Development of Variable Magnetic Field Instrumentation for Transient Absorption Measurements from Femtoseconds to Seconds for Chemical and Biochemical Studies

Thesis submitted for the degree of Doctor of Philosophy by

Joanna A. Hughes

Faculty of Science and Engineering The University of Manchester

2018

Table of Contents List of Tables…………………………………………………………………………..6 List of Figures……………………………..………………………………………...... 7 Abstract……………………………………...………………………………….….....11 Declaration...... 12 Copyright Statement...... 12 List of Abbreviations………………………………………………………………....14 Acknowledgments …………………………...……………………………………….17

Chapter 1. Introduction

1.1 Photochemistry…………………………………………...…………………….20

1.1.1 Photophysics...... …..20 1.1.2 Photochemical reactions ...... …..25

1.2 Radical Pair Chemistry……………………………………………………....26

1.2.1 Radicals…………...... ………………………………………………..27 1.2.2 Properties of spin………………………………………………………..27 1.2.3 Spin-state mixing………………………………………………………...39 1.2.4 Spin Hamiltonian of a Radical Pair……………………………………..31 1.2.5 Radical Pair Mechanism…………………………………..………….....32

1.3 Magnetic Field Effects (MFEs)………………………………...……..…32

1.3.1 Chemical processes……………………………………………………...32 1.3.2 Hyperfine mechanism and the effect of the Zeeman interaction……...…35 1.3.3 The Δg mechanism…………………………...... 36 1.3.4 The Low Field Effect (LFE)…………………………………...... 37 1.3.5 Influence of MF on biological systems…………………………………...37

1.4 References…………..………………………………………..………...... 40

Chapter 2. Experimental methods

2.1 Transient Absorption (TA) Spectroscopy…………………………………..47

2.1.1 TA Artifacts……………………………………………………………...49

2.2 Standard TA setup……………………………………...... ………………..51

2.2.1 Laser System…………………………………………………………….51 2.2.2 Helios/Eos experimental setup……...……………………..…………....53

2.3 Sample preparation…....……………………...... ……………………....55

2.3.1 FAD Sample preparation...... 55 2.3.2 AdoCbl Sample preparation...... 55

2.4 Calculations of the experimental error…………………….………...... …56

2.5 References………….………………………………………………...... ….60

Chapter 3. Development of the MF-generating apparatus

3.1 Neodymium disc magnets….....……………………………………………..63

3.1.1 Magnet and sample holder- version 1……………………………....…..64 3.2.1 Magnet and sample holder- version 2…………………………....……..65

3.2.1.1 Comparison of MF simulations and measurements……..…….66

3.2. Sample cell……………………………………………………...... 68

3.3. MF generating apparatus test system……………………………...... ……..69

3.3.1 Reproduction of previously reported MFE on intramolecular ET in FAD……………...... …69

3.4 References…………...... ……………………….....70

Chapter 4. Investigations of MFEs in flavin adenine dinucleotide

4.1 Photochemistry of FAD…………………………….……………………..…73

4.2 Reported MFEs in flavins………………………….……...... ………..…78

4.3 Investigations of MFEs in FAD………………………..……………………81

4.3.1 Results and discussion…………………….……………………………..81

4.3.1.1 Excited state dynamics of FAD at a range of pH..….....………82

4.3.1.2 MFE investigation in external fields of up to 950 mT………...90

4.4 Summary…………………………………………..…………………………....96

4.5 References…………………………………………………………………...... 96

Chapter 5. Development of Femtosecond – Nanosecond Transient Absorption Instrument for the Investigations of Magnetic Field Effects

