable to mutagenesis studies.

~~1.2.4 Design a redox-active mononuclear nonheme iron binding site in azurin~~

Figure 1.8 Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron protein. (Left) X-ray structure of Fe(II)-M121E/M44K azurin. (Right) Reactivity toward superoxide by Fridovich test.64

Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. McLaughlin et al. the first time engineered a nonheme ironsite in P. aeruginosa azurin.65 The designed mutant binds iron(II) to give a 1:1 complex, which has been characterized by electronic absorption, Mössbauer, and NMR spectroscopies, as well asX-ray crystallography and quantum-chemical computations. However, the iron(II) complex does not react with chemical redox agents to undergo oxidation or reduction. Later, Liu et al. reported spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild- type azurin (wtAz) with Glu and Fe(II), respectively (Figure 1.8, left).64 In contrast to the redox inactive

~~Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein~~

13 exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude (Figure 1.8, right). This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.

~~1.3 Summary~~

The ultimate test is to use this knowledge to design new metallproteins that reproduce the structures and functions of native proteins. Metalloprotein design not only duplicates biochemical and biophysical studies of native metalloproteins but also elucidates structure features that may remain hidden in those studies. The use of small, stable, easy-to-make, and well-characterized blue copper protein azurin as scaffold to design novel metal binding sites has been demonstrated to be a promising and effective way for protein redesign. Not only can its reduction potential be rationally tuned beyond the nature range via secondary coordination sphere engineering, but also designing CuA, type zero copper and nonheme iron sites within such a stable Greek key β barrel scaffold.

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~~57 Savelieff, M. G. & Lu, Y. Cua centers and their biosynthetic models in azurin. J. Biol. Inorg. Chem.~~

~~15, 461-483 (2010).~~

~~58 Hay, M., Richards, J. H. & Lu, Y. Construction and characterization of an azurin analog for the~~

~~purple copper site in cytochrome c oxidase. Proc. Natl. Acad. Sci. U. S. A. 93, 461-464 (1996).~~

~~59 Hay, M. T. et al. Spectroscopic characterization of an engineered purple CuA center in azurin. Inorg.~~

~~Chem. 37, 191-198 (1998).~~

~~60 Lu, Y. in Comprehensive coordination chemistry II (eds Jon A. McCleverty & Thomas J. Meyer)~~

~~91-122 (Pergamon, 2003).~~

~~61 Andrew, C. R. et al. Engineered cupredoxins and bacterial cytochrome c oxidases have similar cua~~

~~sites: Evidence from resonance raman spectroscopy. J. Am. Chem. Soc. 117, 10759-10760 (1995).~~

~~62 DeBeer, G. S. et al. A quantitative description of the ground-state wave function of cua by x-ray~~

~~absorption spectroscopy: Comparison to plastocyanin and relevance to electron transfer. J. Am.~~

~~Chem. Soc. 123, 5757-5767 (2001).~~

~~63 Robinson, H. et al. Structural basis of electron transfer modulation in the purple CuA center.~~

~~Biochemistry 38, 5677-5683 (1999).~~

~~64 Liu, J. et al. Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron~~

~~protein: Preparation and study of Fe(II)-M121E azurin. J. Am. Chem. Soc. 136, 12337-12344~~

~~(2014).~~

~~65 McLaughlin, M. P. et al. Azurin as a protein scaffold for a low-coordinate nonheme iron site with~~

~~a small-molecule binding pocket. J. Am. Chem. Soc. 134, 19746-19757 (2012).~~

~~Chapter 2 A Novel Copper-Sulfenate Complex from Oxidation of a Cavity Mutant of Pseudomonas aeruginosa AzurinI~~

~~2.1 Introduction~~

The presence of cysteine sulfenic acid (Cys-SOH) as a product of post-translational oxidation of the cysteine thiol side chain has been found to play important roles in biology1-7 in the context of metal coordination,8 enzyme/protein regulation,9,10 redox signaling,1,6 and gene regulation.11-14 Unlike its higher oxidation state counterparts, i.e., sulfinic (Cys-SO2) and sulfonic (Cys-SO3) acids (Figure 2.1), the sulfenic acid is highly reactive and requires stabilization in order to operate in a controlled context in biological systems.1-5 The x-ray structure of sulfenic acid form of SarZ, a redox active global transcriptional regulator in Staphylococcus aureus, revealed that cysteine sulfenic acid Cys13-SOH is stabilized through two hydrogen bonds with Tyr 27 and a water molecule (Figure 2.2).15

~~Figure 2.1 List of different products during cysteine oxidation.~~

I Portions of this chapter are previously published and are here reproduced with permission from Sieracki, N. A.; Tian, S.; Hadt, R. G.; Zhang, J.-L.; Woertink, J. S.; Nilges, M. J.; Sun, F.; Solomon, E. I.; Lu, Y. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 924. (N.A.S., S.T., and R.G.H. contributed equally to this work)

Figure 2.2 Cysteine sulfenic acid Cys13-SOH in SarZ. Atoms are colored gray (carbon), red (oxygen), and yellow (sulfur). Water molecules are labeled W1 or W2 and shown as red spheres. Hydrogen bonds are shown as black dashed lines.15

Because of its high reactivity, cysteine sulfenic acid have been observed in few metal-binding sites in proteins.1-7 Nature has evolved proteins to allow metal ions to coordinate sulfenic acids, resulting in interesting functions. For example, nitrile hydratase, a metalloenzyme, which has long found utility in the industrial synthesis of acrylamide,16 features both cysteine sulfenate and a cysteine sulfinate in the active site, both coordinated to the metal center (Fe or Co) (Figure 2.3).17 In a similar evolutionary context is thiocyanate hydrolase,8 which uses a similar Co coordination environment to hydrolyze thiocyanate to carbonyl sulfide, ammonia, and water. In both enzymes, the sulfenic and sulfinic groups are unambiguously required for activity.8,17 It is commonly speculated that such oxidized forms of cysteine in a metal site encourage and stabilize the more oxidized form of redox-active metals, suppressing their potential toxicity in enzymes that do not require the metal center to participate in electron-transfer.18,19 In a more recent example, an iron-coordinated sulfenate was observed when a thiolate-containing substrate analog in isopenicillin N-synthase was unexpectedly oxidized to a coordinated sulfenato group in crystallo, and a connection to the biogenesis of nitrile hydratases and thiocyanate hydrolases was drawn.20

Figure 2.3 The active site of nitrile hydratase contains either Fe or Co. The metal ion is coordinated by two carboxamido nitrogens from the protein backbone and three cysteine derived sulfurs. Two of the sulfur donors are oxygenated through a post-translational modification yielding one sulfenato (RSO-) and - one sulfinato (RSO2 ) donor. The sixth coordination site is the presumed substrate binding site. It may be occupied by nitric oxide in an inactivated form of the enzyme.

~~While Cys-SOH residues coordinated to cobalt and non-heme iron have been observed in the~~

8,20 21 enzymes described above and the corresponding synthetic models reported, the Cys-SOH has not been observed to stably bind other metals in a biological context. Here we demonstrate generation of the first copper-coordinated sulfenic acid complex. The work focuses on type 1 blue copper protein,

~~Pseudomonas aeruginosa azurin. These results and their implication in our understanding of the metal- sulfenate centers related to their biological functions are discussed.~~

~~2.2 Materials and methods~~

~~2.2.1 Site-directed mutagenesis and protein purification~~

Plasmid containing the M121G mutation was constructed by site-directed mutagenesis using wild-type azurin (pET9a) as the template using the Quik-Change mutagenesis procedure with the forward primer 5’-CAC-TCC-GCA-CTG-GGG-AAA-GGT-ACC-3’ and the corresponding complementary reverse primer. The mutation was confirmed via sequencing using a T7 promoter. The protein was expressed in BL-21* E. Coli (Novagen, Madison, WI) and purified according to published procedures.22,23

~~Electrospray ionization mass spectrometry confirmed the identity of the isolated variant (MW = 13871;~~

~~MWcal = 13871.67).~~

~~2.2.2 Spectroscopic measurements~~

UV-vis absorption spectra were obtained at room temperature on an HP Agilent 8453 diode array spectrometer. An extinction coefficient of 9000 M-1cm-1 at 280 nm was used to determine the concentration of purified protein.24 All UV-vis spectra were recorded in 50 mM potassium phosphate pH 7.0 buffer unless otherwise noted. X-band EPR measurements were collected at 30 K on a Varian-122 spectrometer equipped with an Air Products Helitran cryostat and temperature controller, with a collection frequency of 9.05 GHz, power of 0.2 mW and field modulation of 4.0 G. EPR spectra were recorded with 20% glycerol in 50 mM temperature-independent-pH (TIP) buffer (pH 7.0).25

~~2.2.3 Mass spectrometry measurements~~

The mass spectra of the proteins were acquired using a Waters Quattro II spectrometer operating in positive-ion mode. Samples (10 μL of 20 µM protein in ammonium acetate buffer) were injected into a flow of 50 µL/min of 50% CH3CN/H2O mobile phase and integrated over the first minute of detection.

Unless otherwise noted, protein samples were first treated with a 1:10 volume of 1% v/v formic acid immediately prior to injection. Syringe pump mass spectra were obtained by directly injecting 0.2 mM protein in 10 mM ammonium acetate (pH 6.0) into the spectrometer at a rate of 5 ụL/min. The mass spectra were collected from 500-2000 m/z and were deconvoluted using the MassLinx software package with a 1 Da resolution and a 10,000-20,000 Da calculation window.

~~2.2.4 Preparation of Cu(II)-M121G azurin~~

~~Aliquotes of metal-free (apo-) M121G azurin (less than 2ml) in 50 mM ammonia acetate buffer~~

(pH 6.35) were titrated with 2 eq. Cu(II) (from a stock solution of 50 mM CuSO4) and stirred at room temperature for 30 minutes. Excess copper was removed using a PD-10 desalting column containing 8.3 ml of SephadexTM G-25 medium. The buffer was exchanged to 50 mM potassium phosphate buffer (pH

~~7.0) with Amicon® Ultra-4 centrifugal filter devices with a 10 kDa molecular weight cutoff.~~

~~2.2.5 Crystallography of Cu(II)-M121G azurin~~

Crystals of Cu(II)-M121G azurin suitable for X-ray diffraction grew over three days at 4 oC using the hanging drop method, which consisted of suspension of a 2 µL droplet containing 1 μL apo-M121G azurin (1.3 mM) in 100 mM NaOAc buffer (pH 5.6) and 1 µL PEG buffer (25% PEG 4000 containing

100 mM LiNO3, 10 mM CuSO4 and 100 mM Tris (pH 8.0)) above 250 µL well buffer (25% PEG 4000 containing 100 mM LiNO3 10 mM CuSO4 and 100 mM Tris (pH 8.0)). The structure was solved by molecular replacement using AutoMR docked in Phenix.26 The crystal structure of wild type azurin (PDB: 4AZU) was used as a searching model.

~~2.2.6 Preparation of Cu(I)-M121G azurin~~

~~The Cu(II)-M121G azurin (1.0 ml of 2.0 mM protein in 50 mM potassium phosphate buffer (pH~~

~~7.0)) was degassed with argon on a Schlenk line and was then transferred into a glove bag. The Cu(II)-~~

~~M121G azurin was then reduced by slow addition of solid sodium ascorbate containing 10%mol N, N, N’,~~

N’-tetramethyl-p-phenylenediamine (TMPD) as a mediator until the protein solution turned completely colorless. The resulting solution was purified via PD-10 desalting column to remove excess reductant.

~~2.2.7 Trypsin digest and HPLC MS/MS analysis~~

Trypsin digest and HPLC MS/MS analysis were carried by the Protein Science Facility in the Biotechnology Center at the University of Illinois at Urbana-Champaign. Samples were digested with trypsin for 15 minutes at 55 °C using a CEM Liberty microwave digester (CEM Corporation, Matthews, NC). The digested products were analyzed on a Thermo Scientific Velos Pro mass spectrometer with a Dionex RSLCnano Ultimate 3000 UPLC front end using an Acclaim PepMap RSLC (75 micron X 15 cm, C18 2 micron 100 Angstrom) column (Thermo Scientific) with a linear gradient of 1 to 60 % acetonitrile in 0.1 % formic acid over 120 minutes. The data were analyzed using the Mascot database search engine (Matrix Science, London) and searched against a custom database containing the mutant M121G Azurin sequence.

~~2.2.8 DFT calculations~~

~~All DFT calculations were carried out using the Gaussian 09 software.27 For all calculations the~~

B3LYP28 functional was used in the unrestricted format. For the small models, a TZVP29 basis set on all atoms was used for all calculations. For the large models, a split 6-311G(d) (on Cu, ligating atoms, and

O’s on the oxidized thiolate, either bound or unbound)) and 6-31G(d)30-32 (all other atoms) basis set was used. All molecular orbital compositions were determined using the QMForge program33 (Mulliken population analyses), and all orbital surfaces were generated using the β-LUMO program.34 The M121G crystal structure was used for a starting point for all models considered. Geometry optimizations using small models involved Cα constraints, while large model constraints included Cα and protein backbone O and N atoms. All reported energies and single point calculations (e.g., EPR and time dependent DFT

~~(TDDFT)) have been environment corrected using a PCM model (ε = 4.0).35 TDDFT calculations were simulated using the SWizard program revision 4.636,37 using Gaussian band-shapes with half-widths of~~

~~2000 cm-1. Wave function stability checks were carried out to ensure they represented energetic minima.~~

~~2.3 Results and discussion~~

~~2.3.1 UV-vis and crystallographic characterization of Cu(II)-M121G azurin~~

M121G azurin was purified in the metal-free (apo-) form. Treatment of apo-M121G azurin with copper(II) sulfate resulted in a strong blue colored solution. This color remained associated with the protein after size exclusion chromatography via a PD-10 column. The UV-vis spectrum of the protein exhibited a strong absorption at 614 nm with a shoulder at 453 nm, consistent with that of Cu(II)-M121G azurin reported previously38 and similar to those of other Type-1 blue copper proteins.39

~~To elucidate the structural changes of the M121G mutation on azurin, I solved its crystal structure.~~

Analysis of the crystal structure reveals that the geometry and the bond distances around the copper- binding site are quite similar to those of wild type (WT) azurin (Figure 2.4). In each of the two proteins, an equatorial plane is established by two histi2dine imidazolyl ligands and one cysteine thiolate ligand.

The major difference between the two structures is the presence of a crystallographically defined water molecule in the active site of M121G azurin, located 2.92 Å from the copper ion and occupying the Met-

121 axial ligand position in WT azurin. This observation offers structural support for previously described resonance Raman (rR) spectroscopic data suggesting the presence of an axial water ligand in M121G azurin.40 Interestingly, no crystallographically defined water in the axial position to the copper ion was observed in M121A azurin,41 suggesting that, in azurin, the subtle differences in size and hydrophobicity between axial Ala versus Gly may dictate water access to the metal center.

Figure 2.4 X-ray crystal structures showing the active sites of wild type azurin and M121G azurin. (A) Cu(II)-WT azurin (PDB code 4AZU). (B) Cu(II)-M121G azurin (1.54 Å, PDB code 4MFH). (C) Surface accessibility maps generated with a probe sphere radius of 65 pm to simulate O-atom accessibility from crystal structures of Cu(II)-M121G azurin, the water channel to the active site was highlighted. (D) The hydrogen bonding network around the water channel to the active site. (Cu in orange, S in yellow, N in blue, C in cyan and O in red)

~~Table 2.1 Diffraction and refinement data for Cu(II)-M121G azurin crystal structure.~~

Cu(II)-M121G Az Data collection Beamline 21-ID-F Wavelength (Å) 0.97872 Space Group C 2 2 21 Cell dimension 55.75 145.8 97.09 a, b, c (Å) Resolution (Å) 50-1.54 (1.57-1.54) R-merge 0.069 (0.376) I/σI 33.88 (7.22) No. of unique reflections 53091 (2616) Completeness (%) 90.5 (91.1) Redundancy 6.8 (6.5) Refinement Resolution (Å) 29.15-1.54 (1.58-1.54) Reflections in free set 2648 (5%) Rwork/Rfree 0.1704(0.229)/0.1936(0.262) No. reflections 50042 No. atoms 3291 Protein 2916 Cu 6 Water 340 Metal Ion Cu B-factor 13.00 Protein 12.10 Cu 13.38 Water 20.80 ESU(ML) 0.044 Rms. deviations Bond lengths (Å) 0.023 Bond angles (o) 2.123 Ramachandran favored 98 (%) Ramachandran outliers 0 (%)

It is noteworthy that a water channel is also observed in the crystal structure of Cu(II)-M121G azurin, shown in Figure 2.4C. This channel contains four water molecules in a hydrogen bonding network with the protein backbone and side chains (Figure 2.4D), and is not present in the structure of wild type azurin. We investigated whether this channel might allow the Cu(I) site of reduced M121G azurin to react with small molecules. Hydrogen peroxide (H2O2) is a small molecule capable of oxidizing or reducing

42 metal centers. Interestingly, addition of five equivalents of H2O2 to Cu(I)-M121G azurin in 50 mM potassium phosphate buffer (pH 7.0) resulted in formation of a green-colored solution, with a visible electronic absorption spectrum dominated by spectral features at 452 nm, 614 nm and a shoulder peak around 350 nm (Figure 2.5A).

Figure 2.5 Time course of UV-vis and EPR spectra upon formation of the air sensitive species and its decomposition upon air expose. (A) Addition of 5 eq. H2O2 to 0.9 mM Cu(I)-M121G azurin in 50 mM potassium phosphate pH 7.0 under anaerobic condition. (B) Exposure of the resulting green solution to air. (C) A comparison of UV-vis spectra of the green species (in green), the product after air exposure

(in red) and Cu(II)-M121G azurin (in blue). The absorbance was normalized to unchanging spectral contribution at 614 nm. (D) X-Band EPR spectra of H2O2 treated Cu(I)-M121G azurin in 50 mM TIP7 buffer pH 7.0 for 30 min (black), simulations of the air-sensitive species (green), Cu(II)-M121G azurin (blue) and a type II copper site (red).

The color of the green solution persisted for several hours at room temperature in the absence of air. In contrast, upon addition of five equivalents of H2O2 to apo-M121G azurin, Zn(II)-M121G azurin, or

Cu(II)-M121G azurin under the same conditions, no significant change in the UV-vis spectrum was observed over 30 mins (Figure 2.6-A, B, C). These results suggest that the new species in the green- colored solution was a result of a process requiring reduced Cu(I). When Cu(I)-WT azurin was treated with the same amount of H2O2, we observed only reappearance of the intense blue solution with absorbance spectrum typical of Cu(II)-WT azurin (Figure 2.6D). This result indicated that, while Cu(I) was reasonably efficiently re-oxidized by H2O2 to Cu(II) in the WT variant, the efficiency was lost in the

~~M121G variant, resulting in one or more new species.~~

Figure 2.6 UV-vis spectrum upon addition of 5 eq. H2O2 to different protein solutions: (A) apo- M121G Az, (B) Zn(II)-M121G Az, (C) Cu(II)-M121G azurin and (D) Cu(I)-WT azurin in 50 mM KPi buffer pH 7.0. Spectra in blue were obtained before H2O2 addition, while spectra in red were obtained 30 minutes after H2O2 addition.

Figure 2.7 Electrospray mass spectrum upon addition of 5 eq. H2O2 to different protein solutions: (A) CuII-M121G; (B) apo-M121G; (C) ZnII-M121G; (D) CuII-Wild-Type; and (E) CuI-Wild-Type. Spectra in blue were obtained before oxidant addition, while spectra in red were obtained 30 minutes after H2O2 addition. (For M121G azurin, cal. 13871.67 and obs. 13871 Da; for wild type azurin, cal. 13945.81 and obs. 13945 Da)

Interestingly, exposing the above green colored solution (generated by Cu(I)-M121G azurin reacted with H2O2) to air resulted in the rapid loss of the spectral features at 452 nm and 350 nm, while those at 614 nm were largely unaffected (Figure 2.5B). The final spectrum was nearly identical in shape to that of Cu(II)-M121G azurin (Figure 2.5C) albeit with significantly less intensity. This result

~~32 demonstrated that the green colored solution was a mixture of an air-sensitive yellow species and an air- stable blue species, the latter of which resembled authentic Cu(II)M121G azurin.~~

~~2.3.2 EPR investigation of the air-sensitive species~~

To investigate the nature of the copper-containing air-sensitive species, we collected electron paramagnetic resonance (EPR) spectra of frozen reaction solutions at various time points after anaerobic addition of H2O2 to Cu(I)-M121G azurin. The EPR data revealed gradual formation of two major axial

~~Cu(II) species besides Cu(II)-M121G azurin (Figure 2.8A). The exposure to air of the H2O2 treated Cu(I)-~~

~~M121G azurin sample resulted in complete disappearance of one of the major axial Cu(II) species (Figure~~

~~2.8B).~~

Figure 2.8 EPR study and simulation of the air sensitive species. (A) Time-resolved X-band EPR spectra of a solution of 0.6 mM Cu(I)-M121G after addition of 5 eq. H2O2 in 50 mM TIP7 buffer (pH 7.0) along with authentic Cu(II)-M121G (bottom). Reactions were performed anaerobically in an EPR tube and quenched via freezing in liquid N2. Spectra were collected at 30 K and were an average of 10 scans. Microwave frequency = 9.05 GHz; field modulation = 4.0 G; microwave power = 0.2 mW. (B) X-Band EPR spectra of Cu(I)-M121G azurin after incubating with 5 eq. H2O2 for 30 min in 50 mM TIP7 buffer

~~33 pH 7.0 (black), the resulted solution exposed to air (pink) and simulation of the air-sensitive species (green).~~

Simulation of the above EPR data suggests formation of three different Cu(II) species from oxidation of Cu(I) M121G azurin with H2O2: Cu(II)-M121G azurin, an air sensitive Cu(II) species and a normal type II copper species (see Figure 2.5D and spectral parameters in Table 2.2). The air sensitive

Cu(II) species associated with the 452 nm peak in the UV-vis spectra had a lower gz value of 2.169 compared to normal type II copper, indicating a highly covalent interaction. The type 2 copper signal is most likely due to overoxidation caused by extra equivalents of hydrogen peroxide.

~~Table 2.2 Summary of EPR spectral parameters and percentages for select complexes.~~

-4 -1 Species gx, gy, gz Ax, Ay, Az (Cu) (×10 cm ) Percentage Air-sensitive species 2.033, 2.041, 2.169 26.3, 17.8, 139.3 17.5 % Cu(II)-M121G azurin (A) 2.024, 2.102, 2.294 42.3, 17.6, 18.1 10.9 % Cu(II)-M121G azurin (B) 2.025, 2.042, 2.303 77.0, -, 11.5 8.1 % Type 2 Copper Site 2.038, 2.067, 2.276 -, 19.7, 179.9 63.5 %

~~2.3.3 Resonance Raman of the air-sensitive species~~

In an effort to gain more information into the air-sensitive species, the Cu(I)-M121G azurin solutions after reacting with H2O2 were frozen in liquid N2 and analyzed using resonance Raman spectroscopy (rR). rR data using laser excitation into the 452 nm charge transfer (CT) band of the

~~16 18 intermediate species formed from the reaction of Cu(I)M121G Az with H2 O2 and H2 O2 (pH 5.1) are~~

16 given in Figure 2.9 A and B (λex = 457.9 nm). For H2 O2-treated Cu(I)-M121G Az, three well-resolved resonance-enhanced vibrations were observed in the Cu-ligand stretching region at 374, 380, and 407 cm-

1 (Figure 2.9A and Table 2.3), with the 407 cm-1 feature (c) being the most intense. Several features are also resonance enhanced in the higher energy region of the spectrum (~700 – 875 cm-1; Figure 2.9B). This region contains the S(Cys)-Cβ stretch, overtone and combination bands of the fundamentals observed in the lower energy region, and possible fundamental vibrations of the intermediate species. In particular, the 837 cm-1 feature is too high in energy to be related to the fundamental vibrations observed in the lower

18 energy region. No higher energy vibrations are observed. When H2 O2 is used as the oxidant, the lower energy vibrations are observed at 371, 380, and 395 cm-1, which correspond to Δ(16O-18O)’s = 3, 0, and 12 cm-1, respectively. Also, the lower energy feature (labeled a in Figure 2.9A) becomes the most intense.

~~The experimental energies, intensities and isotopic shifts of features (a) and (c) are consistent with a~~

~~Fermi resonance. These values were used to determine the vibrational energies in the absence of this~~

~~16 -1 interaction. For the reaction with H2 O2, features (a) and (c) are predicted to be at 399 and 381 cm ,~~

18 -1 respectively, while for reaction with H2 O2, (a) and (c) are predicted to be at 387 and 383 cm , respectively. In the higher energy region, the 837 cm-1 feature shifts down in energy to 807 cm-1 (Δ = -30

~~-1 18 cm ) when H2 O2 is used as the oxidant. The rR enhanced vibrations observed here, combined with the~~

EPR results, may either reflect an oxidized thiolate bound to Cu(II) or a Cu(II)-OOH species. These possibilities, as well as different Cu(II)-SOxHx species and their binding modes are evaluated using DFT and TD-DFT calculations (vide infra).

Figure 2.9 Resonance Raman spectroscopy of the air sensitive species. (A) Resonance Raman spectra 16 18 of Cu(I) + H2 O2 (red) and H2 O2 (blue) in the low energy region vibrational region (* indicates Raman scattering feature of ice) and (B) the high energy vibrational region (λex = 457.9 nm). (C) Low energy vibrational region using λex = 647.1 nm.

~~Figure 2.9 (cont.)~~

~~Table 2.3 Resonance Raman vibrational frequencies and isotope obtained using λex = 457.9 nm for 16 18 Cu(I) + H2 O2 and H2 O2.~~

~~16 -1 18 -1 16 18 -1 H2 O2 (cm ) H2 O2 (cm ) Δ( O- O) (cm ) a 374 371 3 b 380 380 0 c 407 395 12 SO 837 807 30~~

~~Lastly, the 614 nm absorption band was also investigated on the same samples discussed above~~

~~(λex = 647.1 nm). Using this excitation wavelength, identical rR data (only with variable total intensities)~~

16 18 were obtained upon reaction of Cu(I)-M121G Az with H2 O2 or H2 O2, or upon further exposure of the reaction mixture to air (Figure 2.9C). These data are the same as those obtained by laser excitation into the M121G Az CT band, and indicate that the electronic absorption contributions in the 610 – 630 nm region observed in the UV-vis experiments can be assigned to variable concentrations of authentic Cu(II)-

~~M121G Az, consistent with the EPR results above.~~

~~2.3.4 Capture of sulfenic acid with the selective labeling agent, dimedone~~

Since the above results suggested the presence of an oxidized sulfur at the active site of the air- sensitive species, we tested the hypothesis that a sulfenic acid moiety coordinates a copper ion using dimedone, a well-established agent for the selective detection of sulfenic acid.43,44 After reaction of sulfenic acids with dimedone, the total mass should increase by +138 Da due to irreversible and selective condensation, which can be observed via mass spectrometry.4,45

Addition of dimedone to copper-sulfenate species under normal physiological conditions did not result in any modification of the protein, probably due to a lack of access by the dimedone to the sulfenic group in the folded protein. To overcome this constraint, we added guanidine hydrochloride to the final concentration of 6 M. This procedure resulted an increase of +138 Da to the MW of the M121G Az

(Figure 2.10A, incubation time of Cu(I)-M121G azurin with H2O2 of 30 sec), consistent with a dimedonylated protein. When the incubation time of Cu(I)-M121G Az with H2O2 was increased from 30 sec to 30 min, significantly enhanced intensity of dimedone labeled product was observed (Figure 2.10B).