5.1 Experimental setup development…………..…………………………...…..102

5.2 Software development…………………………………………………….....109

5.3 Further instrument development……………...... ………………..116

5.4 Optical setup test system- Vitamin B12……………...... ………...122

5.4.1 Photochemistry of Vitamin B12………….....……………………….…...122

5.4.2 Results and discussion……………………...... ……………...... 129

5.4.2.1 Photolysis of adenosylcobalamin using the Experimental Setup Selene…………………………………………....130

5.5 Summary……………...... ………………………………………………....131

5.6 References……..………...... …………………………....132

Chapter 6. Investigation of MFE in AdoCbl in MFs up to 900 mT

6.1 Reported MFE studies………………………….………...... ………....138

6.2 TA measurements of free AdoCbl in external MFs of up to 900 mT...... 146

6.2.1 Results and discussion……………………………...... …………….....146

6.2.1.1 MFE studies on photolysis of free AdoCbl up to 200 mT...... 146

6.2.1.2 MFEs in AdoCbl at neutral pH and external fields of up to 900 mT...... 150

6.2.1.3 MFEs in AdoCbl at low pH and external fields of up to 900 mT…………...... 156

6.3 Summary………………………………………………………………...….....159

6.4 References.....………………………………………………………………....159

Chapter 7. Summary, Conclusions and Future Work

7.1 Summary……………………...... …………………………….....165

7.2 Future Work…………………………………………...... …...170

7.3 References……………………………………………...... ………………...172

List of Tables

1.1 The approximate timescales for different transitions………………………...... …25

4.1 The lifetime values of all components in various pH values…...... ………………..87

4.2 Relative rate values for the decay of the second component in applied MFs up to 950 mT...... 94

6.1 Relative rate values for the decay of the third component in applied MFs up to 900 mT...... 159

6.2 Isotropic g-values for some typical organic radicals...... 161

List of Figures

1.1 Different types of radiation that compose the electromagnetic spectrum……...... 22

1.2 Potential energy diagram for the ground state and an excited state of a diatomic molecule………...... ……...... 23

1.3 Jablonski diagram for a hypothetical organic molecule………………………...... 23

1.4 Relative positions of different spectra...... 24

1.5 Energy diagram for the approach of two hydrogen atoms………...... 30

1.6 Vector representation of RP spin states in an applied external MF…………….....31

1.7 Typical plots of percentage MFE on a recombination yield in applied MF………34

1.8 The Zeeman effect…………………………………………………………………36

2.1 Basic scheme of transient absorption……………………………………………....48

2.2 ΔA spectrum...... 49

2.3 Block diagram of the Solstice Ace assembly……………………………………....51

2.4 The experimental amplifier laser setup…………………………………………….52

2.5 Helios/Eos experimental setup…………………………………………………...... 53

2.6 Rates obtained for the fitting of TA time profiles...... 57

2.7 MFE-dependence data spreadsheet...... 59

3.1 An example of N52 neodymium disc magnets……………………………………..64

3.2 Magnet holder- version 1………………………………………………………...... 65

3.3 Magnet holder- version 2……………………………………………………...... 66

3.4 Comparison of computer simulations and measurements of MF....………………..67

3.5 Newly designed and built capillary flow cell………………………………………69

3.6 Time profiles of TA observed at λ= 600 nm with (upper B= 0.2 T) and without (lower B= 0 T) MF…………………………………………………...... 70

3.7 An example of MFE on decay kinetics at 580- 600 nm with and without the application of external MF of 200 mT and in pH= 2.3……....…………...... …72

4.1 The structure of FAD……………………………………………………………….74

4.2 Different redox states of FAD under various physiological conditions...... 74

4.3 Schematic representation of various FAD structures in different pH……………...75

4.4 Photochemical reaction scheme for intramolecular RP formation between flavin (F) and an electron donor (D)………………………………………………………….76

4.5 Basic scheme of the photoinduced ET in FAD………………………………….....77

4.6 pH dependence of the time profile of TA observed in MF= 0 mT at 650 nm……...79

4.7 Time profiles of TA observed at λ= 600 nm with (upper B= 0.2 T) and without (lower B= 0 T) MF……………………………………………………...... 79

4.8 MFE- action spectra on the TA at pH= 2.3 (filled circle), 2.9 (open circle), 3.3 (filled square) and 4.1 (open square)……………………………………………....80

4.9 Normalized UV/Vis spectra of FAD in low pH taken before (blue line) and after (black line) measurements……………………………………………………...... 82

4.10 Time-resolved visible spectroscopy data for FAD………………………...…….83 4.11 EAS and SVD of the residual matrix resulting from global analysis of time-resolved visible spectroscopy data for FAD…………………………………...…84

4.12 Time-resolved visible spectroscopy data for FAD in buffer of various pH……....86

4.13 EAS and SVD of the residual matrix resulting from global analysis of time-resolved visible spectroscopy data for FAD………………………………...…....87

4.14 Comparison of EAS spectra in various pH and lifetime values for separate components in various pH……………………………………………..……...88

4.15 Comparison of EAS spectra of individual components observed in various MF obtained by global fit…………………………………………...... ……………...90

4.16 Comparison of the a) EAS1 and b) EAS2 lifetime values against the Applied MF……………………………………....………...... 91

4.17 Relative rate constants for the decay of the second component against the applied MF………………………………………………………………………....…...94

5.1 Cylindrical mu-metal shielding…………………………………………………...107

5.2 Newly designed and built double capillary flow cell……………………………107

5.3 Ultrafast transient absorption laser instrumentation development-‘Selene’………108

5.4 Ultrafast transient absorption laser instrumentation development-‘Selene’, a breadboard located above the existing Helios setup...... 109

Fig.5.5 Ultrafast transient absorption laser instrumentation development-Selene...... 109

5.6 Decay observed on a shielded path after at 450 nm after the photoexcitation with a laser pulse centered at 375 nm using a) Helios and b) Selene detectors……..111

5.7 Decay observed on a MF path after at 450 nm after the photoexcitation with a laser pulse centered at 375 nm using a) Helios and b) Selene detectors………...…122

Fig.5.8 Signal observed at 450 nm after the photoexcitation with a laser pulse centered at 375 nm on a a) shielded and b) MF path...... 113

5.9 Front panel and block diagram of the communication test for the Selene software…...... 116

5.10 Default settings block diagram for the newly developed software...... 117 5.11 Block diagram of the Selene ‘Save’ Sub-VI...... 118 5.12 Front panel of the newly designed software..………...………………...…...... 120

5.13 Pump and probe beam pulses in traditional TA……………………………...... 121

5.14 In the proposed new scheme the probe beam is modulated to be half the repetition rate of the beam generated by the OPA2……………………………………………...122

5.15 The basic scheme for the modified Selene development………………………..123

5.16 The Selene chopper head…...... 124

5.17 TA traces observed at 450 nm following the excitation of FAD...……………....125

5.18 TA traces observed at 450 nm following the excitation of FAD………………...126

5.19 Chemical structure of selected B12 derivatives………………....………………..128

5.20 a) A scheme of the Co-C bond homolysis upon substrate binding…………..….129

5.21 The photolysis of AdoCbl……………………………………………………..…130

5.22 Absorption spectrum of adenosylcobalamin...... 131

5.23 Normalized UV/Vis spectra of AdoCbl………………………………………….134

5.24 TA traces and regular residuals plots obtained on different days ……………….136

6.1 MF dependency of the CblII quantum yield for the cw photolysis of 200 μM AdoCbl at 20°C………………...... ………………………………………….....146

6.2 a) Overlaid and difference traces acquired at 525 nm for the anaerobic cw-photolysis free AdoCbl b) MF-dependence of the relative, observed rate coefficient from the initial downward phase representing CoII accumulation...... 147

6.3 The aerobic cw-photolysis of 10 µM AdoCbl in the presence and absence of a 190 mT MF titrating EAL apoenzyme………………………...... …..148

6.4 Magnetic field dependence (O) of the aerobic cw-photolysis relative rate of the AdoCbl bound to EAL...... 149

6.5 a) The MF-dependence of the anaerobic cw-photolysis of free MeCbl...... 150

6.6 An example of MFE on the decay observed at 525 nm following the excitation of AdoCbl...... 153

6.7 Comparison of lifetime values and the fitting errors for the a) first b) second and c) third component in external MF of up to 200 mT...... 154

6.8 Comparison of the MFE on photolysis of AdoCbl observed in the a) previous and b) current study...... 155

6.9 The predicted MFE curve obtained using computer software Spinach...... 156

6.10 Comparison of lifetime values against the applied MF...... 157

6.11 Relative rate values for the decay observed at 525 nm following the photolysis of free AdoCbl with a laser pulse centered at 375 nm against the applied MF...... 158

6.12 An example of MFE on the decay observed at 525 nm following the excitation of AdoCbl in buffered solution of pH 2.2...... 161