I A control experiment using Cu -WT azurin after treatment with H2O2 under the same condition showed no labeling peak, and this result was critical in demonstrating that the sulfenic moiety did not form through processes involving unfolded protein.

Figure 2.10 Mass spectra of the air sensitive species. Electrospray mass spectrum before (black) and after (red) dimedone labeling of the air-sensitive species. The Cu(II)-sulfenate-M121G azurin is generated by treating Cu(I)-M121G azurin with 2 eq. H2O2 in 50 mM potassium phosphate pH 7.0 for (A) 30 sec and (B) 30 min.

As azurin contains three native cysteine residues (two of which constitute a disulfide bond), we confirmed dimedone labeling on Cys 112 via tryptic digest and tandem MS/MS analysis of the dimedone- labeled material. The dimedone modified peptide NH2-LKEGEQYMFFCdimedoneTFPGHSALGK-COOH was robustly detected, and was further analyzed via MS/MS. A 241.0105 Da difference was observed between b10 and b11 ions, corresponding to the molecular weight of dimedone labeled cysteine after dehydration (cal. 241.0773 Da). This result confirms that the sulfenic acid modification is isolated to the

~~Cys-112 residue of M121G azurin (Figure 2.11).~~

~~5 8x10 766.43544~~

~~b 7x105 13 y12~~

~~5 6x10 1762.8209 y13 1402.7022~~

5x105 1549.7778 y b9 14 4x105 y Intensity y 9 b y 1680.8713 15 5 8 10 1126.5736 2+ 3x10 b13 2+ y b10 b 913.51529 18 11 b12 1843.9353 5 b 979.471521014.5581 y y5 b 7 16 2x10 y 6 1163.2887 y b5 6 4 b14 1079.4132 1273.6866 1514.6971 1615.7673

~~5 475.37911 848.43511 1971.9394 685.39845 1x10 612.4062~~

~~557.35693 1859.9676 388.30337 0 500 1000 1500 2000 m/z~~

Figure 2.11 MS/MS data of NH2-LKEGEQYMFF(C-dimedone)TFPGHSALGK-COOH formed from trypsin digestion of dimedone treated reaction solution. The precursor mass was m/z (2+) = 1264.5855 (cal. m/z (2+) = 1264.0998).

~~2.3.5 Isotopic enrichment study of Cys-SO3H formation~~

~~To confirm that the oxygen atom on the cysteine residue of M121G was derived from H2O2, we compared the isotope distribution of oxygen found in the oxidized cysteine after treatment of Cu(I)-~~

18 18 M121G azurin with either H2O2 or O-labelled H2O2 (H2 O2). We exposed the green colored solution to air before demetallation and tryptic digestion. The peptide containing Cys112, NH2- EGEQYMFFCTFPGHSALGK-COOH, was analyzed by high resolution MS spectrometry. The data shows that the peptide exhibited a mass increase consistent with addition of three oxygen atoms, suggesting formation of a cysteine sulfonic acid, the most oxidized organic form of sulfur. After comparing the experimental data with the calculated isotopic distribution, a nearly pure isotopic spectrum

18 16 of Cys-S O O2H is formed (Figure 2.12), suggesting that under these conditions H2O2-mediated cysteine oxidation stopped at sulfenic acid, and further oxidation was from air. We note that higher equivalents of

~~H2O2 may produce sulfinic and sulfonic acid directly by further oxidizing Cys-SOH produced in the reaction.~~

Figure 2.12 Calculated and experimental isotopic distributions for product EGEQYMFF(C- + +2 18 SO3H)TFPGHSALGK tryptic peptides [(pep) + 2H ] with progressive O labeling. Experimental 16 18 spectra were obtained with 1 eq. additions of H2 O2 or H2 O2 to Cu(I)-M121G and an aerobic workup.

~~2.3.6 DFT calculations~~

We evaluated several potential structural models for the air-sensitive intermediate species. A small model of the active site (S-M121G-H2O) was created from the M121G crystal structure as a starting point. Optimization of this small model with two N(Imidazole (Im)) and one S(Ethyl (Et)) equatorial ligands and one axial H2O ligand resulted in a structure in reasonable agreement with crystallography

~~(Figure 2.13 and Table 2.4). The axial H2O-Cu distance is ~0.4 Å too short in the DFT structure (2.5 vs.~~

~~2.9 Å). This difference in distance likely reflects the absence of several H-bonding partners to the Cu-OH2 bond, which are present in the X-ray crystal structure.~~

~~Figure 2.13 Small DFT structures for M121G-H2O and models for the air-sensitive species. Atom coloring: Cu, pink; S, yellow; O, red; N, blue; C, grey; H, white.~~

~~Table 2.4 Comparison between experimental bond distances and DFT structures for small M121G models and possible structures for the air-sensitive species.~~

Bond Distances Mulliken Spin Densities (PCM ε = 4.0) Cu-S (Å) Cu-O (Å) Cu-N (Å)a S-O (Å) Cu S/O Cu(II)-M121G 2.2 2.9b 2.0 ------azurin (exp.) S-M121G- 2.18 2.52b 2.04 -- 0.37 0.54/-- H2O 1 2.63 1.92 2.02 1.61 0.31 0.37/0.25 2 2.48 2.09 1.97 1.73 0.52 0.19/0.07 3 2.41 -- 1.93 1.66 0.47 0.32/0.04 4 -- 2.07 1.93 1.72 0.57 0.20/0.05 5 2.53 -- 1.94 1.49c 0.22 0.21/0.38d 6 -- 2.00e 2.01 1.59c 0.61 0.02/0.20d 7 -- 2.04 1.96 1.53c 0.31 0.16/0.38d 8 -- 2.32 1.94 1.55 0.03 0.55/0.40 9 2.77 -- 1.94 1.54 0.07 0.44/0.42

~~Table 2.4 (cont.)~~

Cu-OOH 2.28 1.90 2.15 1.44f 0.45 0.24/0.23 L-M121G- 2.17 2.48 1.99 -- 0.59 0.25/-- H2O 10 -- 1.90 1.98 1.60 0.50 0.16/0.28 11 2.79 -- 1.92 1.54 0.15 0.46/0.34 a Averages of Cu-N(Im) distances. b Axial Cu-OH2 distance. c Average distance for SO2. d Sum over both O atoms. e Average of Cu-O distances. f O-O distance.

~~A number of oxidized thiolate models of the intermediate species have been considered (1 – 7).~~

The relevant optimized bond distances are given in Table 2.4 and Figure 2.13. Mulliken spin densities are also given in Table 2.4 for comparison. Specifically, 1 is a pseudo-side-on sulfenate (Cu(II)-SO-) species.

~~2 – 4 have protonated sulfenates (Cu(II)-SOH) bound to Cu(II) in a pseudo-side-on fashion and through S~~

- or OH, respectively. 5 – 7 contain a sulfinate (SO2 ) group bound to Cu(II) via S, two O’s, and one O, respectively. In order to lock in on a structural model for the intermediate, both TDDFT and frequency calculations have been carried out on 1 – 7 for correlation to experimental data. The TDDFT calculated absorption spectra are shown in Figure. 2.11-A, B (divided for clarity). The best agreement between the experimental and calculated absorption spectrum of the intermediate is given by 1 (red line, Figure

~~2.14A), which shows a CT transition at higher energy. However, 2 (blue line, Figure 2.14A) and 3 (green~~

16 18 line, Figure 2.14A) are also potential candidates. The DFT calculated frequencies and Ox and Ox shifts and the vibrational assignments are given in Table 2.5. For the non-oxidized site, the S-M121G-H2O model gave calculated Cu-S frequencies (408 and 413 cm-1) in good agreement with experiment (Figure

2.9C). Note the Cu-S vibration mixes with N(Im) and S(Et) ligand modes at similar energy. Comparing the vibrations of 1 – 7 to the experimental rR data, 1 gives the best agreement. That is, two vibrations are predicted at 442 and 418 cm-1, which shift down in energy to 431 and 412 cm-1, respectively, upon 18O substitution. An S-O vibration is predicted at 832 cm-1, which shifts down in energy to 803 cm-1 (Δ = 29

~~42 cm-1). Experimentally, the higher energy vibrations are observed at 837 and 807 cm-1 (Δ = 30 cm-1). The~~

~~ν(16O)/ν(18O) ratio is 1.037, in excellent agreement with the harmonic value for 32S16O and 32S18O (1.039).~~

Figure 2.14 Time dependent DFT calculated absorption spectra. TDDFT calculationed absorption spectra for potential air-sensitive species (A) 1 – 4 and Cu(II)-M121G azruin (S-M121G-H2O), (B) 5 – 7 and Cu-OOH species (small models) and (C) 10 -11 and Cu(II)-M121G azurin (L-M121G-H2O).

~~Table 2.5 DFT vibrational frequencies for small M121G models and potential structures of the air- sensitive species.~~

~~Model ν(Cu-S) (cm-1) ν(Cu-S) (cm-1) exp calc M121G- 400 408/413 H2O~~

~~16 -1 18 -1 -1 ν(H2 O2)(cm ) ν(H2 O2)(cm ) Δ(cm ) assignment 1 418/442 412/431 6/11 Cu-O 832 803 29 S-O~~

~~Table 2.5 (cont.)~~

~~2 307/315/379 304/314/377 3/1/2 Cu-S(OH) 713 689 24 S-O 3 303/391 302/390 1/1 Cu-S(OH) 767/791 748/784 19/7 S-O 4 291 284 7 Cu-OH~~

392 389 3 S-Cβ-Cα + Cβ-S-OH 677 654 23 S-O 5 311 308 3 Cu-S 415/418 401/417 14/1 O-S-O 493 487 6 Cu-S 1013/1174 977/1140 36/34 S-O (as)/S-O (s) 6 345 343 2 Cu-O(s) + Cu-N 365 353 12 Cu-O(as) 504/579 492/561 12/18 Cu-O(s) + O-S-O + S-C-C 829/884 803/850 26/34 S-O(as)/S-O(s) 7 248 241 7 Cu-O

~~341 331 10 SO2-Cβ~~

~~376 370 6 Cβ-S-O 447 433 14 O-S-O Cu-OOH 375 374 1 Cu-S 459 437 12 Cu-OOH 891 840 49 O-OH~~

~~Although the intermediate species does not form upon addition of H2O2 to Cu(II)-M121G Az, the frequencies and isotope shifts of the intermediate are reasonably consistent with a Cu(II)-OOH species 46.~~

~~We therefore also considered this possibility. Relevant geometric parameters of the optimized Cu(II)-~~

OOH structure are given in Table 2.4, and the calculated vibrations are given in Table 2.5. The Cu-ligand vibrations and their isotope shifts (Cu-S: 374(1) cm-1; Cu-O: 459(12) cm-1) are in good agreement with experiment; however, the calculated O-OH frequency and shift (891(49) cm-1) are both too high.

~~Furthermore, two intense CT transitions, split in energy by ~4000 cm-1, are predicted by TDDFT but not~~

~~44 observed experimentally (Figure 2.14B). On the basis of the O-OH frequency and isotope shift and the~~

TDDFT predicted absorption spectrum, a Cu(II)-OOH species can be eliminated. Thus, 1, a pseudo-side- on bound Cu(II)-SO- complex, can be considered a likely model of the air-sensitive 450 nm intermediate species.

It is interesting to note that, in addition to 1 (Cu(II)-SO-), two other geometric and electronic isomers exist as energetic minima for a Cu(II)-SO- species. Instead of the pseudo-side-on binding, the SO group can bind through O or S in a monodentate fashion (8 and 9, respectively, Figure 2.13 and Table

2.4). In both cases the SO group reduces Cu to form a Cu(I)-SO• species, in contrast to what is observed experimentally by EPR. 8 is lower in energy than 1 by ~6 kcal/mol, while 9 is higher in energy than 1 by

~3 kcal/mol. Clearly, 8 and 9 are not viable candidates for the air sensitive intermediate due to reduction of the Cu active site. However, SO binding through O with reduction of Cu is favored in these small models. We have therefore considered the role of the protein environment in tuning the relative energies of 1, 8, and 9, as well as the influence of the protein matrix on their respective ground state wave functions (i.e., Cu(II)-SO- vs. Cu(I)-SO•). 47

Figure 2.15 Large DFT structures for M121G-H2O (L-M121G-H2O) and the air-sensitive species: pseudo-side-on (10), O-bound (11), and S-bound (12). Specific second sphere interactions are labeled in the L-M121G-H2O model. Atom coloring: Cu, pink; S, yellow; O, red; N, blue; C, grey; H, white.

~~All three binding modes of SO were optimized in these large models (i.e., SO pseudo-side-on~~

~~(10), O-bound (11), and SO S-bound (12), along with M121G-H2O (L-M121G-H2O)). 10, 11, and 12 are large model analogues of 1, 8, and 9, respectively, in the small models. The relevant geometric~~

~~45 parameters for L-M121G-H2O, 10, 11, and 12 are included in Table 2.4. Their structures are given in~~

Figure 2.15. Large models were therefore constructed with the following second sphere interactions: (1) a negatively oriented dipole near Cu; (2) two amide backbone H-bonds to S (or SO); and (3) a negatively oriented dipole near S. In the large models, both 10 and 11 are found to have Cu(II)-SO- ground states, while in 12, the sulfenate group reduces the Cu. In the large models, 10 and 11 are isoenergetic within

<0.5 kcal/mol, while 12 is ~6 kcal/mol higher in energy. We therefore focus on 10 and 11. The TDDFT predicted UV-Vis absorption spectra for L-M121G-H2O, 10, and 11 are given in Figure 2.14C. From these calculations, the UV-Vis spectrum of 10 best represents the experimental UV-vis data of the intermediate species and is consistent with calculated spectrum of 1, while 11 shows a decrease in energy in the CT transition relative to the L-M121G-H2O model. Experimentally, the CT is found to increase in energy.

~~Table 2.6 DFT vibrational frequencies for large M121G models and potential structures of the air- sensitive species.~~

~~Model ν(Cu-S) (cm-1) exp. ν(Cu-S) (cm-1) calc.~~

~~L-M121G H2O 400 384/403/406~~

~~16 -1 18 -1 -1 ν(H2 O2)(cm ) ν(H2 O2)(cm ) Δ(cm ) assignment 10 (SO) 448/457 432/445 16/12 Cu-O 824 796 28 S-O 11 (O) 387/463/477 381/461/467 6/2/10 Cu-O 820 801/772 19/48 S-O~~

The DFT calculated vibrations are given in Table 2.6. The energies and isotope shifts of 10 are also in better agreement than 11 in terms of the rR vibrations. Specifically, the calculated Cu-O vibrational frequency is higher in 11 than 10 (387, 463, and 477 cm-1 vs. 448 and 457 cm-1). Furthermore, the aggregate 16O/18O shift of the S-O vibration is larger in 10 relative to 11 (34 vs. 28 cm-1, respectively).

Note that the S-O vibration in 11 is mixed approximately equally into two modes, so the average value is used here. Thus, 10 (and its small analog 1), a pseudo-side-on Cu(II)-SO- species, best reproduces the

~~46 experimental absorption and rR data for the intermediate formed in the reaction of Cu(I)M121G Az and~~

~~H2O2.~~

~~It is interesting to note which second sphere interactions allow for the stabilization of a Cu(II)-~~

~~SO- ground state. We systematically removed the second sphere interactions and carried out addition geometry optimizations. The geometric parameters and Mulliken spin densities are given in Table 2.7.~~

Reduction of Cu occurs upon removing both the negatively oriented dipole near Cu and the H-bonds to the sulfenate. Therefore, the secondary protein environment plays an important role in tuning the relative redox couples of the Cu and the sulfenate, allowing for the stabilization of a Cu(II)-SO- species.

~~Table 2.7 DFT structures for large M121G models and potential structures of the air-sensitive species upon removal of specific second sphere interactions.~~

~~10 Cu- H- S- Cu- 11 Cu- H- S- Cu- Dipole bonds Dipole Dipole Dipole bonds Dipole Dipole a /H- /H- bonds bondsb Bond Distances Cu-O 1.93 1.92 1.91 1.92 1.90 1.91 1.96 1.94 1.90 2.19~~

~~Cu-S 2.42 2.40 2.43 2.45 2.43 ------~~

~~Cu-Nc 1.99 1.97 1.99 1.99 1.98 1.98 1.94 1.96 1.97 1.90~~

S-O 1.62 1.62 1.61 1.62 1.61 1.60 1.57 1.59 1.60 1.55 Spin Densities Cu 0.48 0.43 0.47 0.51 0.39 0.44 0.26 0.33 0.49 0.06 O 0.20 0.21 0.23 0.19 0.23 0.30 0.36 0.33 0.29 0.41 S 0.21 0.25 0.25 0.18 0.30 0.19 0.33 0.31 0.16 0.51 a Indicator of which second sphere interaction has been removed. b Only this combination results in reduction of Cu as discussed in the manuscript text. c Average of two Cu-N(His) distance.

~~The ground state wave functions (β-LUMO’s) of both L-M121G-Az and 10 are given in Figure~~

2.16. The calculated Cu(d) and S(p) characters (from Mulliken population analysis) of L-M121G-H2O associated with the Cu(II)-thiolate bond are 56 % and 25 %, respectively. For 10, binding SO- in a pseudo-side-on fashion decreases the Cu(d) character to 48% while the SO- character increases to 35%.

Thus, the Cu(II)-SO- species is more covalent than M121G Az. This is due to the strong σ overlap associated with two bonding interactions between the SO- π* donor and the unoccupied d(x2-y2) orbital in

47 the side-on structure (Figure 2.16, right). Furthermore, the ~450 nm CT band of the intermediate is calculated to be a transition between the bonding and anti-bonding pair of the β-LUMO. Thus, the shift up in energy of this transition in the intermediate relative to the Sp(π) → Cu(d(x2-y2)) (i.e., 600 nm band) of

~~M121G Az reflects the increase in the bonding/anti-bonding interaction of the Cu-SO- unit compared to the Cu-S(Cys) bond in M121G Az.~~

~~Figure 2.16 Comparison of the ground state wave functions (β-LUMO’s) of L-M121G-H2O (Left) and 10 (Right). Results of Mulliken population analyses are indicated.~~

~~In addition, in going from M121G azurin to the intermediate, the g||-value decreases from 2.303 to~~

-4 -1 -4 -1 2.169, and the A||-value increases in magnitude from ~20 x 10 cm to 139 x 10 cm . Note that A|| is negative. The deviation of the g||-value from 2.0023 is inversely proportional to energy of the d(xy) →

~~2 2 d(x -y ) ligand field (LF) transition. The TDDFT calculated energies of this transition in L-M121G-H2O~~

-1 and 10 are ~12,400 and 16,900 cm , respectively, consistent with the larger g|| in the former. However, the energy difference between these two species accounts for only ~60 % of the difference in the g||- values. This indicates that the Cu character in the ground state of 10 must be lower than in L-M121G-H2O, which is consistent with the Mulliken population analyses (Figure 2.16) and spin densities (Table 2.4).

Finally, we note that the increase in the magnitude of A|| has two contributions. First, M121G azurin has a rhombic EPR signal, which is generally associated with dz2/4s mixing. This mixing will reduce the magnitude of A|| in the reference (M121G azurin). Second, the decrease in g|| between M121G azurin and

~~48 the sulfenate species decreases the orbital dipolar contributions to the hyperfine, which is positive and will also contribute to the larger |A|||.~~

~~2.4 Summary~~

In summary, we have shown that anaerobic addition of H2O2 to Cu(I)-M121G azurin resulted in the formation of a side-on Cu(II)-sulfenate species in azurin, as supported by UV-vis, EPR, and rR spectroscopies as well as mass spectrometry and electronic structure calculations correlated to the spectroscopic data. Such an observation is made possible for the first time, mainly due to stabilization of the species by non-covalent secondary sphere interactions. The stabilization and characterization of a copper-sulfenate center in proteins opens a new avenue for the detection and exploration of other similarly reactive species in metallobiochemistry.

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~~Chapter 3 Reversible S-Nitrosylation in an Engineered Azurin and its Application as NO regulator under Physiological ConditionsII~~

~~3.1 Introduction~~

S-nitrosylation, the covalent attachment of nitric oxide (NO) to the thiol side chain of cysteine, has emerged as an important pathway for dynamic post-translational regulation of many classes of proteins involved in important functions such as blood flow regulation,1 muscle contraction,2 cellular trafficking,3 apoptosis regulation,4 and functions related to Alzheimer’s disease.5 Although it is widely accepted that the S-nitrosylation occurs in vivo, the mechanisms by which nitrosothiols (RSNOs) are formed and removed are unclear.6 Since the S-nitrosylation requires one-electron oxidation of the initial complex between NO and a thiol, in the absence of an electron acceptor, such as O2 or a redox-active metal ion, the nitrosothiol will not form. Because the S-nitrosylation that occurs in cells is often enhanced

7 under anaerobic conditions, O2 is not absolutely required for the process. Therefore, redox-active metalloproteins likely play a key role in promoting the S-nitrosylation reactions by using the metal ion as the electron acceptor. Indeed, it has been reported that some heme proteins can catalyze S-nitrosylation at cysteine residues when the heme is in close proximity to the target thiol.8-12

While the S-nitrosylation catalyzed by heme proteins is well known, the same process promoted by copper in proteins is rare in the literature. Copper-zinc superoxide dismutase (SOD) is reported to increase the yield of S-nitrosohemoglobin and nitrosylated hemes by eliminating superoxide8 or by facilitating the transfer of NO from S-nitroso-glutathione (GSNO) to Cysβ93 of oxyhemoglobin in concentrated solutions of the protein.13 The Cu(II)-containing protein ceruloplasmin is also shown to catalyze S-nitrosylation of heparan-sulphate proteoglycan, glypican-114 as well as the formation of GSNO

II Portions of this chapter are previously published and are here reproduced with permission from Tian, S.; Liu, J.; Cowley, R. E.; Hosseinzadeh, P.; Marshall, N. M.; Yu, Y.; Robinson, H.; Nilges, M. J.; Blackburn, N. J.; Solomon, E. I.; Lu, Y. Nature Chem. submitted.

~~54 from NO.15 However, a more recent study contradicted this finding and showed that depletion or~~

16 – supplementation of the ceruloplasmin in plasma failed to alter plasma nitrosothiol levels. Instead, NO2 was observed as the major product of the reaction between NO and ceruloplasmin. It is thus proposed that the Cu(II) in ceruloplasmin can oxidize NO to NO+, which would immediately react with water to form

– NO2 . In addition, the S-nitrosylation of bovine serum albumin and glutathione mediated by free copper ion has been shown in vitro.17 However, since the level of free copper ion is rigorously regulated in vivo and is estimated to be in the attomolar range (10-18 M), such a free copper catalyzed S-nitrosylation observed in vitro is unlikely to occur in vivo.18-21

Protein design is a powerful tool for uncovering essential relationships between protein structures and functions.22 Herein we report the first direct observation of S-nitrosylation of copper-bound cysteine in an engineered red copper center in Pseudomonas aeruginosa azurin (Az), supported by UV-vis absorption (UV-vis), electronic paramagnetic resonance (EPR) and extended x-ray absorption fine structure (EXAFS) spectroscopic techniques. We have also elucidated the reaction mechanism by kinetic studies and density functional theory (DFT) calculations. More importantly, we show that such an S- nitrosylation can proceed in a fast (k = 6.7 × 105 M-1s-1) and efficient (> 90% yield) way under physiological conditions in the presence of micromolar level concentration of either protein or NO, preventing NO inhibition of cytochrome bo3 oxidase activity.

~~3.2 Materials and methods~~

~~3.2.1 Site-directed mutagenesis and protein purification~~

Plasmid containing the M121H/H46E and M121H/H46E/F114P mutations were constructed by site-directed mutagenesis using wild-type azurin (pET9a) as the template for the quick change mutagenesis procedure with the forward primers 5’-CGA AGA ACG TTA TGG GTG AAA ACT GGG

~~TTC TGT CC-3’ (fwd-H46E), 5’-TTC TTC TGC ACT CCG CCG GGT CAC-3’ (F114P), 5’-CAC TCC~~

~~GCA CTG CAT AAA GGT ACC CTG-3’ (fwd-M121H) and the reverse primers 5’-GGA CAG AAC~~

~~CCA GTT TTC ACC CAT AAC GTT CTT CG-3’ (rev-H46E), 5’-GTG ACC CGG CGG AGT GCA~~

~~GAA GAA-3’ (rev-F114P), 5’-CAG-GGT-ACC-TTT-ATG-CAG-TGC-GGA-GTG-3’ (rev-M121H). The mutations were confirmed by sequencing. The proteins were expressed in BL-21* E. coli (Novagen,~~

~~Madison, WI) and purified according to published procedures.23,24 Electrospray ionization mass spectrometry confirmed the identity of the isolated variant (apo-M121H/H46EAz, MWobs = 13944, MWcal~~

~~= 13943.73; apo-M121H/H46E/F114PAz, MWobs = 13894, MWcal = 13893.67).~~

~~3.2.2 UV-vis spectroscopy measurements~~

UV-vis absorption spectra were obtained at room temperature on an HP Agilent 8453 diode array spectrometer. Extinction coefficient of 9000 M-1cm-1 at 280 nm was used to determine apo protein concentration. All UV-vis spectra were recorded in 50 mM sodium 2-(N-morpholino)ethanesulfonate

~~(MES) pH 6.0 buffer unless otherwise stated.~~

~~3.2.3 Mass spectrometry measurements~~

The mass spectra of the proteins were acquired using a Waters Quattro II spectrometer operating in positive-ion mode. Samples (10 μL of 50 µM protein in 50 mM ammonium acetate buffer at pH 6.0) were injected into a flow of 50 µL/min of 50% CH3CN/H2O mobile phase. The mass spectra were collected from 500 to 2000 m/z and deconvoluted using the MassLinx software package with a 1 Da resolution and a 10,000 – 20,000 Da calculation window.

~~3.2.4 Electron paramagnetic resonance (EPR) spectroscopy measurements~~

~~All EPR samples were prepared in the presence of 20 % glycerol in 50 mM MES pH 6.0 buffer.~~

~~X-band EPR spectra were collected at 30 K on a Varian-122 spectrometer equipped with an Air Products~~

~~Helitran cryostat and temperature controller, with a collection frequency of 9.05 GHz, power of 0.2 mW and field modulation of 4.0 G.~~

~~3.2.5 Electrochemical measurements~~

The reduction potential of Cu(II)-M121H/H46EAz and Cu(II)-M121H/H46E/F114PAz were measured by cyclic voltammetry using a CH instruments 617A potentiostat equipped with a picoamp booster and a Faraday cage. A pyrolytic graphite edge (PGE) electrode was polished and 2 µl protein solution was added directly to the electrode. After a short incubation time, the electrode was immersed in different pH buffers with 100 mM NaCl and scanned at varying rates between 10 mV and 500 mV. The reduction potentials were measured against Ag/AgCl and converted to NHE.

~~3.2.6 Collection and analysis of XAS data~~

~~Cu K-edge (8.9 keV) extended X-ray absorption fine structure (EXAFS) and X-ray absorption near edge structure (XANES) data were collected at the Stanford Synchrotron Radiation Lightsource~~

(SSRL) operating at 3 GeV with currents between 100 and 80 mA. All samples were measured on 100 element beamline 9-3 using a Si 220 monochromator with crystal orientation φ = 90° and a Rh-coated mirror located upstream of the monochromator set to 13 keV to reject harmonics. A second Rh mirror downstream of the monochromator was used to focus the beam. Data were collected in fluorescence mode using a high-count-rate Canberra 30-element Ge array detector with maximum count rates below 120 kHz.

A 6 μ Z-1 Ni oxide filter and Soller slit assembly were placed in front of the detector to reduce the elastic scatter peak. Six scans of a sample containing only sample buffer were collected, averaged, and subtracted from the averaged data for the protein samples to remove Z-1 Kβ fluorescence and produce a flat pre-edge baseline. The samples (80 μL) were measured as aqueous glasses (20% ethylene glycol) at

8−10 K. Energy calibration was achieved by reference to the first inflection point of a copper foil (8980.3 eV) placed between the second and third ionization chamber. The data were carefully monitored for photoreduction, and where evident, only the first spectrum of a series was included in the final average. In such cases, a new sample spot was chosen for each spectrum included in the average.

~~Data reduction and background subtraction were performed using the newest version of the~~

57 program modules of EXAFSPAK, which is compatible to data obtained from SSRL.25 Data from each detector channel were inspected for glitches and spectral anomalies before inclusion in the final average.

~~Spectral simulation was carried out using the program EXCURVE 9.2 as previously described.26-28~~

EXAFS data were simulated using a mixed-shell model consisting of imidazole and thiolate-S coordination. The imidazole ring geometry was constrained to ideal values of the internal bond lengths and angles, while the first-shell distance (R) and Debye−Waller factor for the Cu−N(imid) shell

~~(including the single and multiple scattering contributions for the imidazole rings), the Cu−S shells, and~~

E0 were refined. In these preliminary refinements, the imidazole ring outer shell C and N atoms were constrained to move relative to the first shell Cu−N distance so as to maintain the idealized ring geometry.

In practice final outer shell coordinates for acceptable fits deviated by less than the permitted amount from the idealized position. We refined the parameters of the fit as follows: E0, the photoelectron energy

i threshold, Ri the distance from the central metal atom (Cu) to atom i and 2σ2 the Debye-Waller (DW) term for atom i. Coordination numbers were fixed to those previously established from crystal structures whenever possible. The quality of the fits was determined using the least-squares fitting parameter, F, which is defined as:

~~2 6 theory experiment 2 F = (1/N) ∑ik (χi – χi ) and is hereafter referred to as the fit index or fit (FI).~~

~~3.2.7 Reconstitution of M121H/H46EAz and M121H/H46E/F114PAz with Cu(II)~~

~~Aliquots of metal-free (apo-) azurin mutants (1.5-2.0 mL) in 50 mM ammonia acetate buffer (pH~~

2+ 6.35) were titrated with 2 eq. Cu (from a stock solution of 50 mM CuSO4) and stirred at room temperature for 30 minutes. Excess copper was removed using a PD-10 desalting column containing 8.3 ml of SephadexTM G-25 medium. The buffer was exchanged to 50 mM MES buffer (pH 6.0) with

~~Amicon® Ultra-4 centrifugal filter devices with a 10 kDa molecular weight cutoff.~~

~~3.2.8 Crystallography of Cu(II)-M121H/H46EAz~~

Crystals of Cu(II)-M121H/H46EAz suitable for X-ray diffraction grew over three days at 4 oC following reported procedure.29 2 μL apo-M121H/H46EAz (1.2 mM) in 100 mM NaOAc buffer (pH 5.6) was mixed with 2 µL well buffer containing 25% PEG 4000, 100 mM LiNO3, 10 mM CuSO4 and 100 mM Tris at pH 8.0 on silica slides and equilibrated over 250 µL well buffer using hanging drop method at

5 oC. Light green block crystals were obtained in 2-3 days. The crystals were dipped in cryo-buffer containing 70% of well buffer and 30% of glycerol before mounting and frozen in liquid N2 for diffraction analysis. Crystallographic parameters are shown in Table S1. Diffraction data were collected using 1.0750 Å as wavelengths of data collection at the Brookhaven National Synchrotron Light Source

X29 beamline. All data were integrated using the program HKL2000.30 The crystal structure was solved by the molecular replacement method using AutoMR docked in the PHENIX Suite.31 Refinement was performed using phenix.refine in the PHENIX Suite and Coot.32 The crystal structure of wild type azurin

~~(PDB: 4AZU) was used as a searching model.~~

~~3.2.9 Preparation of Cu(I)-M121H/H46E/F114PAz~~

~~Cu(II)-M121H/H46E/F114PAz (1.0 ml of 2.0 mM protein in 50 mM MES buffer, pH 6.0) was degassed and equilibrated with argon on a Schlenk line and was then transferred into a glove bag. Then~~

Cu(II)-M121H/H46E/F114PAz was reduced by slow addition of solid sodium ascorbate containing 10 mol % N, N, N’, N’ – tetramethyl-p-phenylenediamine (TMPD) as a mediator until the protein solution turned completely colorless. The resulting solution was passed through PD-10 desalting column to remove excess reductant.

~~3.2.10 Reaction of Cu(II)-M121H/H46E/F114PAz with nitric oxide (generated from DEA~~

~~NONOate)~~

~~Cu(II)-M121H/H46E/F114PAz solution in 50 mM MES pH6.0 buffer was degassed and equilibrated with argon on a Schlenk line and was then transferred into a glove bag. To 2.0 ml of 0.1 mM~~

Cu(II)-M121H/H46E/F114PAz solution, 1 eq. DEA NONOate (from 50 mM DEA NONOate stock solution in 10 mM NaOH) was added at room temperature under stirring. The color of the solution changed from light green to colorless in 1 minute. The process is monitored with UV-vis spectroscopy by

~~HP Agilent 8453 diode array spectrometer.~~

~~3.2.11 Reaction of Cu(II)-M121H/H46E/F114PAz with nitric oxide (prepared from nitric oxide saturated buffer) with stopped-flow~~

Experiments were performed on an Applied Photophysics Ltd. (Leatherhead, U.K.) SX18.MV stopped-flow spectrometer equipped with a 256 element photodiode array detector. The instrument was prepared for anaerobic stopped-flow by rinsing its lines out several times with buffer that had been degassed by bubbling argon gas through it. Cu(II)-M121H/H46E/F114PAz solution in 50 mM MES buffer (pH 6.0) was degassed and equilibrated with argon on a Schlenk line. The NO saturated buffer was prepared by bubbling NO gas through the buffer using a NO line. Two-syringe mixing was employed to mix equal volumes of 50 μM Cu(II)-M121H/H46E/F114PAz solution with 2 mM NO in 50 mM MES buffer (pH 6.0, NO saturated) for pseudo first-order kinetics or 70 μM Cu(II)-M121H/H46E/F114PAz solution with 0.2 mM NO in 50 mM MES buffer (pH 6.0, diluted from NO saturated buffer) for second- order kinetics. All reported data sets originally consisted of 200 spectra collected over 0.2 s. The integration period and minimum sampling period were both 1 ms.

~~3.2.12 Effect of Cu(II)-M121H/H46EAz on NO-inhibition of E. coli cytochrome bo3 oxidase~~

Oxygen consumption catalyzed by cytochrome bo3 ubiquinol oxidase was measured with a Clark- type O2 electrode interfaced with a chart recorder. In a typical experiment, 2.5 μl of 200 μM cytochrome bo3 oxidase was added to 500 μl air-equilibrated 50 mM Tris buffer (pH 7.0) containing 100 μM ubiquinol-1 and 1 ~ 5 μM Cu(II)-M121H/H46E/F114PAz. After addition of nitric oxide reaching 1 ~ 5

~~μM to inhibit the enzyme, the O2 consumption rates were measured by injecting 2.5 μl of 1 M dithiothreitol (DTT) as reductant.~~

~~3.2.13 Electronic structure calculations~~

The computational model of the red Cu site was built from the crystal structure of M121H/H46E azurin, with each residue truncated at the α carbon. For all geometry optimizations the coordinates of the

α carbons were frozen. In a few cases, small imaginary frequencies (~ -10 cm-1) involving motion of the α carbons resulted from this constraint, however calculated ΔG energies tracked well with ΔE (within 2 kcal/mol), suggesting artifacts due to imaginary frequencies are negligible. All calculations were performed with Gaussian 09 (revision D01)33 using the spin-unrestricted hybrid density functional

B3LYP34-36 and TZVP37,38 basis set on an ultrafine integration grid. PCM solvation (water, ε = 4.0) was used to account for the protein medium. For broken-symmetry singlet states on the Cu(II)-SCys/NO radical coupling coordinate, the Yamaguchi formalism39 was used to eliminate triplet contamination and obtain a

~~푩푺 ퟐ ퟑ ퟐ 푬 − (〈푺 〉푩푺 ∙ 푬) 1 BS 3 corrected energy (ퟏ푬= ퟐ , where E is the corrected singlet energy, E and E are ퟐ − 〈푺 〉푩푺~~

~~2 energies of the broken-symmetry and triplet states, respectively, and 〈S 〉BS is the calculated spin expectation value obtained from the broken-symmetry wave function).~~

푩푺 ퟐ ퟑ ퟏ ퟐ 푬 − (〈푺 〉푩푺 ∙ 푬) 푬 = ퟐ ퟐ − 〈푺 〉푩푺 Individual orbitals were visualized with the program Lumo40 and orbital compositions were analyzed with the program QMForge.41

~~3.3 Results~~

~~3.3.1 Design, spectroscopic, crystallographic and redox studies of an engineered red copper protein in azurin (Cu(II)-M121H/H46EAz)~~

To obtain direct evidence of S-nitrosylation of copper-bound cysteine in proteins and elucidate the mechanism of its formation, we started with wild type, blue copper azurin from Pseudomonas aeruginosa (WTAz), which has shown to be an excellent protein for both modeling other metalloproteins and designing novel ones,29,42,43 and investigated its reaction with NO. It has been reported that WTAz reacted with NO, monitored by the decrease of the 625 nm band characteristic of the blue copper center in

~~WTAz, and a photolabile {CuNO}10 adduct was proposed to form in the process.44,45 However, such~~

{CuNO}10 adduct was only fully formed at very low temperature (77 K).44 At room temperature, in contrast, the intensity of the 625 nm band decreased by only ~15% under NO saturated conditions.44 We tested the reaction of 70 µM of Cu(II)-WTAz with 5 equivalents of NO at pH 7 at room temperature and observed < 8% of the 625 nm band decrease over two hours (Figure 3.1). These results suggest that the reaction between the WTAz and NO is unlikely to be physiologically relevant.

Figure 3.1 UV-vis spectroscopic study of Cu(II)-WTAz reacting with NO. (A) UV-vis spectra of 70 μM Cu(II)-WTAz reacting with 5 eq. NO in 50 mM BisTris pH7.0 buffer. (B) Time trace of the reaction based on monitoring the absorbance at 625 nm.

To elucidate the structural features that promote S-nitrosylation, we sought to re-design WTAz to accommodate a mononuclear red copper center similar to that in nitrosocyanin, found in Nitrosomonas europaea.46 Both WTAz and nitrosocyanin share high structural homology,46 but the electronic and redox properties of the copper centers in these proteins differ significantly. While the blue copper center in azurin displays a strong absorption at 625 nm, assigned as S(Cys)→Cu(II) ligand-to-metal charge transfer

(LMCT) band and a reduction potential (E°) of 310 mV at pH 7,47 the red copper center in nitrosocyanin exhibits a strong absorption at 390 nm and a lower E° (85 mV at pH 7).46 More importantly, while the coordination sphere of Cu(II)-WTAz is saturated (Figure 3.2A), a water molecule was found to coordinate to the copper in Cu(II)-nitrosocyanin (Figure 3.2B), providing a potential site for substrate binding.48 The unique electronic properties of nitrosocyanin, together with the presence of an extra water ligand distinguish this red copper protein from other cupredoxins, and point to possible involvement of this protein in catalytic activities. Since nitrosocyanin is found in an ammonia-oxidizing bacterium, it could conceivably be involved in denitrification pathways.49

Figure 3.2 The Cu(II) binding sites of (A) WTAz (PDB: 4AZU), (B) nitrosocyanin (PDB: 1IBY) and (C) M121H/H46EAz (PDB: 4WKX). Copper atoms are green; oxygen atoms are red; sulfur atoms are yellow; nitrogen atoms are blue; carbon atoms are cyan.

Similar to the blue copper center in WTAz, one Cys and two His ligands are found to coordinate to the red copper in nitrosocyanin. However, a comparison of the blue and red copper centers reveals a rearrangement of the copper ligands. First, the thioether ligand in Az (Met121) is replaced with a histidine imidazolyl in nitrosocyanin (His130). Furthermore, a glutamate (Glu60) is present in nitrosocyanin in

~~63 place of the His46 in Az. Based on these differences, we designed a new Az mutant, M121H/H46EAz,~~

~~(Figure 3.2C).50 Construction, expression, and purification of apo-M121H/H46EAz was carried out~~

~~29 following a reported procedure. Titration of apo-M121H/H46EAz with CuSO4 saturated at 1 equiv.~~

~~Cu(II), and the resulting Cu(II)-M121H/H46EAz showed a strong absorption band at 403 nm (ε = 2300~~

~~M-1cm-1) and a weak and broad band around 750 nm (Figure 3.3 and Figure 3.4) distinct from that of~~

WTAz, but similar to that of nitrosocyanin. The strong absorption band around 390 nm and the weak absorption band around 720 nm in nitrosocyanin have been assigned as a pσ(Cys)→Cu(II) ligand to metal charge transfer (LMCT) and a Cu(II) d-d transition, respectively.46,49 The fact that the 600 nm band in

2 2 WTAz, assigned as pπ(Cys)→Cu(II) LMCT, completely disappeared in this mutant, suggests that the dx -y ground state of Cu(II) only overlaps with pσ orbital of S(Cys) with minimal π interaction, similar to that in nitrosocyanin.

Figure 3.3 Titration of apo-M121H/H46EAz with CuSO4. (A) Aliquots of 50 mM CuSO4 stock solution were added to a solution containing 2 ml of 150 μM apo-M121H/H46EAz in 50 mM NH4OAc pH6.4 buffer under stirring. After stirring for 5 minutes at room temperature, the UV-vis absorption spectra were recorded. (B) Titration curve monitored by the increased absorption at 405 nm versus the equivalents of Cu2+. The visible region absorption bands reached saturation at one molar equivalent of Cu(II), suggesting that the engineered copper center in M121H/H46EAz is mononuclear.

Figure 3.4 Measurement of the extinction coefficient of Cu(II)-M121H/H46EAz. (A) Aliquots of 50 mM CuSO4 stock solution were added to a solution containing 2 ml of 1.0 mM apo-M121H/H46EAz in 50 mM NH4OAc pH6.4 buffer under stirring (the amount of protein was in large excess to ensure complete binding of Cu2+). The UV-vis absorption spectra were recorded after stirring for 5 minutes at room temperature. (B) Extinction coefficient of Cu(II)-M121H/H46EAz at 405 nm is determined by fitting the plot of absorption at 405 nm vs. Cu2+ concentration using Beer’s law A = εlc where l = 1 cm and c = Cu2+ concentration. The extinction coefficient at 405 nm was determined to be 2300 M-1cm-1.

To obtain 3D structure of the Cu(II)-M121H/H46EAz, we crystallized the protein and solved its crystal structure. As shown in Figure 3.2C, the Cu(II)-binding site adopted a more distorted tetrahedral geometry compared to that in WTAz. While the Cu-N(His117) distance remains to be similar to that of

WTAz (2.10 Å), the distance of Cu-N(His121) was 2.40 Å, significantly shorter than the ~ 2.9 Å distance of the original Cu-S(Met121) in blue copper proteins. Consequently, the metal center is further away from the backbone carbonyl oxygen at Gly45 with a distance of 3.45 Å, longer than the ~ 3 Å in WTAz.51

More importantly, the Cu-S(Cys112) distance is 2.28 Å, longer than the ~ 2.1 Å in blue copper proteins, but comparable to that in nitrosocyanin (2.29 Å).48,51 The Cu-S(Cys112) distance in Cu(II)-M121HAz from A. denitrificans is 2.16 Å, and the elongation compared to Cu(II)-WTAz was attributed to the tighter binding of His121 to the Cu(II) center compared to Met121.52 Therefore, the further weakening of the Cu-

S(Cys112) bond in M121H/H46EAz variant may arise from the H46E mutation, and concomitant rearrangement of the first coordination sphere of the metal center. This geometric rearrangement is further supported by the observation of an exogenous water at 3.78 Å from the Cu(II) within hydrogen bonding distance of the coordinating O of Glu46 (2.56 Å) in Cu(II)-M121H/H46EAz (Figure 3.2C). The presence

~~65 of water and its associated hydrogen bonding network has not been observed in other cupredoxins including Cu(II)-WTAz and Cu(II)-M121HAz, but is present similarly in nitrosocyanin (Figure 3.2B).48~~

~~These results suggest that we have engineered the WTAz into a red copper center structurally and spectroscopically comparable to nitrosocyanin. The diffraction and refinement data of Cu(II)-~~

~~M121H/H46EAz crystal structure were summarized in Table 3.1.~~

~~Table 3.1 Diffraction and refinement data for Cu(II)-M121H/H46EAz crystal structure.~~

Cu(II)-M121H/H46EAz Beamline NSLS X29 Wavelength (Å) 1.0750 Space Group P 31 2 1 Unit Cell 47.77 47.77 241.64 90 90 120 Resolution (Å) 41.37-1.939 (2.008-1.939) R-merge 0.11 Mean I/sigma(I) 14.26 (6.91) Unique reflections 24919 (2437) Completeness (%) 99.95 (99.75) R-work 0.1977 (0.2053) R-free 0.2348 (0.2541) Number of non-hydrogen 2130 atoms Macromolecules 1959 ligands 21 Water 126 Protein residues 256 RMS (bonds) 0.007 RMS (angles) 1.03 Ramachandran favored (%) 99 Ramachandran outliers (%) 0 Clashscore 2.99 Average B-factor 19.70 macromolecules 19.40 ligands 21.10 solvent 23.90

~~3.3.2 The design, spectroscopic, and redox studies of Cu(II)-M121H/H46E/F114PAz~~

Given the structural similarity between Cu(II)-M121H/H46EAz and Cu(II)-nitrosocyanin, we used cyclic voltammetry to measure E° of Cu(II)-M121H/H46EAz. Surprisingly, its E° = 400 mV at pH 7 is much higher than nitrosocyanin (85 mV) and is even higher than WTAz (310 mV) and M121HAz (310 mV) at pH 7.47 This large difference in E° prompted us to search for other mutations to better mimic the red copper center by tuning its secondary coordination sphere, as demonstrated previously.53 Since a

~~F114P mutation has been shown to lower the E° of WTAz by 85 mV through deletion of one of the hydrogen bonds to Cys112 and a shift of the copper ion out of the equatorial plane,54 we introduced the~~

~~F114P mutation to M121H/H46EAz. Upon titrating Cu(II) into apo-M121H/H46E/F114PAz, the~~

S(Cys)→Cu(II) LMCT band was observed to shift from 403 nm to 390 nm (Figure 3.5) with a higher extinction coefficient (from ε = 2300 M-1cm-1 to 3500 M-1cm-1, Figure 3.6), consistent with the increased

~~σ donation from S(Cys) to the Cu(II) as a result of the deletion of the hydrogen bond to Cys112.~~

Figure 3.5 Titration of apo-M121H/H46E/F114PAz with CuSO4. Aliquots of 50 mM CuSO4 stock solution were added to a solution containing 2 ml of 120 μM apo-M121H/H46EAz in 50 mM NH4OAc pH7.0 buffer under stirring. The UV-vis absorption spectra were recorded after stirring for 5 minutes at room temperature.

Figure 3.6 Measurement of the extinction coefficient of Cu(II)-M121H/H46E/F114PAz. (A) Aliquots of 50 mM CuSO4 solution were added to a solution containing 2 ml of 1.0 mM apo-M121H/H46EAz in 50 mM NH4OAc pH6.4 buffer under stirring (the amount of protein was in large excess to ensure complete binding of Cu2+). The UV-vis absorption spectra were recorded after stirring for 5 minutes at room temperature. (B) Extinction coefficient of Cu(II)-M121H/H46E/F114PAz at 390 nm is determined by fitting the plot of absorption at 390 nm vs. Cu2+ concentration using Beer’s law A = εlc where l = 1 cm and c = Cu2+ concentration. The extinction coefficient at 390 nm was determined to be 3550 M-1cm-1.

~~More importantly, the E° of M121H/H46E/F114PAz was lowered to 163 mV at pH 7 and 107 mV at pH 8 (Figure 3.7), much closer to that of nitrosocyanin (85 mV at pH 7).~~

Figure 3.7 Cyclic Voltammetry of Cu(II)-M121H/H46E/F114PAz. (A) Cyclic voltammogram of Cu(II)-M121H/H46E/F114PAz recorded at pH7.0. The baseline subtracted spectrum is shown in red. (B) Cyclic voltammogram of Cu(II)-M121H/H46E/F114PAz recorded at pH8.0. The baseline subtracted spectrum was shown in red. The scan rate was 100 mV/s and temperature was 25 oC.

~~To further investigate the electronic structure of the Cu(II)-M121H/H46E/F114PAz, we collected its X-band EPR spectrum (Figure 3.8), which displayed g values of 2.237, 2.038 and 2.037 (Table 3.2).~~

~~-4 -1 Interestingly, the hyperfine coupling constants in the parallel region (A// = 163×10 cm ) are much larger~~

68 than that of WTAz (60×10-4 cm-1),55 but similar to that of nitrosocyanin (144×10-4 cm-1).46 These results suggest that the Cu center in M121H/H46E/F114PAz is similar to red copper center in nitrosocyanin in both structural and electronic properties.

~~g-factor~~

~~2.8 2.6 2.4 2.2 2 1.8~~

~~2300 2700 3100 3500 Magnetic field (Gauss)~~

Figure 3.8 EPR spectrum of Cu(II)-M121H/H46E/F114PAz. Experimental (black) and simulated (red) X-band EPR spectra of Cu(II)-M121H/H46E/F114PAz. Frequency: 9.05 GHz. Power: 0.2 mW. Field modulation: 4.0 G. Simulated parameters are given in Table S2.

~~Table 3.2 Spectroscopic and EPR spin parameters of nitrosocyanin, Cu(II)-WTAz and Cu(II)- M121H/H46E/F114PAz.~~

Copper proteins λ (nm) gx, gy, gz Ax, Ay, Az (Cu) Reduction (× 10-4 cm-1) potential (vs. SHE) Nitrosocyanin 390, 496, 720 2.023, 2.067, -, 20, 144 85 mV (pH7) 2.245 Cu(II)-WTAz 627 2.059, 2.059, -, -, 53 310 mV (pH7) 2.259 Cu(II)- 390, 704 2.037, 2.038, 29, -, 163 163 mV (pH7) M121H/H46E/F114PAz 2.237 107 mV (pH8)

~~3.3.3 S-nitrosylation in the engineered red copper protein~~

To date, the function of nitrosocyanin is still unknown, but experimental evidence and genomic information suggest that nitrosocyanin could be involved in the denitrification process.56,57 Considering the similarities between Cu(II)-M121H/H46E/F114PAz and nitrosocyanin and the observed vacant site

~~69 near the metal center indicated by the distal water molecule, we treated Cu(II)-M121H/H46E/F114PAz with NO, an intermediate in the denitrification process. Upon addition of one equivalent of DEA~~

NONOate, a NO donor which releases 1.5 NO/molecule,58 to 110 µM of Cu(II)-M121H/H46E/F114PAz under anaerobic conditions at pH 6, immediate bleaching of the 390 nm peak was observed, suggesting disruption of the S(Cys)→Cu(II) LMCT band, which could result either from the reduction of Cu(II) to

Cu(I) or modification of the coordinating S of Cys112. An isosbestic point at 347 nm indicated clean conversion to the new species with absorption around 334 nm and 540 nm (Figure 3.9B), which persisted for at least one hour under anaerobic conditions, and then slowly decayed overnight at room temperature.

~~The UV-Vis spectrum of the NO addition product is strikingly similar to that characteristic of nitrosothiols, which typically have an intense band in the 330 – 350 nm region (ε ~ 1000 M-1cm-1,~~

* -1 -1 assigned as n0→π transition) and a weak band in the 550 – 600 nm region (ε ~ 20 M cm , forbidden

* 59 nN→π transition). Electrospray ionization mass spectrometry (ESI-MS) of the reaction mixture revealed a new species in ~ 80% yield with a mass increase of +29 Da compared to apo-M121H/H46E/F114PAz

~~(Figure 3.9C), further supporting S-nitrosylation of the protein.~~

Figure 3.9 UV-Vis absorption, Mass spectrometric and EPR spectroscopic characterization of S- nitrosylation in engineered red copper protein. (A) The UV-Vis spectra of Cu(II)-WTAz, Cu(II)- M121H/H46EAz and Cu(II)-M121H/H46E/F114PAz. (B) Kinetic UV-Vis spectra of 110 μM Cu(II)- M121H/H46E/F114PAz reacting with 1 equivalent DEA NONOate in 50 mM Mes buffer at pH 6. A spectrum was recorded every 10 s. (C) ESI-MS before (black) and after (red) NONOate addition. (D) X- band EPR spectra of Cu(II)-M121H/H46E/F114PAz reacting with different amounts of NO. Inset: EPR integration as a function of NO equivalents.

~~To further verify the S-nitrosylation of the protein, we performed high-resolution ESI-MS using~~

~~15NO as the reactant. As shown in Figure 3.10, the isotopic pattern was shifted to higher molecular weight compared to 14NO, confirming that NO was incorporated into the protein.~~

~~B A RedAz 100 100 Az-14NO SNO Az-15NO 80 80~~

~~60 60~~

~~40 40~~

~~Intensity (%) Intensity Intensity (%)~~

~~20 20~~

~~0 0 926 927 928 929 930 928.4 928.6 928.8 929.0 929.2 929.4 929.6 929.8 M/z (Da) M/z (Da)~~

Figure 3.10 High-resolution mass spectrometric study of the S-nitrosylation reaction in engineered azurin. (A) High-resolution ESI-MS before (black) and after (blue) NONOate addition. The peak with m/z = 15 was chosen because it displayed the strongest intensity. After reacting with NO, the peak was shifted from 927.0588 Da to 928.9903 Da (corresponding to + 28.9725 Da overall shift in averaged whole protein molecular weight), consistent with the observation in low-resolution ESI-MS of S-nitrosylation with + 29 Da. (B) The comparison of isotopic pattern after Cu(II)-M121H/H46E/F114PAz reacting with 14NO (blue) and 15NO (red). After using 15NO, the isotopic pattern was shifted to higher molecular weight compared to 14NO, confirming that NO was incorporated onto protein.