7.1 The effect of increasing MF on a singlet reaction yield for a T-born RP...... 169

7.2 An example of the Halbach array...... 171

Abstract

Cobalamin, also known as vitamin B12 or adenosylcobalamin (AdoCbl) is the largest and the most complex of all types of vitamins and the only one containing cobalt. It plays a key role in normal functioning of the brain and nervous system and is necessary in formation of blood. All biologically active forms of B12 have an unusually labile C- Co to the upper axial ligand, which has bond dissociation energy of 31 kcal/mol (for 5’- deoxyadenosylcobalamin, AdoCbl). Absorption of light below 610 nm will induce homolysis of the C-Co bond to produce a spin-correlated, geminate radical pair (RP). Calculations based on the average hyperfine couplings in the AdoCH2⋅CblII RP suggest that the spin state mixing for a separated pair is likely to be rapid (a period of ~430 ps). One might also expect fast relaxation processes, due to the presence of heavy transition metal, although none were needed to model the saturation of the Zeeman effect by magnetic fields in AdoCbl.

To more fully investigate these phenomena we have developed novel transient absorption (TA) technique that allowed investigations of magnetic perturbation of the cobalamin photoresponse from femtoseconds to seconds. To provide a wide range of magnetic field (MF) strength values, powerful neodymium disc magnets as well as Halbach array were employed. Data of both, computer simulations and measurements is presented. A previously published MFE in a Flavin Adenine Dinucleotide (FAD) intramolecular electron transfer reaction was chosen as a biological test system. The field-dependence was not only reproduced, but also extended by measurements covering a range of MFs up to 1 T.

A newly developed technique was used for TA MFE studies with both free AdoCbl and AdoCbl-dependent ethanolamine ammonia lyase (EAL) on ultrafast timescale. The new optical setup allowed for MF induced changed to be measured directly. By employing lock-in amplifier even very low signals, which in standard techniques are usually overwhelmed by ever present noise, could be detected.

Technical drawings, designs of magnet holder, double capillary flow cell and the optical table layout are all discussed. Data from all experimental chemical systems is presented.

Declaration

No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning.

The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://www.campus.manchester.ac.uk/medialibrary/policies/intellectual- property.pdf), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on presentation of These.

Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so that we may fear less.

― Maria Sklodowska - Curie

Abbreviations and Symbols

HEPES 2-[4-(2-hydroxyethyl)piperazin- 1-yl]ethanesulfonic acid A absorbance AdoCbl adenosylcobalamin Å Ångström, 10-10 m CIDEP chemically induced dynamic electron polarisation CIDNP chemically induced dynamic nuclear polarisation Co-C cobalt-carbon bond Co-R cobalt-variable axial group bond J combined electron/nuclear magnetic moment CW continuous wave CNCbl cyanocobalamin °C degrees Celsius EF(s) electric field(s) EM electromagnetic EMF(s) electromagnetic field(s) 2J(r) electron exchange interaction EPR electron paramagnetic resonance ET electron transfer EAL ethanolamine ammonia lyase ε extinction coefficient FAD flavin adenine dinucleotide FMN flavin mononucleotide FTIR Fourier-transform infrared spectroscopy Hz hertz HRP horseradish peroxidase H-bond hydrogen bond

OHCbl hydroxocobalamin hfc hyperfine couplings HFI hyperfine interaction IR infrared LFE low field effect(s) MARY magnetic effect on reaction yield MF magnetic field B magnetic field (or magnetic induction)

B1/2 magnetic field at half saturation

BS magnetic field at saturation MeCbl methylcobalamin μ micro m milli ml millilitres M molar, moles per litre n nano pH negative logarithm of the proton (H+) concentration Nd:YAG neodymium-doped yttrium aluminum garnet OPA optical parametric amplifier Φ quantum yield RPM radical pair mechanism RP(s) radical pair(s) k rate constant s second S singlet spin-state SVD singular value decomposition T Tesla τ time constant / lifetime TRIR time-resolved infrared

T0/±1 triplet spin-states

TA ultrafast transient absorption (fs – μs) UV ultraviolet UV-vis ultraviolet-visible λ wavelength WLC white light continuum T0 zero-delay time