~~To further probe the S-nitrosylation process in Cu(II)-M121H/H46E/F114PAz, we collected its~~

EPR spectra in the presence of different equivalents of NO under anaerobic conditions. The spectra showed decreasing signal intensity with increasing concentration of NO, indicating the formation of an

~~EPR silent species (Figure 3.9D). Since S-nitrosylation requires a formal one-electron oxidation of NO, the nearby Cu(II) is likely the oxidant, thus forming an EPR silent Cu(I)-(RSNO) product.~~

~~To obtain pseudo-first-order rate constant of this process, we measured the reaction between 50~~

µM of Cu(II)-M121H/H46E/F114PAz and NO in an NO-saturated buffer using a stopped-flow spectrometer (Figure 3.11A). The LMCT band at 390 nm completely vanished within 10 ms, with a pseudo-first-order rate constant of 293 s-1. To slow down the reaction and obtain more data points for accurate kinetic fitting, we repeated this reaction in the presence of 100 µM NO (Figure 3.11B) and obtained a second-order rate constant of 6.7 × 105 M-1s-1.

~~A 0.08 0.06~~

~~0.06~~

~~0.04 k = 293 s-1~~

~~0.04~~

~~0.02~~

~~Absorbance (Au) 0.02 Absorbance at 390 nm (Au) 0.00 400 500 600 700 800 0.01 0.02 0.03 0.04 Wavelength (nm) Time (s)~~

~~B 0.12 0.12~~

~~0.08~~

~~0.08 k = 6.7 * 105 M-1s-1~~

~~0.04 0.04~~

~~Absorbance (Au) Absorbance390 nm (Au) at~~

~~400 500 600 700 800 0.00 0.05 0.10 0.15 Wavelength (nm) Time (s)~~

Figure 3.11 Stopped-flow kinetics of Cu(II)-M121H/H46E/F114P directly reacting with nitric oxide in 50 mM MES buffer pH6.0. (A) Two-syringe mixing was employed to mix equal volumes of 50 μM Cu(II)-M121H/H46E/F114PAz solution with 2 mM NO in 50 mM MES pH6.0 buffer (NO saturated). (B) Two-syringe mixing was employed to mix equal volumes of 70 μM Cu(II)-M121H/H46E/F114PAz solution with 0.2 mM NO in 50 mM MES pH6.0 buffer (diluted from NO saturated buffer). All reported data sets originally consisted of 200 spectra collected over 0.2 s. The integration period and minimum sampling period were both 1 ms. The spectra took at 1 ms are shown in blue and the spectra took at 200 ms are shown in red. The fitting of absorbance changing at 390 nm in pseudo first order reaction kinetics are shown on the right.

~~Interestingly, when the freshly formed Cu(I)-S(Cys)NO species was exposed to the air at room temperature, the original LMCT band at 390 nm was recovered with a yield of ~ 66% after three hours~~

(Figure 3.12), indicating that the S-nitrosylation is reversible. Partial recovery of the LMCT band was also consistently observed when applying vacuum to the solution containing Cu(I)-S(Cys)NO species

~~(Figure 3.13). The slow recovery rate and low recovery ratio under air or in vacuo indicate that the reaction equilibrium highly favors the formation of S-nitrosylation product.~~

Figure 3.12 The reversibility of Cu(I)-SNO species formation towards air exposure. (A) UV-vis spectra of exposing Cu(I)-SNO species to air. Inset: the time-dependent recovery of Cu(II)- M121H/H46E/F114PAz upon air exposure. (B) The comparison between Cu(II)-M121H/H46E/F114PAz (black), Cu(I)-SNO species (red) and the recovery of Cu(II)-M121H/H46E/F114P upon air exposure after Cu(I)-SNO formation (blue).

Figure 3.13 The reversibility of Cu(I)-SNO species formation towards vacuum. UV-vis spectra of Cu(II)-M121H/H46E/F114PAz (black), the adduct of Cu(II)-M121H/H46E/F114PAz with nitric oxide (red) and adduct of Cu(II)-M121H/H46E/F114PAz with nitric oxide after vacuuming at room temperature for 20 min (blue) in 100 mM MES pH 6.0 buffer.

~~To find out what other products were formed in this reaction, we investigated the reaction of the~~

S-nitrosylated protein with O2 using ESI-MS and EPR (Figure 3.14). A peak with +32 Da shift was observed in ESI-MS (Figure 3.14A), suggesting that the protein was oxidized after air exposure. The MS shift is consistent with the oxidation of cysteine (Cys-SH) to cysteine sulfinic acid (Cys-SO2H). Based on

~~ESI-MS result, ~ 30% of the cysteine was oxidized, consistent with 66% LMCT recovery after air~~

~~74 exposure. At the same time, a new type 2 copper signal (accounting for ~ 40% of overall Cu concentration) was observed in EPR, which displayed g values of 2.298, 2.108 and 2.037 with A// = 159 ×~~

10-4 cm-1 (Figure 3.14B). We propose that Cys112 is partially oxidized by the ROS generated from the reaction between reduced Cu(I) and O2, resulting the loss of LMCT and the formation of a new Cu(II) species.

~~Cu(I)-SNO exposed to air for 24 h Cu(II)-M121H/H46E/F114PAz new type 2 copper species~~

~~2400 2800 3200 3600 Magetic field (G)~~

Figure 3.14 ESI-MS and EPR investigation of S-nitrosylated protein with O2. (A) ESI-MS of S- nitrosylated protein after air exposure for 24 h. A peak with +32 Da shift was observed, suggesting that the protein was oxidized after air exposure. The MS shift is consistent with the oxidation of cysteine (Cys-SH) to cysteine sulfinic acid (Cys-SO2H). Based on ESI-MS result, ~ 30% of the cysteine was oxidized, agreeing with 66% LMCT recovery after air exposure (Figure 3.12). (B) EPR spectrum of the S-nitrosylated protein exposed to the air. Two sets of copper signals were observed. One signal was from Cu(II)-M121H/H46E/F114PAz as expected and the other one was from a typical type 2 copper (accounting for ~ 40% of overall Cu concentration), which displays g values of 2.298, 2.108 and 2.037 -4 -1 with A// = 159 × 10 cm .

~~3.3.4 X-ray absorption spectroscopic studies of S-nitrosylation~~

Initial attempts to characterize the NO-treated Cu(II)-M121H/H46E/F114PAz sample with resonance Raman failed, probably due to the photosensitivity of the Cu-RSNO species, as S-nitrosothiols are known to be photosensitive.60 Therefore, to further confirm the oxidation state of the copper site after

~~S-nitrosylation and obtain information on the ligand environment of the site, we collected Cu K-edge~~

~~XAS of Cu(II)-M121H/H46E/F114PAz, Cu(I)-M121H/H46E/F114PAz, and NO-treated Cu(II)-~~

~~M121H/H46E/F114PAz. To ensure the observed Cu(I) feature is not due to Cu(II) reduction upon X-ray~~

~~75 irradiation, only one XAS spectrum was taken at each spot. As shown in Figure 3.15A, an intense edge feature characteristic of Cu(I) was observed in the NO-treated Cu(II)-M121H/H46E/F114PAz sample~~

~~(which was also observed in an authentic sample of Cu(I)-M121H/H46E/F114PAz prepared by adding~~

Cu(I) to the apo-protein), indicating that the metal center was reduced to Cu(I) upon NO addition, consistent with the lack of an EPR spectrum (Figure 3.9D). Furthermore, an XAS edge energy of 8981.29 eV (value of the peak in the first derivative of the XAS) was detected in the NO-treated Cu(II)-

~~M121H/H46E/F114PAz sample, which was distinctly different from the edge energy of Cu(I)-~~

~~M121H/H46E/F114PAz at 8980.48 eV and Cu(II)-M121H/H46E/F114PAz at 8981.54 eV (Figure 3.15B).~~

~~A B 8980.48 8981.29~~

~~1.0 0.006~~

~~(E) 0.8 8981.54  0.004~~

~~0.6~~

~~derivatives 0.002 st 0.4 1~~

~~normalized x~~

~~0.000 0.2~~

~~8960 8980 9000 9020 8972 8974 8976 8978 8980 8982 8984 8986 Energy (eV) Energy (eV)~~

Figure 3.15 XAS investigation of S-nitrosylation process in engineered red copper protein. (A) XANES of Cu(II)-M121H/H46E/F114PAz (red), Cu(I)-M121H/H46E/F114PAz (blue) and the adduct of Cu(II)-M121H/H46E/F114PAz with NO (green). (B) First derivatives of XANES showing the Cu XAS k-edges of Cu(II)-M121H/H46E/F114PAz (red), Cu(I)-M121H/H46E/F114PAz (blue) and the adduct of Cu(II)-M121H/H46E/F114PAz with NO (green).

~~The results of EXAFS fitting and corresponding Fourier transform are shown in Figure 3.16 and~~

~~Table 3.3. The initial values for ligands and their distances were adopted from the crystal structure of~~

Cu(II)-M121H/H46EAz. Having two His ligands at different distances significantly improved the fittings for Cu(II)-M121H/H46E/F114PAz and Cu(I)-M121H/H46E/F114PAz, consistent with the observation that the two His residues are not equivalent in the crystal structure of Cu(II)-M121H/H46EAz (Figure

~~3.2C). The Cu-S distances of 2.21 Å and 2.18 Å were observed in Cu(II)-M121H/H46E/F114PAz (Figure~~

~~3.16A) and Cu(I)-M121H/H64E/F114PAz (Figure 3.16B) respectively. However, the NO treated Cu(II)-~~

~~M121H/H46E/F114PAz was best fitted when 70% of the Cys ligand was moved to 3.98 Å and a new N~~

76 ligand was added at 2.83 Å (Figure 3.16C), suggesting that the weak interaction between Cu(I) ion and S- nitrosothiol is through the N-atom rather than the S-atom. Inclusion of O ligands in the second and third shells from Glu, NO and a presumed water molecule was used to improve the fitting. Only first shell ligands used for EXAFS fittings are shown in Table 3.3.

Figure 3.16 The Fourier transform and fitting of EXAFS data. Fourier transform and EXAFS (inset) for the (A) Cu(II)-M121H/H46E/F114PAz, (B) Cu(I)-M121H/H46E/F114PAz and (C) NO treated Cu(II)- M121H/H46E/F114PAz. Experimental data are shown as solid black lines, and the simulation is shown as red lines. The first shell ligands and distances used for EXAFS fittings are shown on right. Copper atoms are green; oxygen atoms are red; sulfur atoms are yellow; nitrogen atoms are blue; carbon atoms are cyan.

~~Figure 3.16 (cont.)~~

~~Table 3.3 EXAFS fitting parameters.~~

Sample/fit FI Cu-S (Cys) Cu-N(His) Cu-O(Asp) Cu-N (SNO) N R(Å) DW(Å2) N R(Å) DW(Å2) N R(Å) DW(Å2) N R(Å) DW(Å2) Cu(II)- 0.16 1 2.21 0.006 1 1.89 0.008 1 1.95 0.003 N. A. M121H/H46 (e-3) 1 2.12 0.019 E/F114PAz Cu(I)- 0.15 1 2.18 0.008 1 1.99 0.005 1 2.09 0.030 N. A. M121H/H46 (e-3) 1 2.47 0.003 E/F114PAz The adduct 0.32 0.7 3.98 0.012 2 1.96 0.010 1 2.47 0.024 1 2.83 0.004 of Cu(II)- (e-3) 0.3 2.60 0.013 M121H/H46 E/F114PAz with NO

~~3.3.5 DFT calculations and mechanism of S-nitrosylation~~

Density functional theory calculations (B3LYP/TZVP) were performed to understand the mechanism of the cysteine nitrosylation using a model of the red copper site based on the crystal structure of Cu(II)-M121H/H46EAz. Two local minima were located for the optimized Cu(II) site that differ in their core geometry and the orientation of the sulfur p orbital interacting with the Cu dx2-y2. The lowest energy isomer has an Sσ ground state characteristic of red copper (Figure 3.17A) that is observed

49,61 experimentally by the intense observed Sσ → Cu LMCT at ~390 nm. A second isomer with a blue copper Sπ ground state (Figure 3.17B) is higher in energy (ΔE = +2.1 kcal/mol). Similar to the red copper

~~49 protein nitrosocyanin, the Sσ ground state arises from two geometric effects: (1) rotation of the S(Cys)-~~

~~Cβ bond into the Cu equatorial plane, which orients the Sσ orbital into Cu dx2-y2, and (2) the open His-Cu-~~

His angle (138°) which orients dx2-y2 to overlap with Sσ. Water coordination to give five-coordinate Cu(II) was calculated to be uphill (ΔG° = +6.6 kcal/mol), consistent with the crystallography that shows the localized water adjacent to Cu(II) does not form a strong Cu-OH2 interaction.

~~Figure 3.17 Ground state wavefunctions of optimized Cu(II) M121H/H46E azurin of the (A) red copper isomer (Sσ ground state), and (B) blue copper isomer (Sσ ground state, ΔE = +2.1 kcal/mol).~~

~~Two possible mechanisms for S-NO bond formation were evaluated. In the first mechanism~~

(pathway 1 in Figure 3.18), coordination of NO to Cu(II) affords a singlet five-coordinate {CuNO}10 species, which is best described as Cu(I)/NO+ due to the minor Cu character in the two unoccupied NO π* orbitals (< 20%, Figure 3.19). The Cu-NO species was not stable on the triplet spin surface; optimization invariably led to NO• dissociation. Although NO coordination to Cu(II) is calculated to be unfavorable

~~(ΔG° = +12.8 kcal/mol to singlet Cu-NO), the resulting intramolecular electrophilic attack of NO+ on~~

~~S(Cys) occurs with a low additional barrier (ΔG‡ = +5.5 kcal/mol) to give a total reaction barrier of ΔG‡ =~~

+18.3 kcal/mol with respect to separated Cu(II) + NO reactants. The product of this electrophilic NO+ attack is η2-(Cys)S-NO, in which the nitrosocysteine is coordinated side-on to Cu(I) through both N and S atoms. However, this complex is thermodynamically unstable with respect to loss of the coordinated

~~S(Cys)-NO ligand (ΔG° = -16.6 kcal/mol) to afford a trigonal Cu(I) site (consistent with the Cu(I) K-edge,~~

Figure 3.15) and free S(Cys)-NO. Alternative η1 binding modes of S(Cys)-NO to Cu(I) were also calculated to be more stable than the η2 isomer (by ΔG° = -7.4 kcal/mol for the N-bound isomer and -7.8 kcal/mol for the S-bound isomer); however, these isomers are still ~10 kcal/mol higher in energy than

Cu(I) + free S(Cys)-NO (Figure 3.18, bottom right). DFT calculations thus predict loss of the (S(Cys)-NO ligand upon S(Cys) nitrosylation, consistent with EXAFS data that show very weak coordination of the nitrosocysteine product to Cu(I). Each of the optimized isomers with coordinated S(Cys)-NO feature short

~~Cu-N or Cu-S bonds (Cu-N = 2.15 Å and Cu-S = 2.36 Å for the η2-SNO isomer; Cu-N = 2.08 Å for SNO-~~

~~κN; Cu-S = 2.37 Å for SNO-κS) that are not observed by EXAFS.~~

Figure 3.18 Electrophilic and radical pathways for (Cys)S-NO bond formation and possible isomers of the Cu(I)/(Cys)S-NO product. All energies are reported as free energies (ΔG and ΔG‡) at 298 K relative to the Cu(II) starting complex and free NO.

~~Figure 3.19 Frontier molecular orbitals of the {CuNO}10 complex. The minor contribution of Cu to the two unoccupied NO π* orbitals (3% and 17%) suggests the best description is Cu(I) and NO+.~~

Figure 3.20 Frontier α molecular orbitals at several points along the direct SCys-NO reaction coordinate, showing the rotation of the NO π* hole (116α in starting complex) to overlap with the occupied Sπ (112α) and the evolution of the S-N σ and σ* orbitals (104α and 116α in final product). The unoccupied, perpendicular NO π* (115α) is shown for completion. Orbital energies are given in eV.

Figure 3.21 Frontier β molecular orbitals at several points along the direct SCys-NO reaction coordinate, showing the rotation of the occupied NO π* (114β in starting complex) to overlap with the Cu-Sπ hole (115β, which is polarized towards S along the coordinate) and the evolution of the S-N σ and σ* orbitals (104β and 116β in final product). The unoccupied, perpendicular NO π* (115β) is shown for completion. Orbital energies are given in eV.

~~An alternative mechanism was evaluated (Figure 3.18, pathway 2), in which NO reacts directly with S(Cys) to avoid formation of the unfavorable {CuNO}10 intermediate. In this mechanism, the high~~

~~Cu-S(Cys) covalency places significant spin density (0.35 e–) on the coordinated S in the ground state~~

Cu(II) site (Figure 3.17), providing a pathway for direct radical coupling of S(Cys) with NO. This radical coupling reaction was evaluated by scans of decreasing S∙∙∙NO distance on both the triplet and broken- symmetry (BS) singlet spin surfaces. The barrier is lowest on the BS singlet surface, proceeding with a barrier of ΔG‡ = +10.0 kcal/mol, significantly lower than the above electrophilic mechanism (ΔG‡ =

+18.3 kcal/mol) and indicating that it is the favored pathway for S-NO bond formation. The BS singlet transition state has significant spin polarization ( = 0.88) and a long S∙∙∙NO distance of 2.67 Å, indicative of an early transition state. Inspection of frontier molecular orbitals along this reaction coordinate (Figure 3.20 and Figure 3.21) and spin density plots (Figure 3.22) shows that as the NO approaches, the α-spin NO π* hole rotates to overlap with the occupied α Sπ orbital, and the β-spin Cu hole polarizes towards Sπ to overlap with the occupied β-spin NO π*, which results in a doubly occupied

~~S‒NO σ interaction and reduction to Cu(I). Further along the reaction coordinate (Figure 3.22), the BS~~

82 wavefunction converges to an unpolarized singlet ( = 0) at a S∙∙∙NO distance of ~2.3 Å, and optimizes to the S-bound product with (Cys)S‒NO = 1.90 Å. This then leads to the lowest energy isomer with S(Cys)-NO no longer bound to Cu(I) (Figure 3.18, lower right) as described above.

2 Figure 3.22 Spin polarization () and spin density plots along the SCys-NO radical coupling reaction coordinate, showing the rotation of the β-spin NO π* to overlap with the α-spin Sπ to form the S‒NO σ interaction. The transition state is indicated in red, and α and β spin density are shown in blue and green, respectively.

~~DFT calculations have thus identified the most plausible mechanism for S(Cys)-NO formation is the direct radical reaction of NO with S(Cys) (Pathway 2 in Figure 3.18) without formation of a~~

~~{CuNO}10 intermediate. Therefore, rather than oxidizing NO (to NO+) for an electrophilic attack on~~

S(Cys), the role of Cu(II) in this reaction is to activate S(Cys) for radical coupling with NO, a result of the high covalency of the Cu(II)-S(Cys) bond. The S-nitrosylation is calculated to be favorable (ΔG° = -11.0 kcal/mol) with a low barrier (ΔG‡ = +10.0 kcal/mol) consistent with the observed reaction kinetics (ΔG‡ =

~~5 -1 -1 +9.7 kcal/mol for k2 ~5 × 10 M s ). The back reaction (NO release) is calculated to have a barrier of~~

~~ΔG‡ = +21.0 kcal/mol (half-life ~5 min), indicating that S-nitrosylation is slowly reversible at room temperature consistent with the experimental observation.~~

~~3.3.6 Using the engineered red copper protein to regulate NO concentration under physiological conditions by S-nitrosylation~~

The high yield, fast kinetics, and preference of the S-nitrosylated product motivated us to test if the engineered red copper protein could compete with native enzymes for NO. NO is known to inhibit mitochondrial respiration by reacting with the heme-copper center in cytochrome oxidase.43 The effect of

Cu(II)-M121H/H46E/F114PAz on the NO inhibition of E. coli cytochrome bo3 oxidase activity was investigated by measuring O2 reduction rates via a Clark-type O2 electrode (Figure 3.23A). In the presence of 5 μM NO, the O2 reduction rate of 10 nM cytochrome bo3 oxidase decreased by 56% (from

~~O2 consumption rate of 79 μmol/min to 35 μmol/min, Figure 3.23B).~~

Figure 3.23 NO inhibition of E. coli cytochrome bo3 oxidase. (A) Time courses of O2 reduction catalyzed by 10 nM cytochrome bo3 oxidase, 100 μM ubiquinol-1 and 5 mM DTT in the absence of NO (black), in the presence of 1 μM (red), 2 μM (blue), 3 μM (pink), 4 μM (green) or 5 μM NO (navy). (B) O2 consumption rates derived from the slopes of linear regions in O2 concentration curves. From left to right: cytochrome bo3 oxidase only and cytochrome bo3 oxidase with 1 μM, 2 μM, 3 μM, 4 μM or 5 μM NO respectively.

~~The addition of Cu(II)-M121H/H46EF114PAz to the above solution rescued the NO inhibition through NO sequestration (Figure 3.24). 5 μM Cu(II)-M121H/H46E/F114PAz was able to rescue the~~

~~84 inhibition of 5 μM NO on 10 nM cytochrome bo3 oxidase by 29% (from O2 consumption rate of 35~~

~~μmol/min to 67 μmol/min, Figure 3.24 inset), recovering 85% activity of WT cytochrome bo3 oxidase.~~

Figure 3.24 Effect of Cu(II)-M121H/H46E/F114PAz on NO inhibition of E. coli cytochrome bo3 oxidase. Time courses of O2 reduction catalyzed by 10 nM cytochrome bo3 oxidase, 100 μM ubiquinol-1 and 5 mM DTT in the absence of NO (black), in the presence of 5 μM NO (red), 5 μM NO and 1 μM Cu(II)-M121H/H46E/F114PAz (blue), 5 μM NO and 2 μM Cu(II)-M121H/H46E/F114PAz (green), 5 μM NO and 3 μM Cu(II)-M121H/H46E/F114PAz (pink), 5 μM NO and 4 μM Cu(II)-M121H/H46E/F114PAz (navy), or 5 μM NO and 5 μM Cu(II)-M121H/H46E/F114PAz (violet). Inset: O2 consumption rates derived from the slopes of linear regions in O2 concentration curves. Column A: cytochrome bo3 oxidase only; column B: cytochrome bo3 oxidase with 5 μM NO; column C-G: cytochrome bo3 oxidase with 5 μM NO in the presence of 1 μM, 2 μM, 3 μM, 4 μM and 5 μM Cu(II)-M121H/H46E/F114PAz respectively.

The negative control experiments showed that Cu(II)-WTAz and Cu(II)-M121H/H46/F114PAz had no effect on O2 reduction rates of cytochrome bo3 oxidase in the absence of NO and Cu(II)-WTAz had no effect on O2 reduction rates of cytochrome bo3 oxidase in the presence of NO (Figure 3.25). These results demonstrated that the engineered red copper protein is capable of competing with native enzymes for NO binding.

~~) 250~~

~~mmol~~



(

~~200~~

~~Cytochrome oxidase only~~

~~Concetration Cytochrome oxidase + Cu(II)-WTAz~~

~~2 Cytochrome oxidase + Cu(II)-M121H/H46E/F114PAz~~

~~O Cytochrome oxidase + NO Cytochrome oxidase + NO + Cu(II)-WTAz 150 100 150 200 Time (s)~~

Figure 3.25 Negative control of Cu(II)-M121H/H46E/F114PAz on NO inhibition of E. coli cytochrome bo3 oxidase. Time courses of O2 reduction catalyzed by 9.2 nM cytochrome bo3 oxidase, 100 μM ubiquinol-1 and 5 mM DTT in the absence of NO (black), in the presence of 5 μM Cu(II)-WTAz (red), 5 μM Cu(II)-M121H/H46E/F114PAz (blue), 5 μM NO (pink), 5 μM NO and 5 μM Cu(II)-WTAz (green).

~~3.4 Discussion~~

~~Metal ions, particularly Cu ions, are known to play important roles in NO-release regulation from~~

RSNOs.60,63 For example, Cu,Zn-superoxide dismutase catalyzed the decomposition of S- nitrosoglutathione (GSNO) in the presence of GSH.64 Cu(II) thiolate model complexes have been reported to react with S-nitrosothiols to generate NO possibly via a RSNO-κN copper complex suggested by DFT calculations.65 In addition, Cu(II)-doped polymers spontaneously generated NO from RSNOs, presumably via a Cu(I) intermediate.66 Selected RSNO-Cu(I) complexes have been investigated theoretically and found that coordination of the S atom to Cu(I) lengthens the S-N bond and concomitantly strengthens the

N-O bond, thus promoting decomposition.67 On the other hand, Cu(I) binding at N in the -SNO group is found to dramatically shorten and thus strengthen the S-N bond with a concomitant lengthening of the N-

~~O bond, suggesting stabilization of the RSNOs against NO release.68 However, the barrier for interconversion of Cu(I) binding from N to S is relatively small, which makes controlling the binding~~

86 modes between nitrosothiols and copper complexes difficult, and consequently Cu(I)-bound RSNO species are inherently unstable.68 Based on the EXAFS fittings, the Cu-S distance increases from 2.18 Å in Cu(I)-M121H/H46E/F114PAz to 3.98 Å in the corresponding S-nitrosylation species, while a new N ligand was observed at 2.83 Å from the copper center in the S-nitrosylation species. The results indicate that the Cu(I) ion weakly interacts with the N-atom instead of S-atom of the Cys112-SNO species. Such a structure arrangement is consistent with the DFT calculation results that the binding between Cu(I) and

~~Cys112-SNO is energetically uphill. The weak Cu(I)-RSNO interaction prevents the reverse NO-releasing reaction which requires a strong Cu-RSNO bond. Therefore, the weak interaction between Cu(I) and~~

Cys112-SNO species, together with interacting with N-atom rather than S-atom, provide reasonable explanations for the stability of the RSNO species formed in our system near a Cu(I) center, allowing us to observe direct S-nitrosylation in a copper protein for the first time.

Nitrosothiols have been known as one of the two possible naturally occurring forms for storage and delivery of NO in biological systems.59 In our engineered red copper protein, S-nitrosylation could proceed in a fast (k = 6.7 × 105 M-1s-1) and efficient (> 90% yield) way at room temperature at pH 7 even at micromolar level concentration of either protein or NO. The reaction could occur reversibly when NO could be consumed from the system by either vacuum or air. The reversibility of S-nitrosylation reaction together with the physiologically relevant conditions implies potential in vivo functions like NO delivery or regulation for such copper proteins. The results in Figure 3.24 showed that this S-nitrosylation reaction of our engineered red copper protein could be used to rescue NO-inhibition of cytochrome oxidase activity in the presence of micromolar level NO, demonstrating the possibility of NO concentration regulation by copper containing proteins under physiologically relevant conditions.

~~3.5 Summary~~

In summary, we have converted a blue copper center in WTAz into a red copper center that closely mimics that in nitrosocyanin by rational design of the primary coordination sphere (M121H/H46E) first, and then by tuning its reduction potential via deleting a hydrogen bond in the secondary

87 coordination sphere (F114P). The engineered red copper protein exhibits a significantly longer Cu-S(Cys) bond distance (~2.28 Å), lower reduction potential (~107 mV at pH 8) and larger hyperfine splitting in the parallel region (~160×10-4 cm-1) as compared to blue copper proteins, and the electronic properties of the engineered protein are comparable to those of the red copper center in nitrosocyanin. Stoichiometric titration of NO to Cu(II)-M121H/H46E/F114PAz yields nearly quantitative S-nitrosylation product with concomitant reduction of the metal center with a second order rate constant of ~ 105 M-1s-1. The resulting

S(Cys)-SNO containing protein shows a strong absorbance at 334 nm and a weak absorbance around 540 nm, similar to other reported RSNO species. Reduction of the Cu(II) site to Cu(I) during S-nitrosylation process is confirmed by a loss of over 90% of Cu(II) signal in EPR spectrum and a shift of the Cu K-edge to lower energy in XANES. Further EXAFS fitting reveals that the Cu-S distance increases from 2.18 Å in the reduced engineered red copper protein to 3.98 Å in the S-nitrosylation species, indicating Cu(I) interacts with N rather than S atom. DFT calculations have identified the most plausible mechanism for

S(Cys)-NO formation as the direct radical reaction of NO with Cu(II)-coordinated S(Cys) enabled by the high covalency of the Cu(II)-S(Cys) bond. Our results provide the first direct evidence of S-nitrosylation of copper-bound cysteine in a protein, and show that such a reaction can modulate solution NO concentration under physiologically relevant conditions to prevent NO inhibition of cytochrome oxidase activity.

~~3.6 References~~

~~1 Singel, D. J. & Stamler, J. S. Chemical physiology of blood flow regulation by red blood cells.~~

~~Annu. Rev. Physiol. 67, 99-145 (2004).~~

~~2 Xu, L., Eu, J. P., Meissner, G. & Stamler, J. S. Activation of the cardiac calcium release channel~~

~~(ryanodine receptor) by poly-s-nitrosylation. Science 279, 234-237 (1998).~~

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~~Chapter 4 A Novel Purple Cupredoxin from Nitrosopumilus maritimus Containing a Mononuclear~~

~~Type 1 Copper Center with an Open Binding SiteIII~~

~~4.1 Introduction~~

Cupredoxins are copper-containing proteins that exhibit redox properties important for electron transfer (ET) function in many important biological processes from photosynthesis to respiration.1,2 The most prominent member of this class of proteins is the type 1 copper (T1Cu) proteins, which contains a copper center coordinated by one Cys and two His in a trigonal plane and axial interactions above and sometimes below the plane.3-5 For several decades, numerous cupredoxins have been studied and almost all of them contain a coordinately saturated T1Cu center and perform ET function. The only exception so far is nitrosocyanin, which is in a cupredoxin scaffold but contains an additional water ligand.6 However, spectroscopic and x-ray crystallographic studies suggested that nitrosocyanin contains a type 2 copper

~~(T2Cu) center, similar to many copper enzymes.7~~

With modern techniques for the isolation of microbes and genomic sequencing, previously unknown but important new organisms are being discovered. An example of such an organism is the archaeon Nitrosopumilus maritimus (N. mar.), which was discovered in a filter in the Seattle aquarium.8

This and similar strains have since been shown to exist throughout the oceans in the world.9-25 What is interesting about N. mar. is that it is missing the genes coding for cytochromes c, the most ubiquitous class of ET proteins, and appears to have replaced them by cupredoxin-like proteins. N. mar. has been shown to grow and oxidize ammonia even at the low ammonia concentrations found in the ocean.8,12 Therefore, it is a major player in converting dissolved ammonia in the oceans to nitrite, a critical step in the global nitrogen cycle.11,12,22,24

III Portions of this chapter are here reproduced with permission from Hosseinzadeh P.; Marshall, N. M.; Tian, S.; Hemp, J.; Mullen, T.; Gao, Y-G.; Nilges, M. J.; Robinson, H.; Gennis, R. B.; Lu, Y. J. Am. Chem. Soc. to be submitted. (P.H., N.M.M. and S.T. contributed equally to this work)

Given the important role that N. mar plays in the global nitrogen cycle, and its unusual replacement of heme-based ET proteins with copper-based counterparts, isolating, purifying and characterizing some of the cupredoxins from the N. mar would expand our under-standing of the interplay between heme-based and cupredoxin-based biochemical processes. Here we report the recombinant expression, purification and characterization of a small cupredoxin protein from N. mar., called Nmar_1307 hereafter. Spectroscopic and x-ray crystallographic studies show that the Nmar_1307 contains a T1Cu center that is coordinatively unsaturated. This open binding site prompted us to investigate its reactivity towards small substrates like

- NO, which lead to the discovery of reversible NO oxidation/NO2 reduction activity by the protein, suggesting a possible role for the protein in the ammonia oxidation pathway or in detoxification of byproducts of such pathway in N. mar.

~~4.2 Materials and methods~~

~~4.2.1 Cell culture and expression of Nmar_1307~~

The protein coding DNA sequence of Nmar_1307 was cloned into the pET-22b periplasmic expression vector (Novagen) from genomic DNA. Nmar_1307 natively has an N-terminal helix that anchors the protein to the cell membrane. These membrane associated helices were omitted from the cloned sequence here in order to result in solubly expressed protein.

Rosetta strain E. coli (Novagen) were then transformed with the cloned sequences and cell stocks were made. This strain of E. coli was used because the codon usage of N. mar is very different from E. coli and Rosetta cells have extra translational machinery and tRNAs to compensate for this. Transformed cells were then used to streak LB plates with 100 mg/L ampicillin. These plates were incubated at 37°C overnight to obtain single colonies. Single colonies were then used to inoculate 5 mL LB cultures with 100 μg/mL of ampicillin, which were then incubated at 37°C until an OD600 of 0.6-0.8 was achieved. A 2 L portion of

2xYT media was then inoculated with 1 mL of the 5 mL culture and 100 mg/L of ampicillin. The 2 L cultures were then incubated at 37°C overnight, typically reaching an OD600 of around 1.5 after 18 hours.

In the morning, cells were induced with 100 mg/L of Isopropyl β-D-1-thiogalactopyranoside (IPTG) and allowed to express protein at 37°C for an additional 4 hours. After expressing for 4 hours, proteins were extracted via the osmotic shock procedure outlined below.

~~4.2.2 Osmotic shock procedure for periplasmically expressed Nmar_1307~~

The volumes in the procedure outlined below were scaled based on the volume of the original culture. After harvesting the E. coli by centrifugation, the cells were re-suspended in 1/8 the volume of the original culture of a solution containing 20 % w/v sucrose, 50 mM tris HCl buffer at pH 8.0, and 5 mM ethylenediaminetetraacetate (EDTA). The cells were incubated in this solution at room temperature for 1 hour and re-harvested. The supernatant was discarded and the cells were re-suspended in 1/8 the volume of the original culture of a solution with 4 mM NaCl and 1 mM dithiothreitol (DTT) to lyse the periplasmic membrane. Cells were left in this solution for 10 minutes at 4°C to effectively lyse the periplamsic membrane. Solids were then removed by centrifuging and the supernantant was collected. The pH of the supernatant was then lowered to 4.0 by slowly adding a solution of 500 mM sodium acetate at pH 4.0 (1/10 the volume of the supernatant). Lowering the pH in this way caused more solids to precipitate, which were again removed by centrifuging and the supernatant was collected.

~~4.2.3 Column purification of Nmar_1307 and reconstitution with copper~~

After extracting the protein from the cells by the osmotic shock procedure above, the pH of the N. mar_1307 supernatant solution was raised to 6.0 by slow addition of aqueous NaOH. The supernatant was then applied to a FF Q-sepharose column (50 mL, GE Healthcare) equilibrated in 50 mM sodium acetate buffer at pH 6.0 by adding the beads directly to the supernatant and letting it shake on an orbital shaker at

~~4°C overnight. The protein was eluted from the column by a NaCl gradient on an Akta basic FPLC (GE~~

~~Healthcare). The desired protein typically eluted at around 300 mM NaCl.~~

N. mar._1307 was then reconstituted with copper by adding a slight excess of CuSO4 to the partially purified protein solution. Full incorporation took several hours to complete. It was believed that the slow

incorporation of copper compared to some other cupredoxin proteins may have been due to binding of another exogenous metal in the metal binding site, but first dialyzing the protein versus an excess of EDTA to remove exogenous metal did not increase the copper incorporation rate. The increase in absorbances at

412 and 560 nm were monitored to estimate copper incorporation. Full copper incorporation was assumed to be the point at which no further increase in the visible absorbances was seen upon addition of more copper. Copper incorporated protein was then separated from non-copper bound protein and other contaminants by re-applying the protein to a Q-sepharose column and re-eluting it with a NaCl gradient.

~~The purity of the final protein solution was assessed with SDS-Page and electrospray ionization mass spectrometry (ESI-MS).~~

~~4.2.4 Electrochemical characterization of Cu(II)-Nmar_1307~~

The redox potential of the copper site in Nmar_1307 was measured by a modified protein film voltammetry (PFV) techniques reported previously.26 Pyrolitic graphite edge electrodes were modified by addition of 10mM DDAB to the surface. After the DDAB film was dried, the electrode was rinsed and dried and the protein was added to the surface. The CV values were recorded at scan rates from 200 mV/s in 100 mM Tris buffer pH 8 with 100 mM NaCl.

~~4.2.5 Spectroscopic characterization of Cu(II)-Nmar_1307~~

ESI-MS was collected on a Waters Quattro II Tandem Quadrupole/Hexapole/Quadrupole instrument by adding formic acid to samples to ionize. UV-visible spectra of N. mar._1307 were collected on a Carey 3E (Varian) in temperature independent (TIP) pH 7.0 buffer. For pH studies of the UV-visible absorbances, the protein was exchanged into mixed buffer consisting of 50 mM sodium phosphate, 50 mM sodium acetate, 40 mM MES, 40 mM MOPS, 40 mM CAPS and 100 mM NaCl at the various pH values.

The protein was brought to each pH by exchanging it into the proper buffer with a pd-10 size exclusion column (GE Healthcare). EPR spectra were taken at 30 K in TIP 7, or UB at different pH (4, 7 or 9) buffer as a glass with 20 % glycerol. Spin counting was done by comparing the total area under the signal of the

~~protein containing samples to standard samples of CuSO4 dissolved in water. Spectra were collected on a~~

~~Varian 122 spectrometer with an Air Products Helitran cryostat.~~

~~4.2.6 Crystallization of Cu(II)-Nmar_1307~~

Crystals of Cu(II)-Nmar_1307 were grown by the hanging drop vapor diffusion method. Protein solution was prepared in 50 mM sodium acetate buffer at pH 6.0 to a concentration of about 2 mM. A 2 μL portion of this protein solution was then mixed with 2 μL of a well buffer solution consisting of 0.1 M

~~TrisHCl at pH 8.0, 20mM CuSO4, 0.1M LiNO3 and varying amounts of polyethylene glycol (PEG). The highest quality crystals formed from wells with 35 % w/v PEG 4000 after about 2 months at 4°C.~~

~~4.2.7 Activity tests~~

~~Cu(II) loaded Nmar_1307 protein solution in 50 mM Tris pH 8.0 buffer was degassed in a Schlenk line and transferred to an anaerobic chamber (Coy Laboratories). 5 eq. of Proli NONOate solution (from a~~

~~50 mM stock solution) was added to 2000 μl 250 μM Cu(II)-Nmar1307 solution in 50 mM Tris pH8.0 buffer. The UV-vis spectra were monitored by an Agilent 8453 photodiode array spectrometer located~~

-1 -1 inside the chamber. The extinction coefficient at 560 nm (ε560nm = 1770 M cm ) was used to calculate the concentration of Cu(II)-Nmar1307 solution. The concentration of PROLI NONOate (Cayman Chemical,

~~-1 -1 MI) was determined by ε252nm = 8400 M cm .~~

~~EPR spectra were collected at 30 K on an X-band Varian E-122 spectrometer at the Illinois EPR~~

Research Center equipped with an Air Products Helitran Cryostat and EIP frequency counter. The EPR samples were prepared as described above except 0.8 mM protein concentration was used for better resolution. Glycerol was added to a final 20% v/v and the solution was quickly frozen in liquid nitrogen.

~~4.2.8 Griess assay~~

~~The Griess reagent kit for nitrite quantitation containing N-(1-naphthyl)ethylenediamine dihydrochloride, sulfanilic acid and nitrite standard solution was purchased from Life technologies and~~

~~stored refrigerated at 4 oC, protected from light. Sodium nitrite solutions with concentrations between 1-~~

~~100 μM were prepared by diluting the nitrite standard solution with Millipore water. Equal volumes of N-~~

~~(1-naphthyl)ethlyenediamine and sulfanilic acid were mixed to form the Griess reagent. A mixture of 10~~

μL of Griess reagent, 30 μL of the nitrite-containing sample and 260 μL of Millipore water were incubated for 30 minutes at room temperature. To obtain the standard curve, concentration of nitrite was plotted against the absorbance of the mixture at 548 nm, measured relative to the reference sample containing 10

~~μL Griess reagent and 290 μL Millipore water.~~

~~To investigate whether the protein produces nitrite, Cu(II)-Nmar_1307 was mixed with 5 eq. Proli~~

NONOate solution in 100 mM Tris pH8.0 according to the procedure described above. After the solution turned colorless, 30 μL of the solution was mixed with 10 μL of Griess reagent and 260 μL of Millipore water. In the meantime, a solution of only Proli NONOate in 100 mM Tris pH8.0 was used as a control which was also incubated with Griess reagent at room temperature for 30 min. The absorbance at 548 nm was then collected and nitrite concentrations determined by comparison of the sample absorbances to those from the standard plot.

~~4.3 Results and discussion~~

~~4.3.1 Recombinant expression of apo-Nmar_1307 in E. coli~~

We began the project by inspection of the amino acid sequence of the N. mar_1307, which suggested that it contains an N-terminal transmembrane helix (Figure 4.1). In order to obtain a water-soluble protein for bio-chemical and biophysical studies, we cloned the N. mar_1307 gene without this transmembrane helix. This soluble copper-containing domain was expressed in E. coli and purified by using two consecutive ion exchange chromatographic steps and then via a size exclusion chromatography (Figure

~~4.2). An electrospray ionization mass spectrometry of the purified protein showed a peak with a mass of~~

~~10,746±1 Da (Figure 4.3), which is within the experimental error of the calculated mass of copper-free apo-~~

~~100~~

~~Nmar_1307 (10,747 Da). No other major peak was observed in the mass range, indicating that Nmar_1307~~

~~was successfully expressed and purified in its copper-free (apo) form.~~

Figure 4.1 Structure-based sequence alignment of Nmar_1307 and a number of other cupredoxins proteins. The initial sequence shown in a red box is the membrane part that we excluded to express the protein in a soluble form.

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~~-35 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 0 20 40 60 80 100 120 ml~~

Figure 4.2 Purification of Nmar_1307 via Q-FF and SEC columns. (A) FPLC trace of metal free Nmar_1307 using Q-FF column. The blue line indicates absorption at 280 and the green line represents the concentration of buffers. Collected fractions are shown in red box. (B) After metal reconstitution, the holo- protein is loaded into a second Q-FF column. (C) the SEC trace of Cu(II)-Nmar_1307.

~~101~~

~~100 100 10746.017476~~

~~% %~~

~~0 0 500 750 1000 1250 1500 1750 2000 8000 10000 12000 14000 16000 M/Z mass~~

~~Figure 4.3 ESI-MS spectra of Cu(II)-Nmar_1307. The distribution of peaks with different m/z values (Left); the calculated mass of Nmar_1307 from the distributed m/z peaks (right).~~

~~4.3.2 Preparation and UV-vis, EPR characterization of Cu(II)-NMar_1307~~

Addition of Cu(II) to the apo-Nmar_1307 converted the colorless protein to one with a strong purple color with peaks at 413 nm and 558 nm with extinction coefficients of 1450 and 1770 M-1cm-1, respectively, as determined by ICP-MS (Figure 4.4, left). This UV-vis spec-trum is very similar to those of type 1 green copper proteins.27,28 To corroborate this conclusion, we collected EPR spectrum of the same

-4 -1 protein, which displays Az = 61.3 x 10 cm that is well within the range of those of typical type 1 copper proteins (< 70 x10-4 cm-1). Interestingly, the UV-visible and EPR spectra of the resulting protein are strongly dependent on the pH at which the Cu titration occurs (Figure 4.4, right and Table 4.1).

~~102~~

~~560 nm 0.06 412 nm~~

~~0.04~~

~~abs (AU) 0.02~~

~~Experimental data 0.00 Simulation~~

~~400 500 600 2000 2250 2500 2750 3000 3250 3500 3750 Wavelength (nm) magnetic field /Gauss~~

~~Figure 4.4 UV-vis (left) and EPR (right) spectra of Cu(II)-Nmar_1307 in 50 mM Tris buffer pH 8.0.~~

~~Table 4.1 EPR simulation parameters of Cu(II)-Nmar_1307.~~

~~species gx, gy, gz Ax, Ay, Az~~

~~(10-4 cm-1)~~

~~51.5 % species 1 2.0330, 67, 9.7, 61.3~~

~~2.0561,~~

~~2.2741~~

~~48.5% species 2 2.0567, 47.3, 0.3, 92.7~~

~~2.0493,~~

~~2.2659~~

~~4.3.3 X-ray crystallography study of Cu(II)-Nmar_1307~~

To obtain the geometry information of this novel copper site, we determined the three dimensional structure of Cu(II)-Nmar_1307 by X-ray crystallography (Figure 4.5, see Tables 4.2 and 4.3 for relevant crystallographic). The protein crystallized with 4 molecules in the asymmetric unit, with each of the four

~~103~~

molecules being the same except for different conformations of a few surface residues that display a high degree of freedom. As shown in Figure 4.5A, the Cu(II)-Nmar_1307 displays an over-all structure that is typical of the cupredoxin fold observed in T1Cu proteins. The Figure 4.5B indicates that the copper is coordinated to one Cys (Cu-S distance = 2.25 Å) and two His in a trigonal plane (Cu-N distances are 2.01

Å and 2.07 Å respectively) in a trigonal plane, which closely resemble those of other cupredoxin. However, based on sequence homology with other cupredoxin proteins, the axial ligand above the trigonal plane commonly found in other cupredoxins is occupied by Arg89 in the same position of Cu(II)-Nmar_1307.

The structure of Cu(II)-Nmar_1307, however, clearly shows that Arg89 rotates away from the copper creating an open coordination site (Figure 4.5B). The next closest possible axial ligands are the Oδ from

Asn11 or the Oη from Tyr20, both which are > 4 Å from the copper center and thus is much too long to be considered as an axial ligand. Instead, an extra electron density at 2.25 Å from the T1Cu can be assigned as a water molecule within the Cu coordination sphere. This water is 2.54 Å and 2.97 Å away from potential hydrogen bonding groups of Asn11 and Tyr20, respectively. Therefore, unlike most T1Cu proteins that contain a Met, Leu or Gln residue in the axial position above the trigonal plane, the copper site in

Nmar_1307 is occupied by a water ligand. An opening binding site with water was also observed in nitrosocya-nin. However, the Cu-S distance (2.29 Å) in nitrosocyanin6 is longer than the normal Cu-S distance (~ 2.1 Å) in T1Cu center centers,3 making it a type 2 copper (T2Cu) center. Therefore, the T1Cu center in Cu(II)-Nmar_1307 observed here is the first T1Cu center with an open-binding site containing a water ligand.

~~104~~

~~A B~~

~~Tyr20~~

~~Arg89~~

~~Water Cys83 Ans11~~

~~His86 His43~~

Figure 4.5 Crystal structure of Cu(II)-Nmar_1307. (A) Full structure with the backbone shown in ribbon representation and the primary copper ligands in stick. (B) Zoom in of the copper binding site with nearby amino acids shown in ball-and-stick representation. The coordinating water is shown as a red ball.

On the other side of the trigonal plane, a backbone carbonyl oxygen atom from Pro42 is located near the copper center as in some cupredoxins, such as azurin, where computational work has shown that there is a strong ionic interaction between the copper and the dipole from the oxygen.29 However, the distance between the oxygen and copper in Cu(II)-Nmar_1307 is more than 4 Å, suggesting that any ionic contribution would be weak.

~~Table 4.2 Data collection and refinement statistics of Cu(II)-Nmar_1307 crystal.~~

Resolution range (Å) 45.87 - 1.6 (1.657 - 1.6) Space group P 41 21 2 Unit cell 68.887 68.887 184.45/ 90 90 90 Unique reflections 59603 (5817) Completeness (%) 99.97 (99.74) Mean I/sigma(I) 17.74 (5.31) Wilson B-factor 18.70 R-work 0.1703 (0.2032) R-free 0.1918 (0.2411) Number of non-hydrogen atoms 3609 Macromolecules 3012 Ligands 6 Water 591 Protein residues 384 RMS(bonds) 0.006 RMS(angles) 1.07

~~105~~

~~Table 4.2 (cont.) Ramachandran favored (%) 97 Ramachandran outliers (%) 0 Clashscore 2.57 Average B-factor 22.90 Macromolecules 20.80 Ligands 24.10 Solvent 33.40~~

~~Table 4.3 Relevant bond length near the Cu binding site of Cu(II)-Nmar_1307.~~

Atoms Chain A (Å) Chain B (Å) Chain C (Å) Chain D (Å) Average distance (Å) γ S Cys83-Cu 2.23 2.26 2.26 2.24 2.25 δ N His43-Cu 1.97 2.03 2.00 2.04 2.01 δ N His86-Cu 2.05 2.05 2.07 2.09 2.07 OPro42-Cu 4.24 4.29 4.21 4.28 4.25 δ O Asn11-Cu 3.95 3.90 3.95 3.88 3.92 δ O Asn11-Owater 2.56 2.54 2.51 2.56 2.54 η O Tyr20-Cu 4.92 4.77 4.85 4.84 4.85 η O Tyr20-Owater 3.05 2.96 2.95 2.92 2.97 Owater-Cu 2.24 2.21 2.26 2.27 2.25

~~4.3.4 Redox property of Cu(II)-Nmar_1307~~

The redox properties of the T1Cu center in Cu(II)-Nmar_1307 were investigated by cyclic voltammetry (CV). As shown in Figure 4.6, a current response that was dependent on protein was observed at 484±9 mV vs. standard hydrogen electrode (SHE) in 100 mM TrisHCl, pH 8.0 The unusually large separation between the oxidative and reductive peak in the CV spectra (100 mV) suggests a high reorganization energy associated with redox process which can be attributed to the loss of water in the reduced state. The same water coordination and loss thereof have been observed previously in nitrosocyanin.6

~~106~~

~~) 6~~



~~( 4~~

~~current 0~~

-2

~~-4 200 300 400 500 600 700 800 Potential (mV)~~

Figure 4.6 Cyclic voltammogram of Cu(II)-Nmar_1307 at 100 mM Tris pH 8.0 supplemented with 100 mM NaCl. The CV was obtained at scan rates of 200 mV/s. The red trace shows the peaks after subtraction of baseline capacitance.

~~4.3.5 Potential functions of Cu(II)-Nmar_1307.~~

The open coordination site and binding of a solvent molecule directly to the copper suggests that this protein could display an activity toward small molecules. Since the N. mar is known to play a role in

- the nitrogen cycle, the ability of Cu(II)-Nmar_1307 to oxidize NO to NO2 was tested. Exposure of 0.25 mM of Cu(II)-Nmar_1307 to 10 molar equivalents of NO at pH 8.0 resulted in slow bleaching of the visible absorption bands, suggesting reduction of the Cu(II) to Cu(I) (Figure 4.7A). This reduction is confirmed by an observed decrease of Cu(II) EPR signals upon reaction with NO (Figure 4.7B). In contrast, no reaction was observed between NO and wild type azurin as reported in literature.30 To find out about the product of

~~- the corresponding oxidation of NO, Griess assay was used to measure NO2 produced in the process (Figure~~

~~4.8).~~

~~107~~

~~A B~~

~~0.4 0.3 0.3 0.2~~

~~0.1~~

~~0.2 absorbance (AU) 0.0 Nmar1307 0 20 40 60 1min time (min) 2min 5min 0.1 30min~~

~~absorbance (AU)~~

~~400 600 800 1000 2400 2800 3200 3600 Magnetic Field (G) wavelength (nm)~~

Figure 4.7 Kinetic UV-vis and EPR spectra of Cu(II)-Nmar_1307 reacting with NO at pH8.0. (A) Time course of UV-vis spectra upon bleaching the LMCT bands of 250 μM Cu(II)-Nmar_1307 with 10 equivalents of NO in 50 mM Tris buffer pH8.0 under anaerobic condition. (B) X-band EPR spectra of 0.4 mM Cu(II)-N.mar1307 reacting with 10 equivalents of NO in 50 mM Tris pH8.0 collected at 30 K: Cu(II)- Nmar_1307 before reaction (black), 1 min (red), 2 min (blue), 5 min (pink) and 30 min (green) after reacting with NO.

~~108~~

~~A B 0.30 0.3 0 uM 0.25 20 uM 40 uM 0.20 0.2 60 uM 80 uM 0.15 100 uM 0.10~~

~~absorbance (Au) absorbance 0.1 Absorbance at 548 nm 548 at Absorbance 0.05~~

~~0.00 0.0 400 500 600 700 800 0 20 40 60 80 100 wavelength (nm) Nitrite concentration (mM) C~~

~~0.10~~

~~0.05 Nmar1307~~

~~absorbance (Au) absorbance control~~

~~0.00~~

~~400 600 800 wavelength (nm)~~

- Figure 4.8 NO2 detection by Griess assay after Cu(II)-Nmar1307 reacting with NO. (A) and (B) show - - standard curve of Griess reagent for NO2 production. (C) Production of NO2 in buffer control (27.9 μM) - compared with Cu(II)-Nmar_1307 (40.2 μM) after 30 min time. The background NO2 production in buffer is due to side reaction of NONOate.

The reaction with NO is highly pH dependent. Com-plete reduction of Cu(II) to Cu(I) by NO was observed only at pH > 7.0. At lower pH, the bleaching of the ligand-to-metal charge transfer (LMCT) bands is much slower (Figure 4.9). At pH 8.0, the midpoint potentials of the Cu(II)/Cu(I) (Em=484 mV) is much

~~- - higher than that of NO2 /NOaq (Em=200mV vs. SHE), making it possible for the Cu(II) oxidize NO to NO2 under this condition.~~

~~109~~

~~0.4~~

~~pH6~~

~~0.2 pH7~~

~~pH8 Absorbance at 560 nm 0.0 0 10 20 30 40 50~~

~~time (min)~~

~~Figure 4.9 The NO oxidation activity is highly pH-dependent. The graph shows the rate of decrease in LMCT band of Cys to Cu(II) upon reaction with NO at three different pH values.~~

- Since it is known that decreasing pH will increase the Em of the NO2 /NOaq redox couple by ~120 mV per pH unit, at lower pH such as pH 6.0, the redox reaction will favor the opposite direction of Cu(I)

~~- - being oxidized by NO2 . Indeed, upon treatment of reduced Cu(II)-Nmar_1307 with NO2 at pH 6.0, the~~

~~Cu(II) species formation was observed (Figure 4.10).~~

~~0.06 412 nm 560 nm 0.04 0.04~~

~~0.02 Absorbance (Au)~~

~~Absorbance (Au) Absorbance 0.00~~

~~0.00 400 600 800 1000 0 4000 8000 12000 Wavelength (nm) Time (s)~~

~~Figure 4.10 Kinetic UV-vis spectra (Left) and absorbance traces monitored at 412 and 560 nm (Right) of Cu(II)-Nmar_1307 reacting with NO at pH6.0.~~

~~110~~

~~4.4 Summary~~

A novel cupredoxin isolated from Nitrosopumilus maritimus, called Nmar_1307, was recombinantly expressed in E. coli and characterized with different spectroscopic methods. The protein displays a strong purple color due to strong absorptions around 413 nm (1450 M-1cm-1) and 558 nm (1770

-1 -1 -4 -1 M cm ) in the UV-vis electronic spectrum and a small hyperfine coupling constant (A|| < 100x10 cm ) in the EPR spectrum, typical of T1Cu center. X-ray crystal structure at 1.6 Å resolution con-firms that it contains T1Cu center with a Cu atom coordinated by two His and one Cys in a trigonal place and a short

~~Cu(II)-SCys bond (2.25 Å). In contrast to the coordinately saturated T1Cu center observed in other cupredoxins, the Nmar_1307 contains a unique T1Cu center with an open-binding site containing water.~~

Both UV-vis absorption and EPR spectroscopy studies suggest that the Nmar_1307 can oxidase NO to nitrite, whose activity is attributable to the unusually high reduction potential (484±9 mV vs. SHE) of the copper site. Since the NO oxidation activity of the Cu(II)-Nmar_1307 is slow and so far we have not been able to find conditions to make the protein perform turnovers, we are not uncertain whether the NO oxidation activity discovered here is relevant to its biological function in Nitrosopumilus maritimus.