Acknowledgements

There have been a great number of people who have helped me throughout my PhD, and I couldn’t have asked for more support than I have received from them. To my supervisor Alex, I would like to thank for his guidance and all the science discussions we’ve ever had. I will always admire his patience with students and his ability to remain calm no matter the circumstances. Since the very beginning of my PhD Dr. Samantha Hardman has helped me a great deal in understanding the spectroscopy techniques as well as data analysis, and I would never be able to finish my project without her help. I will definitely miss our conversions about optics, and obviously the frequency of the beams. I am also very impressed with her thoughtfulness and the ability to foreseen the moments, when I was about to lose my patience and have a slight meltdown. I am also grateful for all of her help with working with strong magnets, which allowed me not to lose any of my fingers. To Dr. Darren Graham I’d like to think for his brilliant ideas during the process of instrument development- I am particularly grateful for finding a solution to my problem with the computer software- the wrong cable should probably have been my first thought and would have saved me a month! On the same note, I’d like to thank Darren’s postdoc Dr. Morgan Hibberd, who helped me with the software. To my external supervisor, Dr. Jonathan Woodward I’d like to thank not only for allowing me to spend time with his group in Tokyo, but also for his help in understanding the most complex aspects of my project. Finally I’d like to thank Professor Nigel Scrutton for giving me the position and to Marie Curie Action for their funding. On a personal note, the time doing my PhD has been a fantastic time in my life, not only because of the work I’ve done, but mostly because it made me realize that I’m capable of doing some pretty awesome things. I’d love to thank my friends- Ines, Zhalgas, Ana and Priscilla, who definitely kept me from going crazy. I’d like to thank my parents in Poland and my in-laws here in England for their support and understanding during stressful times. Finally, I’d like to thank my husband Neil- I know that living with me is not always easy and I’m quite unbearable when deadlines are catching up. I appreciate you haven’t left!

Chapter 1

Introduction

Although it has been known for many years that migratory birds and other animals use Earth’s magnetic field (MF) in order to navigate, the process of detecting it and passing the information to the brain remains unclear.1 The MF generated by the Earth is so low, that the magnetic energy of most molecules is much smaller than the thermal energy kB of the system and it is considered that unless energy at least equal to that is supplied, no chemical reaction will take place.2 From a thermodynamical point of view, MFs should not be able to affect living organisms at all and the theory remained controversial for many years. The situation however changed in the 1970s,3 when a series of magnetic field effect (MFE) studies were reported with explanations ranging from the presence of magnetite to photochemical reactions. MFs may have a significant effect on enzymatic reactions as enzyme catalytic cycles containing radicals as reaction intermediates have been identified as an example of potential carriers of biological field sensitivity. It is apparent that there is a potentially harmful interaction, the mechanism of which remains unclear. Until today the only plausible theory explaining the magnetosensitivity of chemical reactions remains the Radical Pair Mechanism (RPM), in which a bond breaks creating two radicals in a close proximity called the radical pair (RP).3 The unpaired electrons on both radicals have an intrinsic property, called spin, which can be imagined as a tiny, spinning charge generating the magnetic moment. It has been determined that every electron can have only two possible conformations, up or down. In large MFs there are in total four possible combinations of these pairs, three separate states when spins are parallel called the triplet (T) states and one state when they are antiparallel- the singlet (S) state. These states can interconvert in the process known as spin state mixing.1 When the external field is none or low, the interconversion is driven by the hyperfine interactions (HFI). The mixing can take place only when HFI constants for two radicals are not identical; the bigger the difference between the two values, the more efficient the interconversion. Due to the Pauli Exclusion Principle,4 only S-state can recombine back to the ground state. As the reaction proceeds radicals can separate