~~Nonetheless, we have discovered Nmar_1307 as a novel cupredoxin that has an open binding site at the~~

T1Cu center. Out results suggest that T1Cu cupredoxins can have a wide range structural features, including an open-binding site containing water, making this class of proteins an even more versatile for their biological functions.

~~4.5 References~~

~~1 Lu, Y. in Comprehensive coordination chemistry II (eds Thomas J. McCleverty & Jon A. Meyer)~~

~~91-122 (Pergamon, 2003).~~

~~2 Liu, J. et al. Metalloproteins containing cytochrome, iron–sulfur, or copper redox centers. Chem.~~

~~Rev. 114, 4366-4469 (2014).~~

~~111~~

~~3 Gray, H. B., Malmstrom, B. G. & Williams, R. J. Copper coordination in blue proteins. J. Biol.~~

~~Inorg. Chem. 5, 551-559 (2000).~~

~~4 Randall, D. W., Gamelin, D. R., LaCroix, L. B. & Solomon, E. I. Electronic structure contributions~~

~~to electron transfer in blue cu and cua. J. Biol. Inorg. Chem. 5, 16-29 (2000).~~

~~5 Solomon, E. I., Szilagyi, R. K., DeBeer George, S. & Basumallick, L. Electronic structures of metal~~

~~sites in proteins and models: Contributions to function in blue copper proteins. Chem. Rev. 104,~~

~~419-458 (2004).~~

~~6 Lieberman, R. L., Arciero, D. M., Hooper, A. B. & Rosenzweig, A. C. Crystal structure of a novel~~

~~red copper protein from nitrosomonas europaea. Biochemistry 40, 5674-5681 (2001).~~

~~7 Arciero, D. M., Pierce, B. S., Hendrich, M. P. & Hooper, A. B. Nitrosocyanin, a red cupredoxin-~~

~~like protein from nitrosomonas europaea. Biochemistry 41, 1703-1709 (2002).~~

~~8 Konneke, M. et al. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437,~~

~~543-546 (2005).~~

~~9 Foesel, B. U. et al. Nitrosomonas NM143-like ammonia oxidizers and nitrospira marina-like nitrite~~

~~oxidizers dominate the nitrifier community in a marine aquaculture biofilm. FEMS Microbiol. Ecol.~~

~~63, 192-204 (2008).~~

~~10 Ingalls, A. E. et al. Quantifying archaeal community autotrophy in the mesopelagic ocean using~~

~~natural radiocarbon. Proc. Natl. Acad. Sci. U. S. A. 103, 6442-6447 (2006).~~

~~11 Labrenz, M. et al. Relevance of a crenarchaeotal subcluster related to candidatus nitrosopumilus~~

~~maritimus to ammonia oxidation in the suboxic zone of the central baltic sea. ISME J 4, 1496-1508~~

~~(2010).~~

~~12 Martens-Habbena, W., Berube, P. M., Urakawa, H., de la Torre, J. R. & Stahl, D. A. Ammonia~~

~~oxidation kinetics determine niche separation of nitrifying archaea and bacteria. Nature 461, 976-~~

~~979 (2009).~~

~~13 Martínez-García, M. et al. Ammonia-oxidizing crenarchaeota and nitrification inside the tissue of~~

~~a colonial ascidian. Environ. Microbiol. 10, 2991-3001 (2008).~~

~~112~~

~~14 Matsutani, N. et al. Enrichment of a novel marine ammonia-oxidizing archaeon obtained from sand~~

~~of an eelgrass zone. Microbes Environ. 26, 23-29 (2011).~~

~~15 Mincer, T. J. et al. Quantitative distribution of presumptive archaeal and bacterial nitrifiers in~~

~~monterey bay and the north pacific subtropical gyre. Environ. Microbiol. 9, 1162-1175 (2007).~~

~~16 Moin, N. S., Nelson, K. A., Bush, A. & Bernhard, A. E. Distribution and diversity of archaeal and~~

~~bacterial ammonia oxidizers in salt marsh sediments. Appl. Environ. Microbiol. 75, 7461-7468~~

~~(2009).~~

~~17 Nelson, K. A., Moin, N. S. & Bernhard, A. E. Archaeal diversity and the prevalence of~~

~~crenarchaeota in salt marsh sediments. Appl. Environ. Microbiol. 75, 4211-4215 (2009).~~

~~18 Porat, I. et al. Characterization of archaeal community in contaminated and uncontaminated surface~~

~~stream sediments. Microb Ecol 60, 784-795 (2010).~~

~~19 Quaiser, A., Zivanovic, Y., Moreira, D. & Lopez-Garcia, P. Comparative metagenomics of~~

~~bathypelagic plankton and bottom sediment from the sea of marmara. ISME J 5, 285-304 (2011).~~

~~20 Siboni, N., Ben-Dov, E., Sivan, A. & Kushmaro, A. Global distribution and diversity of coral-~~

~~associated archaea and their possible role in the coral holobiont nitrogen cycle. Environ. Microbiol.~~

~~10, 2979-2990 (2008).~~

~~21 Urakawa, H., Martens-Habbena, W. & Stahl, D. A. High abundance of ammonia-oxidizing archaea~~

~~in coastal waters, determined using a modified DNA extraction method. Appl. Environ. Microbiol.~~

~~76, 2129-2135 (2010).~~

~~22 Walker, C. B. et al. Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification~~

~~and autotrophy in globally distributed marine crenarchaea. Proc. Natl. Acad. Sci. U. S. A. 107,~~

~~8818-8823 (2010).~~

~~23 Woebken, D., Fuchs, B. M., Kuypers, M. M. M. & Amann, R. Potential interactions of particle-~~

~~associated anammox bacteria with bacterial and archaeal partners in the namibian upwelling system.~~

~~Appl. Environ. Microbiol. 73, 4648-4657 (2007).~~

~~113~~

~~24 You, J., Das, A., Dolan, E. M. & Hu, Z. Ammonia-oxidizing archaea involved in nitrogen removal.~~

~~Water Res. 43, 1801-1809 (2009).~~

~~25 Zeng, Y., Li, H. & Jiao, N. Phylogenetic diversity of planktonic archaea in the estuarine region of~~

~~east china sea. Microbiol. Res. 162, 26-36 (2007).~~

~~26 Hwang, H. J., Ang, M. & Lu, Y. Determination of reduction potential of an engineered cua azurin~~

~~by cyclic voltammetry and spectrochemical titrations. J. Biol. Inorg. Chem. 9, 489-494 (2004).~~

~~27 Solomon, E. I. Spectroscopic methods in bioinorganic chemistry: Blue to green to red copper sites.~~

~~Inorg. Chem. 45, 8012-8025 (2006).~~

~~28 Solomon, E. I. et al. Copper active sites in biology. Chem. Rev. 114, 3659-3853 (2014).~~

~~29 Nar, H., Messerschmidt, A., Huber, R., van de Kamp, M. & Canters, G. W. Crystal structure~~

~~analysis of oxidized pseudomonas aeruginosa azurin at ph 5·5 and ph 9·0. J. Mol. Biol. 221, 765-~~

~~772 (1991).~~

~~30 Gorren, A. C. F., de Boer, E. & Wever, R. The reaction of nitric oxide with copper proteins and the~~

~~photodissociation of copper-no complexes. Biochim. Biophys. Acta 916, 38-47 (1987).~~

~~114~~

~~7 8 9 Chapter 5 From {FeNO} to {Fe(NO)2} to {Fe(NO)2} : Stepwise Nitrosylation of the Biological Nonheme Iron Proteins Observed in an Engineered Mutant of Pseudomonas aeruginosa Azurin~~

~~5.1 Introduction~~

Nitric oxide (NO) plays important roles in a variety of biological processes, such as neurotransmission,1 transcriptional regulation,2-6 cytotoxicity,7,8 immune response signaling,9 and blood pressure regulation.10 Metalloproteins are the primary targets for NO inside of cells.11-14 Dinitrosyl iron

9 15 complexes (DNICs) having a {Fe(NO)2} core given by Enemark-Feltham notation, formed by non heme iron proteins directly reacting with NO, have been detected in a vast array of animal tissues and cell cultures.12,14,16,17 Interestingly, if we consider the situation of a mono-nuclear non heme iron protein reacting

~~7 8 with two molecules of NO, an {FeNO} species should be formed at first and then {Fe(NO)2} species after~~

9 coordinating the other NO. The {Fe(NO)2} species is finally formed by one electron reduction. The origin of the additional electron on the iron atom has not been explicitly identified, but it is suggested that a third

NO serves as an electron donor in a one-electron reduction.14 Although considerable efforts have been made to develop molecular models of biologically prevalent nitrosyl iron complexes,14,18-23 either {FeNO}7 or

9 {Fe(NO)2} complexes were isolated and there is no report of capturing all three species in identical host complexes. Such an achievement not only allows us to elucidate changes to the structure of the iron center during stepwise nitrosylation, but also clarify the factors that control their chemical reactivities.

8 Furthermore, {Fe(NO)2} species has been proposed as an intermediate during the catalysis of nitric oxide reductases (NORs) from denitrifying bacteria which catalyze the two-electron reduction of NO to

~~24,25 nitrous oxide (N2O), an essential step on the biological denitrification pathway. Three possible mechanisms of NO reduction have been proposed: trans, cis-FeB and cis-heme b3 mechanisms (Figure~~

~~5.1).26 Several experimental observations support a trans-mechanism, including the characterization of an~~

27 (FeCO)2 heme/non-heme dicarbonyl complex at the active site of B. a. qCuANOR. Rapid freeze quench samples from single turnover reactions in cNOR have revealed EPR signals consistent with the trapping of

~~S = 1/2 low-spin heme {FeNO}7 and S = 3/2 non-heme {FeNO}7 species.28 However, in many heme~~

~~115~~

29-31 containing proteins, ferrous-ntirosyl complexes are unreactive species. The cis-FeB mechanism has been strongly favored by Thomson and coworkers because it does not involve the formation of a heme b

{FeNO}7 species, which they view as a potential dead-end product.32-35 In this mechanism model, the role of the heme iron would be limited to electron transfer, although interactions with the putative hyponitrite complex may participate in the N-O bond cleavage. To the best of our knowledge, such a proposed

~~8 {Fe(NO)2} intermediate has never been observed in neither native enzymes nor model complexes.~~

Figure 5.1 Schematic representation of the trans, cis-heme b3, and cis-FeB mechanisms. In the proposed cis-heme b3 mechanism, after one NO is bound to the heme b3 site, a free NO electrophilically attacks this heme bound NO. In the cis FeB mechanism, two molecules of NO bind to the FeB site, whereas the trans mechanism suggests one NO binds to heme b3 and the FeB site simultaneously for the N-N bond formation to occur.

~~7 8 In this chapter, the first observation of stepwise nitrosylation from {FeNO} to {Fe(NO)2} to~~

~~9 {Fe(NO)2} in an engineered non heme iron site in blue copper protein azurin was reported. The one-~~

~~8 9 electron reduction of {Fe(NO)2} to {Fe(NO)2} species is achieved by adding either excess amount of NO~~

8 or dithionite as a reductant. Our results not only elucidate the electronic structure of {Fe(NO)2} species and serve as spectroscopic references, but also enhance our knowledge about fundamental reaction mechanisms of endogenous NO.

~~116~~

~~5.2 Materials and methods~~

~~5.2.1 Materials and chemicals~~

~~(NH4)2Fe(SO4)2, Na2IrCl6, dithionite and chelex-100 sodium form were purchased from Sigma-~~

Aldrich Chemical Co. and used without further purification. All the primers for site-directed mutagenesis were purchased from IDT Inc. and used without further purification. DEA and PROLI NONOates were purchased from Cayman Chemical Co. and used without further purification. The water used in all experiments was purified by a Milli-Q system (Millipore, Bedford, MA, USA). All buffers were incubated with Chelex-100 beads and filtered through a Millipore 0.2 μm filter membrane before use. All other chemicals were purchased from Fisher Scientific Inc. and used without further purification.

~~5.2.2 Site-directed mutagenesis and protein purification.~~

Plasmid containing the M121H/H46E mutations was constructed by site-directed mutagenesis using wild-type azurin (pET9a) as the template for the quick change mutagenesis procedure with the forward primers 5’-CGA AGA ACG TTA TGG GTG AAA ACT GGG TTC TGT CC-3’ (fwd-H46E), 5’-

~~CAC TCC GCA CTG CAT AAA GGT ACC CTG-3’ (fwd-M121H) and the reverse primers 5’-GGA CAG~~

~~AAC CCA GTT TTC ACC CAT AAC GTT CTT CG-3’ (rev-H46E), 5’-CAG-GGT-ACC-TTT-ATG-~~

CAG-TGC-GGA-GTG-3’ (rev-M121H). The mutations were confirmed by sequencing. The protein was expressed in BL-21* E. Coli (Novagen, Madison, WI) and purified according to published procedures.{Sieracki, 2014 #40} Electrospray ionization mass spectrometry confirmed the identity of the isolated variant (apo-M121H/H46EAz, MWobs = 13944, MWcal = 13943.73).

~~5.2.3 UV-vis spectroscopy measurements.~~

~~117~~

~~5.2.4 Mass spectrometry measurements.~~

~~5.2.5 Electron paramagnetic resonance (EPR) spectroscopy measurements.~~

~~X-band EPR spectra were collected on a Varian-122 spectrometer equipped with an Air Products~~

~~Helitran cryostat, EIP frequency counter and temperature controller with a collection frequency of 9.20 ~~~

9.55 GHz, power of 0.2 mW and field modulation of 4.0 G. All EPR samples were prepared in the presence of 20 % glycerol in 50 mM BisTris pH 7.0 buffer and quickly frozen in liquid nitrogen unless otherwise stated.

~~5.2.6 Fe(II) titration.~~

Apo M121H/H46EAz in 25 mM Tris buffer pH 7.8 was degassed in a Schlenk line and transferred to an anaerobic chamber (Coy Laboratories). Aliquots of degassed (NH4)2Fe(SO4)2 stock solution with known concentration were added to a solution containing 1 ml of 468 μM apo M121H/H46EAz in 25 mM

Tris pH 7.8. UV-vis absorption spectra were recorded on an HP Agilent 8453 diode array spectrometer inside anaerobic chamber. Spectra were corrected for dilution and baseline. The dissociation constant (KD) is determined by fitting the plot of absorbance at 330 nm as a function of Fe(II) concentration using the following equation:

~~1 2 퐴 = 퐴푚푎푥 × {[(푃 + 푀 + 퐾퐷) − [(푃 + 푀 + 퐾퐷) − (4 × 푃 × 푀)]2} ÷ (2 × 푃)~~

~~2+ where P = protein concentration, M = Fe concentration and KD = dissociation constant. A dissociation constant (Kd) of 8.1 ± 1.0 μM was obtained.~~

~~118~~

~~5.2.7 Determination of extinction coefficient of Fe(II)-M121H/H46EAz.~~

Tris pH 7.8 with a final concentration of Fe(II) less than 500 μM in the end. UV-vis absorption spectra were recorded on an HP Agilent 8453 diode array spectrometer inside anaerobic chamber. Spectra were corrected for dilution and baseline. Based on the relatively small KD of Fe(II)-M121H/H46E and the presence of excess apo M121H/H46EAz in solution, we correlated the concentration of Fe(II)-M121H/H46EAz with the concentration of Fe(II) added. An extinction coefficient for Fe(II)-M121H/H46EAz at 330 nm is determined by fitting the plot of absorbance at 330 nm as a function of Fe(II) concentration using Beer’s law A = εlc where l = 1 cm and c = Fe(II) concentration. An extinction coefficient (ε) of 971 ± 6 M-1cm-1 was obtained.

~~5.2.8 Fe(II) incorporation.~~

Apo M121H/H46EAz in 25 mM Tris buffer pH 7.8 was degassed in a Schlenk line and transferred to an anaerobic chamber (Coy Laboratories). Two equivalents of (NH4)2Fe(SO4)2 from 50 mM degassed stock solution were slowly added into the protein solution during stirring. After stirring for 30 minutes, the mixture was run down through a PD-10 desalting column (pre-equilibrated with 50 mM BisTris pH 7.0 buffer) to remove excess Fe(II). The extinction coefficient of 971 M-1cm-1 at 330 nm was used to determine the concentration of Fe(II) incorporated protein.

~~5.2.9 Mössbauer spectroscopy.~~

Mössbauer data were collected on an SEE Co. Mössbauer spectrometer, equipped with a constant acceleration transducer that uses a 100 mCi 57Co/Rh γ-ray source from Cyclotron Instruments. Mössbauer spectra were recorded using a Cryo Industries closed-cycle cryostat, with a fixed 500 G magnetic field perpendicular to the γ-ray beam. Data analysis was done by using nonlinear least squares simultaneous fitting of multiple spectra to spin Hamiltonian model or fitting of a spectrum with Lorentzian lines.

~~119~~

57 57 Fe(II)-M121H/H46EAz was prepared according to the procedure described above except using FeCl2 instead of (NH4)2Fe(SO4)2. All Mössbauer samples were flash frozen in liquid nitrogen without adding glycerol.

~~5.3 Results~~

~~5.3.1 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by UV-vis spectrum~~

~~Purification of the apo form of M121H/H46EAz was achieved following reported procedure.36~~

Titrating apo-M121H/H46EAz with (NH4)2Fe(SO4)2 resulted in accumulation of a S(Cys)→Fe(II) charge- transfer band center at 330 nm, similar to the LMCT bands reported in the other two engineered non heme iron sites in azurin respectively (Figure 5.2A).37,38 The absorbance at 330 nm reached plateau after adding one equivalent of Fe(II) salt (Figure 5.2B), indicating the mononuclear iron binding site. The disscociation constant (Kd) was determined by measuring the absorbance at 330 nm upon adding Fe(II) salt. By assuming one-to-one complex formation, fitting the absorbance changes at 330 nm as a function of the total Fe(II) concentration resulted in a Kd value of (8.1 ± 1.0 μM) (Figure 5.2C). Based on the strong binding affinity between apo-M121H/H46EAz and Fe(II), the extinction coefficient at 330 nm of Fe(II)-M121H/H46E was measured to be (971 ± 6) M-1cm-1 by titrating a known amount of Fe(II) ions into a large excess of apo-

M121H/H46EAz (Figure 5.2D). It is worth noting that the extinction coefficient of Fe(II)-M121H/H46EAz is significant lower than the other two reported non heme iron sites in Az, which are 1800 and 1610 M-1cm-

~~1 respectively.37,38 This result suggests weaker LMCT interaction between S(Cys) and Fe(II) which is consistent with the longer S(Cys)-M bond distance observed in the mutant M121H/H46EAz.~~

~~120~~

~~0 eq A 0.1 eq B 0.5 0.6 0.2 eq 0.3 eq 0.4 eq 0.5 eq 0.6 eq 0.4 0.7 eq 0.8 eq 0.4 0.9 eq 1.0 eq 1.1 eq 0.3 1.2 eq 1.5 eq 2.0 eq 2.5 eq 0.2 3.0 eq 4.0 eq 0.2~~

~~Absorbance (Au) Absorbance 5.0 eq Absorbance at 330 nm 330 at Absorbance 0.1 0.0~~

~~300 400 500 600 0 1 2 3 4 5 Wavelength (nm) Equivalents of Fe2+~~

0.6 Equation y = a + b*x 0.5 D Weight No Weighting C Residual Sum 2.8931E-5 0.5 of Squares Pearson's r 0.99988 NewFunctio 0.4 Model Adj. R-Square 0.99973 n (User) Value Standard Erro y=M*((P+x+ 0.4 E Intercept 0.01554 0.00148 K)-(((P+x+K) E Slope 9.71325E- 5.60804E-6 Equation 0.3 ^(2))-(4*P*x) )^(0.5))/(2*P) 0.3 Reduced 2.10572E-5 Chi-Sqr 0.2 Adj. R-Squa 0.99914 0.2 Value Standard Err M 0.5144 0.00238

~~Absorbance at 330 nm 330 at Absorbance~~

~~Absorbance at 330 nm 330 at Absorbance E 0.1 E K 8.0994 0.97724 0.1~~

~~0 500 1000 1500 2000 2500 0 50 100 150 200 250 300 350 400 450 Fe2+ concentration (uM) Fe2+ concentration (uM)~~

Figure 5.2 Fe(II) titration and extinction coefficient of Fe(II)-M121H/H46E. (A) UV-vis absorption spectral of titrating 0.1 – 5.0 eq. of Fe2+ to apo-M121H/H46E in 50mM BisTris pH7.0. (B) Titration curve monitored by the increased absorbance at 330 nm versus the equivalents of Fe2+. (C) The dissociation 2+ constant (KD) is determined by fitting the plot of Abs at 330 nm vs. Fe concentration using the following 1 2 equation: 퐴 = 퐴푚푎푥 × {[(푃 + 푀 + 퐾퐷) − [(푃 + 푀 + 퐾퐷) − (4 × 푃 × 푀)]2} ÷ (2 × 푃) where P = 2+ protein concentration, M = Fe concentration and KD = dissociation constant. (D) Extinction coefficient of Fe(II)-M121H/H46EAz at 330 nm is determined by fitting the plot of Abs at 330 nm vs. Fe2+ concentration using Beer’s law A = εlc where l = 1 cm and c = Fe2+ concentration.

~~We next studied the NO binding properties of Fe(II)-M121H/H46EAz by UV-vis spectroscopy.~~

Upon adding 1 eq. DEA NONOate to Fe(II)-M121H/H46EAz solution at pH 7, the solution turned from colorless to yellow. The resulted species shows strong absorptions at 337 and 425 nm, and a weak absorption at 650 nm (Figure 5.3A). Interestingly, when 2 eq. DEA NONOate was added to Fe(II)-

M121H/H46EAz solution, after shortly forming the resulted species as above, a new species was formed with the absorption featuring of the 650 nm peak shifted to 720 nm (Figure 5.4). No covalent modifications were observed in ESI-MS for both products, suggesting the formation of the adduct of Fe(II)-

~~121~~

~~M121H/H46EAz with NO. Considering the reductive nature of NO, we assume that the 650 nm species is reduced to the 720 nm species by excess amount of NO.~~

~~0.8 A B~~

~~0.4 0.6~~

~~0.4~~

~~0.2~~

~~0.2 (Au) Absorbance~~

~~Absorbance (Au) Absorbance~~

~~0.0 0.0~~

~~400 600 800 1000 400 600 800 Wavelength (nm) Wavelength (nm)~~

Figure 5.3 UV-Vis spectrum characterization of dinitrosyl iron complexes formation in engineered non heme iron site in Az. (A) Kinetic UV-Vis spectra of Fe(II)-M121H/H46EAz reacting with 1 eq. Proli NONOate in 50 mM BisTris buffer pH 7. UV-Vis spectra of Fe(II)-M121H/H46EAz (black), intermediate 8 8 (green) and isolated {Fe(NO)2} species (blue). (B) Kinetic UV-Vis spectra of isolated {Fe(NO)2} being 8 9 reduced with 1 eq. dithionite. UV-Vis spectra of isolated {Fe(NO)2} species (blue) and {Fe(NO)2} species (red). A 2.0 B 1.8

~~1.6 Abs at 650 nm 0.12 Abs at 720 nm 1.4~~

~~1.2~~

~~1.0 0.08~~

~~0.8~~

~~Absorbance (Au) Absorbance 0.6~~

~~Absorbance (Au) Absorbance 0.04 0.4~~

~~0.2~~

~~0.0 0.00 400 600 800 1000 0 1000 2000 3000 4000 Wavelength (nm) Time (s)~~

Figure 5.4 UV-Vis spectrum characterization of dinitrosyl iron complexes formation in engineered non heme iron site in Az. (A) Kinetic UV-Vis spectra of Fe(II)-M121H/H46EAz reacting with 2 eq. DEA 8 NONOate in 50 mM BisTris buffer pH 7. UV-Vis spectra of Fe(II)-M121H/H46EAz (black), {Fe(NO)2} 9 species (blue) and {Fe(NO)2} species (red). (B) The time traces of absorbance at 650 nm (black) and 720 nm (red).

~~To test this hypothesis, we first generated the 650 nm species by controlling the NO amount and then reduce it with a different reductant. When we reduced the 650 nm species with 1 eq. dithionite, 650~~

122 nm peak shifted to 720 nm and the UV-vis features of the resulted species exactly matched the 720 nm species we observed with excess amount NO, confirming that the 720 nm species is indeed the reduced product of the 650 nm species (Figure 5.3B). We also tried to oxidize the 720 nm species back to the 650 nm species with Ir(IV) salt, but no absorption changes were observed (Figure 5.5). To confirm that the 650 and 720 nm species remain protein-bound, both species were submitted to buffer exchange using centrifugal filters with a 10 kDa molecular weight cutoff. The yellow solutions did not pass through the membrane, and thus remained in the protein fraction with the UV-vis features still observed.

~~A 0.12 B 0.10 Abs at 720 nm~~

~~0.08~~

~~0.08 0.06~~

~~0.04~~

~~Absorbance (Au) Absorbance Absorbance (Au) Absorbance~~

~~0.02~~

~~0.00 0.00 600 800 1000 0 1000 2000 3000 4000 Wavelength (nm) Time (s)~~

Figure 5.5 UV-Vis spectra characterization of oxidizing dinitrosyl iron complexes with Na2IrCl6 in engineered non heme iron site in Az. (A) Kinetic UV-Vis spectra of adding 3 eq. Na2IrCl6 to the 9 9 {Fe(NO)2} species in 50 mM BisTris buffer pH 7. UV-Vis spectra of the {Fe(NO)2} species (red) and the final spectrum (blue). (B) The time traces of absorbance at 720 nm.

~~5.3.2 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by cwEPR spectrum~~

~~To further investigate the electronic structures of the two species observed via UV-vis, we carried out electron paramagnetic resonance (EPR) studies of both species. The starting material, Fe(II)-~~

M121H/H46EAz in the absence of NO, had no detectable EPR signal (Figure 5.6A). Upon addition of 2 eq. of DEA NONOate, the mixture of Fe(II)-M121H/H46EAz and DEA NONOate solutions were quenched at different time points by fast freeze in liquid nitrogen then EPR spectra were collected at 40 K. The EPR spectra showed a major peak at g ~ 2.0 and a minor peak at g ~ 4.0 increasing with time (Figure 5.7A). An

EPR spectrum was recorded after buffer exchange to remove low molecular mass species while the colored solution stayed above 10K MWCO membrane; the major peak at g ~ 2 was still observed in the protein

123 fraction (Figure 5.7B), indicating the EPR signals raised from protein bound iron complexes. Later we measured temperature-dependent EPR spectra showing that the intensity of the g ~ 2 signal increased with decreasing temperature (Figure 5.8). The EPR spectrum recorded at 5 K revealed a high-spin (S = 3/2)

~~{FeNO}7 species with g ~ 4.0 (Figure 5.6A). A power saturation measurement was also carried out obtained~~

P1/2 = 1.6 mW at 40 K (Figure 5.9), suggesting that the g ~ 2 signal is metal ion associated rather than a free radical. The EPR spectrum at g ~ 2 was simulated as a low-spin iron complex with an S = 1/2 ground state resulting in gx = 2.040, gy = 2.034 and gz = 2.012. The gav = 2.03 is a very characteristic EPR signal for identifying DNICs in both synthetic model complexes and protein-bound DNICs.39 It has been described

9 15 as a {Fe(NO)2} species based on Enemark-Feltham notation with an S =1/2 ground state. Another possibility is a low-spin {Fe(NO)}7 species which also has an S = 1/2 ground state. However, there was no

15 change upon NO labeling (Figure 5.6C), indicating the hyperfine coupling constant between Fe and NNO was very small, similar to other observed DNICs.40 In contrast, reported low-spin {Fe(NO)}7 species exhibited larger N hyperfine coupling which has been explained by the spin density is mainly located on

~~22,23,41 9 the NO nitrogen. Therefore, it is concluded that the EPR signal rises from a {Fe(NO)2} species. The~~

~~9 EPR spectrum of the {Fe(NO)2} species was also collected at room temperature (Figure 5.6D). Instead of~~

9 an averaged g value was observed in low molecular weight {Fe(NO)2} species due to fast tumbling of the molecule,42 the g tensor still stayed anisotropic which further proved that it was actually bound to protein.

It is interesting to note that two species were observed in UV-vis spectroscopic study when excess amount of NO was added (Figure 5.4) and the later species with 720 nm band was the reduced product of the beginning species with 650 nm band (Figure 5.3B). However, only one protein-bound species was observed in EPR spectra with signal intensity increase matching the formation of the later species, indicating the beginning species with 650 nm band was EPR silent. To test the hypothesis, one half equivalent of DEA

NONOate was added to the Fe(II)-M121H/H46EAz solution and similar UV-vis features were observed, but no ERP signal was detected (Figure 5.6B). However, after reducing with one equivalent dithionite, the

~~9 EPR signal corresponding to the {Fe(NO)2} species was observed (Figure 5.6B). Because Fe(II)-~~

~~M121H/H46EAz has a d6 electron configuration and both species are formed by adding NO only, the~~

~~124~~

~~8 beginning species with 650 nm was likely to be {Fe(NO)2} species based on its EPR silent nature and~~

~~9 redox relationship with the {Fe(NO)2} species.~~

~~A B Fe(II)-M121H/H46EAz Fe(II)-M121H/H46EAz + 0.5 eq. Proli NONate Fe(II)-M121H/H46EAz + 0.5 eq. Proli NONate + 1 eq. dithionite~~

~~1200 1600 2000 2400 2800 3200 3600 3200 3250 3300 3350 Magnetic Field (G) Magnetic Field (G)~~

~~C 15NO D 14NO Fe(II)-M121H/H46E + 4.5 eq. NO at r.t.~~

~~3150 3200 3250 3300 3350 3250 3300 3350 3400 3450 Magnetic Field (G) Magnetic Field (G)~~

Figure 5.6 cw-EPR investigation of dinitrosyl iron species formed in engineered non heme iron Az. (A) X-band cw-EPR spectra of Fe(II)-M121H/H46EAz (black), {FeNO}7 species formed by adding 1.0 eq. Proli NONOate to Fe(II)-M121H/H46EAz (red). (B) X-band cw-EPR spectra of Fe(II)-M121H/H46EAz 8 (black), {Fe(NO)2} species formed by adding 1.0 eq. Proli NONOate to Fe(II)-M121H/H46EAz (blue) and 9 8 {Fe(NO)2} species formed by adding 1 eq. dithionite into {Fe(NO)2} species (red). (C) X-band cw-EPR 14 9 15 9 9 spectra of {Fe( NO)2} (red) and {Fe( NO)2} species (black). (D) X-band cw-EPR spectra of {Fe(NO)2} at room temperature. Conditions: microwave frequency, 9.20~ 9.55 GHz; modulation, 2 G; power, 0.2 mW; temperature, 30K.

~~125~~

~~A before adding NO B 1min 2min 5min Fe(II)-M121H/H46E + 3 eq. NO after buffer exchange 10min 30min 1h~~

~~1000 2000 3000 4000 Magnetic Field (G) 1500 2000 2500 3000 3500 Magnectic field~~

9 Figure 5.7 EPR spectra study of the {Fe(NO)2} species formation. (A) Time-dependent EPR spectra of 9 9 the {Fe(NO)2} species. (B) The EPR spectra of {Fe(NO)2} species after three times buffer exchange using Amicon® Ultra-4 centrifugal filter devices with a 10 kDa molecular weight cutoff.

~~20K 30K 40K 50K 60K 70K 80K 100K~~

~~3150 3200 3250 3300 3350 Magnetic Field (G)~~

~~9 Figure 5.8 The EPR spectra of the {Fe(NO)2} under different temperature.~~

~~126~~

~~A 0dB B 5dB 1 10dB 15dB 20dB 25dB 30dB 35dB 40dB 45dB EPRPowerSa Model turation (User )y=K/((1+(2^(2 Equation /B)-1)*(x/P))^( B/2)) 0.1 Reduced 7.55907E-4 Chi-Sqr Adj. R-Squar 0.99539~~

~~Normalized I/sqrt(P) Normalized Value Standard Erro G P 1.5941 0.12973 G B 1.2251 0.14045~~

~~3150 3200 3250 3300 3350 0.01 0.1 1 10 100 Magnetic Field (G) Microwave power (mW)~~

9 9 Figure 5.9 EPR power saturation study of the {Fe(NO)2} species. (A) EPR spectra of the {Fe(NO)2} species in 50 mM BisTris pH 7, recorded at 30 K under varying microwave power from 0 dB to 45 dB. Microwave frequency = 9.244 GHz, modulation amplitude = 2 G. (B) Plots of intensity/√푃 vs. microwave 퐼 power (mW) of the sharpest peak at g ~ 2.04. The plot is analyzed by using the equation: = √푃 퐾 2 푏 where I = peak intensity, K = constant, P = microwave power in mW, b = measure of ( ) 푃 [1 +(2 퐵 −1)×( )]2 푃1/2 homogeneity of the line shape with a value varying between 1 and 3, P1/2 is the microwave frequency power at which the amplitude of the EPR signal is one-half of its unsaturated value.

~~9 5.3.3 The characterization of {Fe(NO)2} species using ENDOR and HYSCORE~~

~~The absence of the hyperfine structure associated with 14NO and similar EPR spectra of 14NO and~~

15 9 NO labeled {Fe(NO)2} species indicate that the principal values of the hyperfine tensor are not larger than the corresponding line width. Electron nuclear double resonance (ENDOR) spectroscopy, which has

~~43 9 been used to investigate other DNICs, was applied to determine the protein-bound {Fe(NO)2} species’~~

9 hyperfine tensors and to obtain the structure information. The Q-band ENDOR spectra of the {Fe(NO)2} species were recorded at 30 K. Two kinds of resonances were observed when irradiated at gz, corresponding to distinct nuclei. The resonance centered at 51.4 MHz (set as zero), the Larmor frequency of 1H nucleus at

1206.9 mT, arises from hydrogen nuclei (Figure 5.10A). Two pairs of 1H-ENDOR signals were observed, suggesting that two different protons interacted with the electron spin with A// = 5.0 and 7.8 MHz respectively which is most likely from two β hydrogens of Cys112 ligand. The resonances found between

1 and 15 MHz are due to nitrogen hyperfine interactions (Figure 5.10B). However, because of the quadrupolar interaction, the pattern of 14N resonances is not amenable to analysis by itself.44 To differentiate

~~127~~

9 the resonances due to NNO from the resonances due to NHis, ENDOR spectra of the {Fe(NO)2} species enriched in 15N were recorded under the same conditions as the unenriched sample (Figure 5.10B). The resonances at 9.2 and 11.9 MHz were still present, meaning that they are not due to NNO hyperfine interaction and most likely from a His ligand. The resonances around 6.0 MHz completely vanished and new bands at 3.6 and 7-8 MHz were observed, suggesting they were from the hyperfine interactions of NNO

9 nucleus. The ENDOR spectra of the {Fe(NO)2} species irradiated at different magnetic fields were also collected and simulated (Figure 5.11). The parameters used for ENDOR simulation were summarized in

Table 5.1. Based on the simulation, the averaged N hyperfine interaction from His was 12.0 MHz. It is significantly larger than the averaged N hyperfine interaction from NO which was 6.6 MHz. This explains why very minimal change was observed in CW-EPR when replacing 14NO with 15NO. Our results are in good agreement with the hyperfine coupling constants observed in a Fe(NO)2(adenine)(cysteine methyl ester) complex at room temperature under physiological conditions with A14N-adenine = 12.3 MHz, A14NO =

~~45 5.9 MHz and AβH-cysteine = 5.9 MHz.~~

~~7.6 MHz A B 5.0 MHz~~

~~14N 15N~~

~~-8 -4 0 4 8 2 4 6 8 10 12 14 16 18 20 22 24 26 MHz MHz~~

Figure 5.10 Pulsed EPR investigation of dinitrosyl iron species formed in engineered non heme iron 1 9 Az. (A) Q-band H-ENDOR spectra of {Fe(NO)2} species formed in engineered non heme iron Az 9 collected at g//. (B) Q-band N-ENDOR spectra of {Fe(NO)2} species formed in engineered non heme iron 14 9 15 9 14 9 Az collected at g//, {Fe( NO)2} (black) and {Fe( NO)2} (red). (C) HYSCORE spectra of {Fe( NO)2} 15 9 and {Fe( NO)2} species taken at the gz postion.

~~128~~

~~Figure 5.10 (cont.)~~

~~-12 -8 -4 0 4 8 12 -12 -8 -4 0 4 8 12 C D 12 12 12 12~~

~~8 8 8 8~~

~~4 4 4 4~~

~~0 0 0 0~~

~~MHz MHz~~

~~-4 -4 15 -4 -4 N 15N~~

~~-8 -8 14 -8 -8 N 14N~~

~~-12 -12 -12 -12~~

~~-12 -8 -4 0 4 8 12 -12 -8 -4 0 4 8 12 MHz MHz~~

~~A B~~

~~11933 G 11931 G~~

~~11955 G 11948 G~~

~~12009 G 12001 G~~

~~12039 G 12031 G~~

~~12069 G 12061 G~~

~~5 10 15 20 25 5 10 15 20 25~~

~~MHz MHz~~

Figure 5.11 N-ENDOR investigation of dinitrosyl iron species formed in engineered non heme iron 14 9 Az. (A) Q-band N-ENDOR spectra of {Fe(NO)2} species collected at different magnetic fields at 30 K 15 15 9 (black). The simulated spectra were shown in red. (B) Q-band N-ENDOR spectra of {Fe( NO)2} species collected at different magnetic fields at 30 K. The simulated spectra were shown in red.

~~14 9 15 9 The HYSCORE spectra of the {Fe( NO)2} and {Fe( NO)2} species (Figure 5.10 C and D) were~~

~~14 collected at gz. The visual inspection of the HYSCORE gave Azz = 13.6 MHz for NHis hyperfine interaction~~

~~129~~

~~14 and Azz = 3.3 MHz for NNO, consistent with the Azz = 13.8 MHz and 3.1 MHz observed in ENDOR respectively. The simulated HYSCORE spectra were shown in Figure 5.12.~~

~~-12 -8 -4 0 4 8 12 A B 12 12~~

~~8 8~~

~~4 4~~

~~0 0~~

~~MHz -4 -4 15N~~

~~-8 -814N~~

~~-12 -12~~

~~-12 -8 -4 0 4 8 12 MHz~~

Figure 5.12 HYSCORE investigation of dinitrosyl iron species formed in engineered non heme iron 14 9 Az. (A) Simulated HYSCORE spectrum of the {Fe( NO)2} species. (B) Simulated HYSCORE spectrum 15 9 of the {Fe( NO)2} species.

~~Table 5.1 The simulation parameters for the 14N-ENDOR fittings.~~

x y z α β γ A (14N His) 9.7 11.1 15.3 16 32 -19 Q (14N His) 0.59 0.42 -1.01 8 20 2 A(14N NO) 0.5 3.5 15.7 109 -95 -114 Q (14N NO) -0.01 0.54 -0.53 110 -94 -101 A(14N NO) 0.5 3.5 15.7 -109 -95 114 Q (14N NO) -0.01 0.54 -0.53 -110 -94 101 g 2.040 2.034 2.012 1) Hyperfine and quadrupole coupling in MHz. For 15N hyperfine couplings, multiply by 1.402. 2) Euler angles rotate the diagonal A matrix into the coordinate system in which g is diagonal. The Euler angles that diagonalize A are given by -γ, β, -α. 3) The coupling matrixes for the two NO’s are assumed to be symmetric about the yz plane.

~~130~~

~~5.3.4 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by Mössbauer spectrum~~

~~The zero-field Mössbauer spectrum of 57Fe(II)-M121H/H46EAz measured at 30 K consisted of a quadrupole doublet (Figure 5.13A). The related Mössbauer parameters, δ = 0.93 mm/s and ΔEQ = 2.69~~

8 mm/s, are characteristic of a high-spin (S = 2) Fe(II) site. The Mössbauer spectrum of the {Fe(NO)2} species also exhibited a quadrupole doublet feature with Mössbauer parameters δ = 0.64 mm/s and ΔEQ =

1.82 mm/s (Figure 5.13B), which are significantly different from the high-spin Fe(II) starting material. It is in the general range of values reported for intermediate-spin (S = 1) ferrous species, which is be consistent with being EPR silent.46,47 The spin S = 1 could be interpreted via the high-spin ferrous center (S = 2) antiferromagnetically coupled with either two spin S = ½ NO molecules or one spin S = 1 NO- while the

+ 9 other nitric oxide being NO (S = 0). The Mössbauer spectrum of the {Fe(NO)2} species revealed two distinct components (Figure 5.13C). The first component, accounting for 72 % of the iron in the sample, exhibited a paramagnetic hyperfine structure with δ = 0.25 mm/s and ΔEQ = -1.32 mm/s fitted with S = ½

9 spin Hamiltonian. It is consistent with the S = ½ EPR signal of the {Fe(NO)2} species. The second component, accounting for 28 % of the iron in the sample, consist of a quadrupole doublet which shared similar Mössbauer parameters as high-spin Fe(II) starting material. The Mössbauer and EPR parameters of

~~8 9 Fe(II)-M121H/H46EAz, {Fe(NO)2} species and {Fe(NO)2} species are summarized in Table 5.2.~~

~~131~~

~~-4 -3 -2 -1 0 1 2 3 4 A~~

~~= 0.93 mm/s, EQ = 2.69 mm/s (86%, blue)~~

~~= 1.19 mm/s, EQ = 3.54 mm/s (9%, red)~~

~~= 0.04 mm/s, EQ = 0.57 mm/s (5%, magenta)~~

~~B -4 -3 -2 -1 0 1 2 3 4~~

~~= 0.64 mm/s, EQ = 1.82 mm/s (82%, blue)~~

~~= 0.06 mm/s, EQ = 0.20 mm/s (18%, red)~~

~~-4 -3 -2 -1 0 1 2 3 4 C~~

~~= 0.25 mm/s, EQ = -1.32 mm/s (72%, blue)~~

~~= 0.89 mm/s, EQ = 2.61 mm/s (28%, red)~~

Figure 5.13 Zero-field Mӧssbauer spectra of 57Fe enriched non heme iron Az samples. (A) Mӧssbauer spectrum of 57Fe(II)-M121H/H46EAz in 50 mM BisTris pH 7 at 30 K. (B) Mӧssbauer spectrum of 57 8 57 9 { Fe(NO)2} species in 50 mM BisTris pH 7 at 25 K. (C) Mӧssbauer spectrum of { Fe(NO)2} species in 50 mM BisTris pH 7 at 25 K. The experimental spectra were fitted (solid curve drawn through the data) with the parameter set of Table 5.2. The solid curves above the experimental spectra show the contribution of each iron species.

~~132~~

~~Table 5.2 EPR and Mössbauer parameters for all the species at 30 K~~

Spin state g values Isomer shift δ Quadrupole -1 (mm·s ) splitting ΔEQ (mm·s-1) Fe(II)- S = 2 - 0.93 2.69 M121H/H46EAz 8 {Fe(NO)2} S = 1 - 0.64 1.82 species 9 {Fe(NO)2} S = 1/2 2.037, 2.029, 0.25 -1.32 species 2.009

~~5.3.5 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by NRVS~~

To further explore the coordination and geometry information of these nitrosyl iron species, we utilized 57Fe nuclear resonance vibrational spectroscopy (NRVS) to characterize the core vibrations that involved motions of the iron atom.48 NRVS has demonstrated to be useful in identification of EPR-silent protein-bound dinitrosyl iron complexes.49,50 The NRVS spectrum of 57Fe(II)-M121H/H46EAz contained four well-resolved peak in the region between 200 and 500 cm-1 (Figure 5.14A), corresponding to Fe-His,

~~Fe-Cys and Fe-Glu vibrations. This coordination sphere is consistent with the crystal structure of Cu(II)-~~

~~M121H/H46EAz. The NRVS spectrum of the {FeNO}7 species showed one well-resolved peak at 520 cm-~~

~~1 and a series of weak signals between 300 and 400 cm-1 (Figure 5.14B), which was best fitted as one His,~~

~~9 one Cys, one Glu and two NO molecules. The NRVS spectrum of the {Fe(NO)2} species exhibited three~~

~~-1 9 well-resolved peaks at 420, 520 and 590 cm (Figure 5.14C). While labeling the {Fe(NO)2} species with~~

15NO, the three peaks shifted to 410, 510 and 580 cm-1 respectively (Figure 5.14C), indicating that vibrations in this region most likely originate from the symmetric and asymmetric stretching modes of the

~~N-Fe-N unit.51 The similar isotopic labeling shift pattern was also observed in synthetic model complexes.49~~

~~133~~

~~A B~~

Figure 5.14 Nuclear resonance vibrational spectroscopy (NRVS) investigation of dinitrosyl iron species formed in engineered non heme iron Az. (A) The NRVS spectrum of 57Fe-M121H/H46EAz in 50 mM BisTris pH 7. (B) The NRVS spectrum of {57FeNO}7 species in 50 mM BisTris pH 7 (top). (C) The 57 9 NRVS spectrum of { Fe(NO)2} in 50 mM BisTris pH 7. The experimental spectra were shown above and DFT calculated vibrational density of states (VDOS) were shown at bottom.

~~5.3.6 Stepwise nitrosylation of Fe(II)-M121H/H46EAz investigated by XAS~~

~~To investigate the oxidation states and elucidate the structures, we collected XANES and EXAFS~~

~~8 9 of Fe(II)-M121H/H46EAz, the {Fe(NO)2} and {Fe(NO)2} species (Figure 5.15). The Fe K-edge of Fe(II)-~~

~~8 9 M121H/H46EAz, {Fe(NO)2} and {Fe(NO)2} species were 7119.6, 7122.6 and 7121.3 eV respectively,~~

~~134~~

~~9 8 consistent with the result that {Fe(NO)2} was the one-electron reduction product of {Fe(NO)2} species~~

~~(Figure 5.15A and 5.16A). The close Fe K-edge pre-edge intensities among Fe(II)-M121H/H46EAz,~~

~~8 9 8 9 {Fe(NO)2} and {Fe(NO)2} species suggested that both {Fe(NO)2} and {Fe(NO)2} species shared similar~~

52 9 distorted Td environment as Fe(II)-M121H/H46EAz. The pre-edge of {Fe(NO)2} species was at 7112.8 eV (Figure 5.15A, inset), which was lower than the characteristic range of 7113.4 – 7113.8 eV for the pre-

~~9 53 9 edge peaks of anionic {Fe(NO)2} DNICs, further confirming the neutral character of {Fe(NO)2} species~~

~~8 with one Cys and one His coordination as suggested by pulsed EPR study. Interestingly, the {Fe(NO)2}~~

9 species had the same pre-edge as the {Fe(NO)2} species, suggesting that the {Fe(NO)2} core managed to minimize the Fe1s → Fe3d transition energy changes upon one-electron reduction. The comparison between

8 9 the EXAFS spectra of {Fe(NO)2} and {Fe(NO)2} species showed that the Fe-NO bond distances became shorter while the Fe-S (Cys) bond distance stayed intact upon reduction (Figure 5.15B). This observation could be interpreted via the one-electron reduction happening at NO ligands rather than the iron center resulting in stronger Fe-NO electrostatic interactions. The anionic NO ligand has been suggested by

~~9 computational study that the electronic structure of the mononuclear {Fe(NO)2} DNICs is best described~~

~~II - 9 III - 9 54 as a resonance hybrid of {Fe (NO)(NO )} and {Fe (NO )2} . The EXAFS fitting parameters are shown in Table 5.3.~~

~~Fe-N (NO) A B 1.6 Fe-Cys~~

~~1.4 Fe-O (NO) 1.2~~

~~1.0 Fe=O? Fe-His~~

~~0.8~~

~~0.6~~

~~0.4 7100 7110 7120~~

~~Energy (eV) 0.2 Fourier Transform (relative intensity) (relative Transform Fourier 0.0 7100 7120 7140 7160 7180 7200 1 2 3 4 5 6 Energy (eV) r (angstrom)~~

~~Figure 5.15 XAS investigation of dinitrosyl iron species formed in engineered non heme iron Az. (A) 8 9 XANES of Fe(II)-M121H/H46EAz (black), {Fe(NO)2} species (red) and {Fe(NO)2} species (blue). (B)~~

~~135~~

~~8 9 EXAFS spectra of Fe(II)-M121H/H46EAz (black), {Fe(NO)2} species (red) and {Fe(NO)2} species (blue). The labels show their characteristic Fe-His, Fe-NO and Fe-Cys bonds.~~

~~25 A B Experimental Simulation 4~~

~~20 2~~

~~chi (k)~~

~~3 k -2 15 -4~~

-6

~~Chi (k) Chi 3 10 5 10~~

~~wave number FT K FT~~

~~7100 7110 7120 7130 7140 0 1 2 3 4 5 6 Energy (eV) r (angstrom)~~

~~C 18 D Experimental Experimental 16 Simulation Simulation 8 4 20 6 14 2 4~~

~~Chi (k) Chi~~

~~3 0 2 12 K 0 15 (k) chi~~

~~-2 3 k -2 10 -4 Chi (k) Chi -4~~

~~8 5 10 -6 chi (k) chi~~

~~3 10 5 10~~

~~FT K FT wave number~~

~~6 wave number FT k FT 4 5 2~~

~~0 0 -2 0 1 2 3 4 5 6 0 1 2 3 4 5 6 r (angstrom) r (angstrom)~~

Figure 5.16 EXAFS investigation of dinitrosylation in an engineered nonheme iron protein. (A) The 8 9 K-edge of Fe(II)-M121H/H46EAz (black), {Fe(NO)2} species (red) and {Fe(NO)2} species (blue). 8 Fourier transform and EXAFS (inset) for the (B) Fe(II)-M121H/H46EAz, (C) {Fe(NO)2} species and (D) 9 {Fe(NO)2} species. Experimental data are shown as solid black lines and the simulation is shown by dashed red lines. Parameters used to fit the data are listed in Table 5.3.

~~136~~

~~Table 5.3 EXAFS fitting parameters~~

Sample/fit FI Fe-S (Cys) Fe-N(His) Fe-O(Asp) Fe-N (NO) Fe – O (NO) N R(Å DW(Å2 N R(Å) DW(Å N R(Å DW(Å2) N R(Å DW( N R(Å) DW(Å ) ) 2) ) ) Å2) 2) Fe(II)- 2.2 1 2.34 0.003 2 2.08 0.003 1 1.97 0.004 N. A. N. A. M121H/H4 1 3.09 0.008 6EAz 8 {Fe(NO)2} 16. 1 2.33 0.005 2 2.50 0.03 N. A. 2 2.03 0.004 2 3.00 0.003 species 7 9 {Fe(NO)2} 21. 1 2.29 0.006 1 2.015 0.003 N. A. 2 1.68 0.01 2 2.98 0.004 species 7

~~0.2 2.34 0.003 0. 2.09 0.003 0.25 1.97 0.004 N. A. N. A. 5 5 0.25 3.09 0.008~~

8 1) The EXAFS fitting of {Fe(NO)2} species was improved with addition of 40 % O ligand at 1.72 Å. The FI increases by ~ 5 – 10 upon removal of this ligand. 9 2) The EXAFS fitting of {Fe(NO)2} species was done with mixing 20% Fe(II)-M121H/H46EAz with the 9 {Fe(NO)2} species.

~~5.4 Summary~~

~~Figure 5.17 Schematic representation of the stepwise nitrosylation in an engineered nonheme iron protein. The ground spin state of each species is labeled in blue below the structures.~~

~~In summary, we have successfully engineered a non heme iron binding site in blue copper protein azurin. The interaction of NO with Fe(II)-M121H/H46EAz produces three nitrosyl-iron species: {FeNO}7~~

~~8 9 with S = 3/2 ground state, {Fe(NO)2} with S = 1 ground state and {Fe(NO)2} with S = 1/2 ground state~~

~~7 8 (Figure 5.17). The {FeNO} and {Fe(NO)2} species can be prepared by controlling the amount of NO added. The NRVS study of {FeNO}7 species suggests that one of the two His is replaced by NO. In the~~

~~8 9 presence of excess amount NO or dithionite, the {Fe(NO)2} species is reduced to the {Fe(NO)2} species.~~

9 15 The pulsed EPR and NRVS studies of the {Fe(NO)2} S=1/2 species with the aid of NO labeling reveal that the iron atom is coordinated by one Cys, one His and two NO molecule in a tetrahedral geometry. To the best of our knowledge, this is the first time that stepwise nitrosylation and the unambiguous conversion

~~8 9 of {Fe(NO)2} to {Fe(NO)2} core is observed in a protein scaffold and the iron ligands of both dintirosyl-~~

~~137 iron centers are positively identified by spectroscopic studies. Our results not only provide structural~~

~~8 information about the unusual {Fe(NO)2} species but also enhance our understanding of the pathological and physiological role of nitric oxide.~~

~~5.5 References~~

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~~- 19 Tsai, F.-T. et al. Dinitrosyl iron complexes (DNICs) [L2Fe(NO)2] (L = thiolate): Interconversion~~

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~~23 Li, M. et al. Tuning the electronic structure of octahedral iron complexes [FeL(X)] (L = 1-alkyl-~~

~~4,7-bis(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclononane, X = Cl, CH3O, CN, NO). The S =~~

~~1/2 ⇌ S = 3/2 spin equilibrium of [FeLPr(NO)]. Inorg. Chem. 41, 3444-3456 (2002).~~

~~24 Wasser, I. M., de Vries, S., Moënne-Loccoz, P., Schröder, I. & Karlin, K. D. Nitric oxide in~~

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~~Rev. 102, 1201-1234 (2002).~~

~~25 Zumft, W. G. Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme–~~

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~~26 Moenne-Loccoz, P. Spectroscopic characterization of heme iron-nitrosyl species and their role in~~

~~no reductase mechanisms in diiron proteins. Nat. Prod. Rep. 24, 610-620 (2007).~~

~~27 Lu, S., Suharti, de Vries, S. & Moënne-Loccoz, P. Two NO molecules can bind concomitantly at~~

~~the diiron site of NO reductase from bacillus azotoformans. J. Am. Chem. Soc. 126, 15332-15333~~

~~(2004).~~

~~28 Kumita, H. et al. No reduction by nitric-oxide reductase from denitrifying bacterium pseudomonas~~

~~aeruginosa: Characterization of reaction intermediates that appear in the single turnover cycle. J.~~

~~Biol. Chem. 279, 55247-55254 (2004).~~

~~29 Moore, E. G. & Gibson, Q. H. Cooperativity in the dissociation of nitric oxide from hemoglobin.~~

~~J. Biol. Chem. 251, 2788-2794 (1976).~~

~~30 Traylor, T. G. & Sharma, V. S. Why nitric oxide? Biochemistry 31, 2847-2849 (1992).~~

~~31 Zhao, Y., Brandish, P. E., Ballou, D. P. & Marletta, M. A. A molecular basis for nitric oxide sensing~~

~~by soluble guanylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 96, 14753-14758 (1999).~~

~~32 Butler, C. S., Seward, H. E., Greenwood, C. & Thomson, A. J. Fast cytochrome bo from escherichia~~

~~coli binds two molecules of nitric oxide at CuB. Biochemistry 36, 16259-16266 (1997).~~

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~~33 Watmough, N. et al. The dinuclear center of cytochrome bo 3 from escherichia coli. J. Bioenerg.~~

~~Biomembr. 30, 55-62 (1998).~~

~~34 Grönberg, K. L. C. et al. A low-redox potential heme in the dinuclear center of bacterial nitric oxide~~

~~reductase: Implications for the evolution of energy-conserving heme−copper oxidases.~~

~~Biochemistry 38, 13780-13786 (1999).~~

~~35 Grönberg, K. L. C., Watmough, N. J., Thomson, A. J., Richardson, D. J. & Field, S. J. Redox-~~

~~dependent open and closed forms of the active site of the bacterial respiratory nitric-oxide reductase~~

~~revealed by cyanide binding studies. J. Biol. Chem. 279, 17120-17125 (2004).~~

~~36 Sieracki, N. A. et al. Copper–sulfenate complex from oxidation of a cavity mutant of pseudomonas~~

~~aeruginosa azurin. Proc. Natl. Acad. Sci. U.S.A. 111, 924-929 (2014).~~

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~~a small-molecule binding pocket. J. Am. Chem. Soc. 134, 19746-19757 (2012).~~

~~38 Liu, J. et al. Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron~~

~~protein: Preparation and study of Fe(II)-M121E azurin. J. Am. Chem. Soc. 136, 12337-12344~~

~~(2014).~~

~~39 Vanin, A. F., Serezhenkov, V. A., Mikoyan, V. D. & Genkin, M. V. The 2.03 signal as an indicator~~

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~~nitrosyl adduct of substrate-bound mouse cysteine dioxygenase by electron paramagnetic~~

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~~143~~

~~Chapter 6 Mechanistic Studies into Adenovirus Serotype 2 Inactivation by Free Chlorine via Expression of Major Capsid Proteins in E.coli~~

~~6.1 Introduction~~

Adenovirus have a non-enveloped, icosahedral virion that consists of a core containing linear double-stranded DNA enclosed by a capsid.1 The capsid is composed of 252 capsid proteins, 240 of which are hexons and 12 of which are pentons. Each penton projects a single fiber that varies in length for each serotype.2,3 As important human pathogens, adenoviruses induce a variety types of illnesses such as respiratory illness, conjunctivitis, cystitis and gastroenteritis involving almost every human organ system.1

~~Although these diseases are acute and self-limiting in immune-competent individuals, they cause significant morbidity in AIDS, cancer, and organ transplant patients with compromised immune systems.4-6~~

~~Adenoviruses present in drinking water sources in both industrialized and developing countries.7~~

There is no method to detect infectious adenoviruses in drinking water currently. Compared to other virus prevalent in drinking water, adenoviruses show high resistance to monochloramine and low pressure ultraviolet light.8,9 On the other hand, free chlorine has been shown to be efficient towards adenovirus disinfection.10,11 However, the mechanism of how disinfectants inactivate the adenovirus is still poorly understood. Efforts have been made to study virus inactivation mechanism using a model virus such as bacteriophage MS2.12-17 Most of the studies directly using adenovirus only contained kinetic data or quantification of protein and DNA damage rather than understanding the mechanism from the molecular level.9,18-21 A systematic understanding of virus inactivation would potentially direct the development and optimization of treatments and strengthen our understanding of virus adaptation to environmental stressors.

In our previous study, we fully characterized the effects of pH and temperature on the kinetics of adenovirus serotype 2 (Ad2) inactivation with free chlorine.22 In an effort to elucidate the molecular components of

Ad2 targeted by free chlorine during the inactivation process, we found that although free chlorine inactivated Ad2 virons lost the ability to form plaques, they retained their ability to bind to Coxsackie- adenovirus receptors (CAR) for both recombinant CAR proteins in vitro and A549 cell monolayers.23 In

144 addition, DNA isolated from free chlorine inactivated Ad2 still could be amplified by PCR and PCR signals decay more slowly than level of inactivation, indicating that genome damage was not the cause of disinfection. The lack of E1A viral proteins were observed during infection of A549 host cell with inactivated Ad2 virions. These results indicate that the inactivation of Ad2 by free chlorine occurs via damaging proteins that govern endosomal lysis and nuclear delivery. Endosomal lysis is triggered by the low endosomal pH and subsequent release of the penton bases and protein VI, which disrupt the endosome.24 After released from the endosome, the virion with loose capsid, which is comprised mostly of hexon proteins with the dsDNA inside, attaches to the outer nuclear membrane of the host cell and viral

DNA is transferred into nucleus. Thus, disruption of either the penton or hexon protein structure by chlorine could have an impact on overall viability by inhibiting these steps that take place within the cell.

We first attempted to detect the damage to Ad2 penton and hexon directly after free chlorine disinfection using Mass spectrometry. However, we only got less than 50% sequence coverage even with high concentration virus stock, similar to the results reported before.7 To overcome this problem, we decided to study the recombinant proteins instead of Ad2 virus. In this Chapter, the first time expression of

Ad2 penton and hexon proteins in E.coli was reported. We located and tracked chemical modifications by free chlorine with trypsin digestion MS/MS study. For all three major capsid proteins of Ad2, over 90% whole sequence coverage is achieved. This approach allowed us to monitor the modifications at the position of amino acid sidechains and provide insight into the mechanism of Ad2 inactivation by free chlorine.

~~6.2 Materials and methods~~

~~6.2.1 Construction of plasmid for the synthesis of adenovirus fiber, penton and hexon proteins~~

The genes of adenovirus fiber, penton and hexon were codon optimized for expression in E. coli and synthesized into pUC57 cloning vector by GenScript (Piscataway, NJ). The genes were subcloned into pET-29b(+) vector at Ndel/Xhol cloning sites. The pET-29b(+) vector carries His-Tag at C-terminal and genes for Kanamycin resistance. The gene sequences were confirmed via sequencing using a T7 promoter.

~~145~~

~~The gene sequences of adenovirus fiber, penton and hexon with C-terminal His-Tag are available in the supporting information.~~

~~6.2.2 Expression of the adenovirus fiber, penton and hexon proteins~~

The pET-29b(+) vectors containing the adenovirus fiber, penton and hexon genes were transformed into BL-21(DE3) E. Coli (Novagen, Madison, WI) via heat shock. Mix 1 μl of DNA plasmid into 50 μl of competent cells in a falcon tube. Place the mixture on ice for 30 min. Heat shock the falcon tube by placing the bottom of the tube into a 42 oC water bath for 30 seconds. Put the tube back on ice for 2 minutes. Add

~~250 μl SOC media and grow in 37 oC shaker for 45 minutes. Plate 100 μl of the transformation solution onto a LB agar plate containing Kanamycin as antibiotic. Incubate the plate at 37 oC overnight.~~

~~A single colony of cells from the plate was dropped into 100 mL growths of super broth (SB) media~~

o with 50 μl 100mg/mL Kanamysin. Shake at 37 C till the media becoming cloudy (OD600 > 0.4). Isopropyl- beta-D-thiogalactopyranoside (IPTG) with concentration of 0.4 mM was added to induce the protein expression for 3 hours. The cells were harvested by centrifugation at 9,000 rpm for 10 minutes.

~~6.2.3 Purification of the adenovirus fiber, penton and hexon proteins~~

The pelleted cells were resuspended in 25 mM Tris pH8.0 with 1% Triton X-100 lysis buffer by orbital shaking in 4 oC cold room for 30 minutes. The mixture was sonicated for 18 minutes (6 seconds pulses on and 12 seconds pulses off with an amplitude of 70) using Misonix ultrasonic liquid processor.

~~The lysate was separated into supernatant and pellet factions by centrifugation at 13,000 rpm for 20 minutes.~~

~~The pellet was resuspended in 8 M urea/100 mM NaH2PO4/10 mM Tris pH8.0 at 5 ml per gram wet weight.~~

~~Shake the mixture at room temperature gently to avoid foaming for 60 minutes. The mixture was centrifuged at 13,000 rpm for 20 minutes to pellet the cellular debris. Add 1 ml of the 50% Ni-NTA slurry~~

(from Qiagen) to 4 ml lysate and mix gently by shaking at 200 rpm on a rotary shaker for 60 minutes at room temperature. Load lysate-resin mixture carefully into an empty column with bottom cap still attached.

~~Remove the bottom cap and let the remaining solution flow through the column. Wash twice with 4 ml 8~~

~~M urea/100 mM NaH2PO4/10 mM Tris pH6.3 (wash buffer). Elute the recombinant protein 4 times with~~

~~146~~

~~0.5 ml 8 M urea/100 mM NaH2PO4/10 mM Tris pH5.9 (elution buffer A), followed by 4 times with 0.5 ml~~

~~8 M urea/100 mM NaH2PO4/10 mM Tris pH4.5 (elution buffer B). Collect fractions and analyze by SDS-~~

~~PAGE.~~

Combine all the factions containing demanded proteins into a dialysis tubing with 10,000 Da molecular weight cut-off. The protein solution was dialyzed against 2 M urea/25 mM Tris pH8.0 overnight at 4 oC. The resulting solution was further dialyzed again 25 mM Tris pH8.0 for 24 hours at 4 oC to further remove the urea and refold the proteins.

~~6.2.4 Transmission electron microscopy (TEM) investigation~~

~~TEM was carried out in part in the Frederick Seitz Materials Research Laboratory Central Research~~

~~Facilities, University of Illinois. The adenovirus hexon solution was allowed to bind to the copper grid~~

(Fomvar/Carbon 200 mesh, Copper) for 10 minutes. The excess was washed off and the grid was soaked in 1% glutaraldehyde in PBS buffer for 20 minutes. Finally, the copper grid was soaked in 2% uranyl acetate for 12 seconds to serve as a negative stain. The samples was observed using JEOL 2100 Cryo TEM at 120 kV.

~~6.2.5 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot~~

~~Precast 4 – 20% gradient SDS-PAGE gels and 2× Laemmli sample buffer were purchased from~~

Bio-Rad Laboratories, Inc. Dithiothreitol (DTT) was added into Laemmli buffer to a final concentration 54 mg/ml before use. Place 10 μl protein sample in the bottom of an Eppendorf tube. Dilute the protein sample with equal volume of Laemmli buffer. Heat the mixture in the boiling water bath for 5 min. Spin down in the bench-top microcentrifuge at 10,000 rpm for 2 min. Remove the comb from the ready-gel and immerse the gel in the running buffer (196 mM glycine/0.1 % SDS/25 mM Tris pH 8.3) of the inner chamber. Load

~~15 μl of the sample into the well of the ready-gel. The gel was run at constant voltage at ~120 V for an hour.~~

After stopping the gel, disassemble the gel cassette and remove the gel. Place the gel in Millipore water and gently shake it at room temperature 3×15 min to remove SDS. The gel was stained with Thermo ScientificTM

~~147~~

~~ImperialTM Protein Stain at room temperature for an hour. Place the gel in Millipore water afterwards to remove the background color.~~

~~Proteins were separated by 12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Blocking buffer was used before probing the membrane with primary and secondary antibodies.~~

~~The 6×His-tagged proteins were detected by probing membranes with mouse monoclonal anti-His antibody~~

~~(1:1,000 GE Healthcare, Piscataway, NJ) followed by HRP-conjugated goat anti-mouse IgG antibody~~

~~(1:5,000 Thermo Scientific, Pierce, Rockford, IL). Membranes were incubated with SuperSignal West~~

~~Femto reagents (Thermo Scientific Pierce, Rockford, IL), and chemiluminescent signals were captured.~~

~~6.2.6 Inactivate major capsid proteins with free chlorine~~

~~The major capsid proteins of AD2 in 25 mM Tris pH8.0 were directly used after dialysis. 10 equivalents of sodium hypochlorite was added to the protein solution under stirring at room temperature.~~

~~After incubating the mixture for 5 min, 15 equivalents of sodium thiosulfate to quench the reaction. The samples were submitted to trypsin digestion and HPLC MS/MS study without further purification.~~

~~6.2.7 Trypsin digest and HPLC MS/MS analysis~~

~~Trypsin digest and HPLC MS/MS analysis were carried by the Protein Science Facility in the~~

~~Biotechnology Center at the University of Illinois at Urbana-Champaign. Samples were digested with trypsin for 15 minutes at 55 °C using a CEM Liberty microwave digester (CEM Corporation, Matthews,~~

~~NC). The digested products were analyzed on a Thermo Scientific Velos Pro mass spectrometer with a~~

~~Dionex RSLCnano Ultimate 3000 UPLC front end using an Acclaim PepMap RSLC (75 micron X 15 cm,~~

C18 2 micron 100 Angstrom) column (Thermo Scientific) with a linear gradient of 1 to 60 % acetonitrile in 0.1 % formic acid over 120 minutes. The data were analyzed using the Mascot database search engine

~~(Matrix Science, London) and searched against a custom database containing the adenovirus protein sequences.~~

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~~6.3 Results~~

~~6.3.1 Adenovirus fiber, penton and hexon expression in E. coli~~

E.coli is one of the most attractive because of its fast growth, easily achieved high cell density, readily available and inexpensive media, well-characterized genetics and availability of large number of cloning vectors and host strains.25-27 For all three major capsid proteins of AD2, only fiber was reported to express in E. coli.28 Penton was expressed in Baculovirus infected cells and hexon was directly purified from AD2 propagated in HeLa S3 cells.29,30 For the fiber protein expressed in E. coli, the procedure required multiple steps of column purification. Recombinant DNA techniques permit the construction of fusion proteins in which specific affinity tags are added to the protein sequence of interest; the use of these affinity tags simplifies the purification of the recombinant fusion proteins by employing affinity chromatography methods.31 The genes of adenovirus fiber, penton and hexon with His-Tag at C-terminal were codon optimized for expression in E. coli and cloned into pET-29b(+) vector at Ndel/Xhol cloning sites. First, the overexpression of AD2 fiber was investigated in different media at 37 oC (Figure 6.1). Interesting, the overexpression was only observed in super broth. Later, the protein overexpression was optimized with different IPTG concentration and induction time at 37 oC (Figure 6.2). Inducing with 1.0 mM IPTG didn’t show distinctively higher overexpression than 0.4 mM IPTG. However, longer induction time significantly improved the yield as the the overexpressed protein bands became much darker (Figure 6.2). The western blot was employed to further confirm where the desired protein were after each step of purification (Figure

~~6.3 and 6.4). The 6×His-tagged proteins were detected by probing membranes with mouse monoclonal anti-~~

His antibody followed by HRP-conjugated goat anti-mouse IgG antibody. The results showed that the Ad2 fiber, penton and hexon were exclusively formed in inclusion bodies. No His-tagged protein was detected in the supernatant after sonication and the supernatant after washing with 1% Triton X-100/25 mM Tris pH

~~8.0 buffer. The bands at desired positions were observed in western blot after 8 M urea extraction, indicating the proteins could be extracted from the cell debris and re-solubilized.~~

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~~Figure 6.1 SDS-PAGE gel investigation the overexpression of AD2 fiber protein in different media.~~

~~Figure 6.2 SDS-PAGE gel investigation the overexpression of AD2 penton and hexon under different concentrations of IPTG and induction time.~~

~~150~~

Figure 6.3 The western blot of Ad2 fiber expressed in E. coli. Line 1: cell suspension; Line 2: supernatant from cell pellet; Line 3: supernatant after sonication; Line 4: Supernatant after washing the pellet with 1% Triton X-100/25 mM Tris pH8.0 buffer; Line 5: solid pellet after 8 M Urea extraction; Line 6: soluble proteins in 8 M urea. Expected molecular weight for AD2 fiber: 63.0 kDa.

Figure 6.4 The western blot of Ad2 hexon expressed in E. coli. Line 1: cell suspension; Line 2: supernatant from cell pellet; Line 3: supernatant after sonication; Line 4: Supernatant after washing the

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Figure 6.4 (cont.) pellet with 1% Triton X-100/25 mM Tris pH 8.0 buffer; Line 5: solid pellet after 8 M Urea extraction; Line 6: soluble proteins in 8 M urea. Expected molecular weight for AD2 hexon: 109.1 kDa.

~~Figure 6.5 (cont.)~~

~~152~~

~~Figure 6.5 (cont.)~~

Figure 6.5 Purification of AD2 fiber, penton and hexon proteins expressed in E. coli. (A) SDS-PAGE gel study of AD2 fiber after each purification step. Lanes: 1, supernatant after cell lysis; 2, supernatant after washing with 1% Triton X-100/25 mM Tris pH 8.0 buffer; 3, supernatant after extracting with 8 M urea solution; 4, supernatant after adding Ni-NTA slurry to 8 M urea extraction solution; 5, the flow-through of eluting the Ni-NTA column with wash buffer; 6 - 7, the flow-through of eluting the Ni-NTA column with elution buffer A; 8 - 9, the flow-through of eluting the Ni-NTA column with elution buffer B. Desired molecular weight for AD2 fiber: 63.0 kDa. (B) SDS-PAGE gel study of AD2 penton after eluting with elution buffers. Lanes: 1 - 4, the flow-through of eluting the Ni-NTA column with elution buffer A; 5 - 8, the flow-through of eluting the Ni-NTA column with elution buffer B. Desired molecular weight for AD2 penton: 63.3 kDa. (C) SDS-PAGE gel study of AD2 hexon after each purification step. Lanes: 1, supernatant after extracting the cell debris with 8 M urea solution; 2, supernatant after adding Ni-NTA slurry to 8 M urea extraction solution; 3, the flow-through of eluting the Ni-NTA column with wash buffer; 4 - 7, the flow-through of eluting the Ni-NTA column with elution buffer A; 8 - 11, the flow-through of eluting the Ni-NTA column with elution buffer B. Desired molecular weight for AD2 hexon: 109.1 kDa.

After optimizing the expression conditions, large scale expression of AD2 fiber, penton and hexon proteins were carried out. Each step in the purification procedure was monitored by SDS-PAGE gel (Figure

6.5). After adding the Ni-NTA slurry into the 8 M urea extraction solution, the bands containing desired proteins either vanished (Figure 6.4A, line 4) or significantly decreasing in intensity (Figure 6.5C, line 2), indicating the binding of His-tagged proteins with Ni-NTA column. The elution of His-tagged proteins were controlled by tuning the pH of elution buffers. As shown in Figure 6.5, the AD2 fiber was eluted only

153 at lower pH but penton and hexon could be eluted at both pH conditions. To remove the large amount of urea from the protein solution and renature the desired the proteins, a stepwise dialysis was applied. First, the protein in 8 M urea was dialyzed against 2 M urea/25 mM Tris pH 8.0 overnight. Then the resulted solution was further dialyzed against 25 mM Tris pH 8.0 to completely remove the urea. Minimum precipitate was observed during the dialysis. Compared to previously reported expression methods, the current method shows great advantage that the overexpression is achieved in E. coli system and the purification only requires to run column once.

Figure 6.6 The TEM images of recombinant AD2 hexon protein expressed in E. coli. (A) Large view of the particles assembled by recombinant AD2 hexon. (B) Zoom in view of the particles assembled by recombinant AD2 hexon.

It has been proven that recombinant fiber protein trimerized spontaneously during synthesis in E. coli but folded more bulky than the native protein.28 To investigate folding and assembly properties of recombinant AD2 hexon protein, we carried out a TEM study and the images were shown in Figure 6.6.

Particles with diameter around 90 nm were observed, similar to the size of native adenoviruses. However, the edges and vertices of icosahedral structure were not well resolved. These results indicate that the recombinant AD2 hexon is well folded and able to at least partially maintain the native assembling structure.

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~~6.3.2 Mechanism investigation of adenovirus inactivation by free chlorine~~

To investigate the mechanism of human AD2 inactivation by free chlorine, the recombinant fiber, penton and hexon were treated with 10 equivalents of sodium hypochlorite in 25 mM Tris pH 8.0 for 5 min at room temperature. The reactions were quenched by adding 15 equivalents of sodium thiosulfate under stirring. The samples were directly submitted to trypsin digestion and HPLC MS/MS study without further purification. The MS/MS data were analyzed using the Mascot database search engine and searched against a custom database containing the adenovirus protein sequences. The recombinant proteins exhibited significantly higher sequence coverage than directly using high concentration adenovirus stock (Table 6.1).

~~The sequence coverage rates are 62%, 90% and 85% for fiber, penton and hexon respectively.~~

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~~Table 6.1 The sequence coverage of trypsin digestion – MS/MS studies of recombinant AD2 fiber, penton and hexon expressed in E.coli. The detected peptide sequences are highlighted in red.~~

Proteins are most likely to be major targets for reaction with free chlorine due to their abundant and highly reactive side chains. The order of reactivity for various side-chains is reported to be: Met > Cys >>

His > Trp > Lys >> Try ~ Arg > Gln ~ Asn.32,33 The high sequence coverages in MS/MS studies enabled us to detect modifications at amino acid sidechains of the entire proteins. Interestingly, besides methionine

~~156 and cysteine oxidation, significant amount of tyrosine oxidations were detected while there were quite amount of more reactive residues left (Table 6.2).~~

~~Table 6.2 Modified amino acid sidechains by MS/MS studies.~~

~~Modified AA sidechains~~

~~Ad2 hexon Met6, Met7, Met13, Met96, Met326, Met332, Met398, Met520, Cys532, Met807~~

~~6.4 Discussion~~

The fiber protein contains the host binding sites and is responsible for binding to the host cell surface receptor protein CAR.34-37 The crystal structure of Ad12 knob with CAR D1 shows that the majority of amino acid residues contributing to the adenovirus-CAR interaction are found in the AB loop. The interactions includes three hydrogen bonds formed by residues Asp415AD12 with Lys125CAR and Lys123CAR,

38 Leu426AD12 with Tyr85CAR, Lys429AD12 with Glu58CAR (Figure 6.7A). However, these residues are not modified based on MS/MS studies, possible due to their poor reactivity towards free chlorine. The residues responsible hydrophobic interaction such as Pro417 and Pro418 were also intact during disinfection. The detected modifications were shown in Table 6.2. Flexibility of the fiber shaft is important to orientate the virion towards the host cell surface. Interestingly, Met373 and Tyr367 were oxidized to methionine sulfoxide and chlorotyrosine in high yield. Once the fiber forms trimer, six oxidized residues locate on the same plane of the fiber shaft. Originally the hydrophobic Met373 points to inside (Figure 6.7B). However, after free chlorine treatment, the formation of more hydrophilic methionine sulfoxide may affect the flexibility of fiber shaft dramatically.

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Figure 6.7 Visualizations of adenovirus fiber interacting with CAR receptor. (A) Visualizations of knob domain from adenovirus serotype 12 in complex with domain 1 of its cellular receptor CAR (PDB: 1KAC). (B) Visualizaitons of modified amino acid sidechains in protein structures of fiber trimer (A, PDB: 1QIU). Each monomer is in red, blue and silver; the Met192 and Tyr196 are shown from horizontal (left) or vertical view (right).

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Our previous results indicate that the inactivation of Ad2 by free chlorine occurs via damaging proteins that govern endosomal lysis and nuclear delivery. The crystal structure of penton shows the formation of a pentamer that buries approximately 26 % of the total surface area of each monomer.39 The stability gained in pentamer formation is due the burying of hydrophobic surface which is unlikely to be affected due its large interaction area and burying deep inside. The penton interacts with the integrins on the host cell surface and is thus responsible for initialization of endocytosis.40 The RGD loop of Ad2 penton consists of around 80 glycine and alanine rich residues. This RGD motif is required for attachment to integrins on the host cell surface which then triggers receptor mediated endocytosis. Without integrin attachment, the virus cannot enter the host cell. However, there were no modifications detected in this region. Interestingly, endosomal lysis is triggered by the low endosomal pH and subsequent release of the penton bases and protein VI, which disrupt the endosome.24 The pKa values for phenol groups in tyrosine,

2-chlorotyrosine and 2,6-dichlorotyrosine are 9.99, 8.56 and 6.79 respectively, suggesting the pKa of certain tyrosine sidechain would decrease after free chlororine treatment. Our hypothesis is that such tyrosines could not be protonated at low pH condition so that the release of penton is prohibited.

After released from the endosome, the virion with loose capsid, which is comprised mostly of hexon proteins with the dsDNA inside, attaches to the outer nuclear membrane of the host cell and viral DNA is transferred into nucleus. Thus, disruption of hexon protein structure by chlorine could have an impact on overall viability by inhibiting these steps that take place within the cell. The crystal structure of hexon with protein VIII shows that Met6 and P8 are important for the hydrophobic interaction between the protein VIII helix and the hexon.3 The oxidation of M6 would result in more hydrophilic methionine sulfoxide. We therefore assume that damage to M6 contributes to a weaker interaction between hexon and protein VIII, causing structural alternations in the capsid structure and thereby influencing virus shape and functionality in a more general way.

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~~6.5 Summary~~

In summary, the first time expression of human Ad2 penton and hexon proteins in E.coli was achieved. The 6×His-tag at the C-terminal enables convenient production of all three major capsid proteins in high quality. As a result, around 90% whole sequence coverages were obtained with recombinant proteins.

~~The modifications at reactive amino acid sidechains were detected with trypin digestion – MS/MS method.~~

The mechanistic study of adenovirus inactivation by free chlorine would potentially direct the development and optimization of water treatments and strengthen our understanding of virus inactivation mechanism.

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