from each other and form free radicals, at which point the mixing of the states can no longer take place.5,6,7 If the process of mixing is enhanced, the population of the S-state, thus the ground state can be increased, assuming the generated RP is in the T-state, but the opposite is true for S-born RPs.8 For most organic radicals, the differences in HFI constants are small, a couple of mT, but there are systems, where the difference is much bigger. An example of such system is adenosylcobalamin (AdoCbl), commonly known as vitamin B12. AdoCbl is the largest and most complex of all vitamins and the only one containing cobalt, it is necessary for normal functioning of the brain, and in the process 2 of blood formation. Deficiency of vitamin B12 leads to anaemia and other haematological diseases. In this case, photoexcitation leads to a bond breaking resulting in the formation of a S-born RP consisting of the adenosyl and Cbl(II) radicals, the hyperfine constants of which are 2.7 and 15.9 mT respectively.9 Due to the big difference between the hyperfine constants, the spin state mixing is expected to be ultrafast and very efficient and as reported previously takes place within 430 ps. However, due to presence of a heavy transition metal, the spin orbit couplings are also expected to be large, resulting in ultrafast relaxation processes which can disrupt the spin state mixing. This is a biological system, which provides perfect conditions for the observations of MFEs. Here we have used transient absorption (TA) spectroscopy, a technique for observing processes occurring on fast timescales, in which an ultrafast laser pulse passes through a sample and initiates the reaction and, then a second pulse, called the probe, investigates the subsequent changes. In order to provide a wide range of MFs that can be easily changed during the measurements neodymium disc magnets were employed and provided variable MF in the range of up to 950 mT. The MF generating apparatus has been designed and constructed and numerous tests and experiments have been performed using the intramolecular electron transfer (ET) in flavin adenine dinucleotide (FAD) as a test system.10,11 We have designed a new TA technique, in which changes induced by a MF can be observed more directly. In the new setup, an additional path was created, in such way that two samples are excited and probed at the same time, but one of them is subjected to the influence of externally applied MF, whilst the one is shielded from its influence. In order to increase our signal to noise ratio, single point detectors and a lock-in amplifier were employed. Using new instrumentation required the creation of new data 19

acquisition software that could be used for the experiments; this was done using the visual programming language LabVIEW. Using this novel experimental set-up the influence of MFs on dynamics of AdoCbl has been investigated in external fields up to

950 mT.

1.1 Photochemistry

Photochemistry is an important part of science focusing on one of the most fundamental natural processes- how light interacts with different molecules and what effects on them it might have.12 Although light is crucial for life on Earth, it can often be destructive and lead to processes that cannot always be reversed. Photochemical reactions are caused by absorption of light and it is crucial to understand its basic principles and properties before we fully understand all the implications of the influence they may have on the biological world.

1.1.1 Photophysics

Scientists have always been fascinated by light and its nature, but only since the mid-nineteenth century have they begun to understand this most fundamental natural phenomenon. Initial theories were created by Isaac Newton,13 who described light as a stream of particles, while according to the theory of Huygens14 light was a wave. Both theories have long existed alongside one another, but neither one of them explained all properties of light. The corpuscular theory could not explain why separate beams interfere with each other, because individual particles should not interact with each other, but the answer is rather simple when we think of light as a wave. The situation remained unclear until the beginning of nineteenth century, when the phenomenon of light diffraction and interference, typical for waves, were discovered and the corpuscular theory seemed to have been defeated. Further discoveries of Maxwell have only confirmed it; he established that the speed of light in the vacuum is equal to the speed of electromagnetic field propagation, which meant that light is a form of electromagnetic (EM) radiation itself. Maxwell postulated that it is characterized by its wavelength (λ), frequency (v) and the velocity of light (c= 3⋅108 m/s) related to each other as shown in equation below. c= v⋅ λ Eq.1.1

However plausible and widely accepted the theory seemed at the time, the beginning of twentieth century brought the arguments back. Scientists reinvigorated the previously rejected corpuscular theory, when they observed that during the process of emission and absorption light acts like a stream of particles, which Albert Einstein called quanta15 or photons; each one of them was said to possess energy that depends on its frequency and the Planck constant (h= 6.63⋅10-34 J⋅s), related by the expression: E= h⋅ v Eq.1.2 An unusual situation arose, when two supposedly incompatible approaches combined into one provided the final explanation. Coupling both equations (Eq.1.1 and Eq.1.2) resulted in a new theory which states that the energy of a photon is determined by its wavelength, speed and the Planck constant related as shown: ℎ푐 E= Eq.1.3 휆 The equation above underlies the theory of wave-particle duality of light and until today it is still considered to be one of the most surprising and important discoveries of physics in the twentieth century. The majority of the energy received by the Earth comes from the Sun in the form of EM radiation of ultraviolet, visible or infrared wavelengths. Different types of radiation depending on their wavelength are shown in Fig.1.1

Fig.1.1 Different types of radiation that compose the electromagnetic spectrum and their respective frequency, wavelength and relation with energy. Higher frequencies (or shorter wavelengths) carry higher energy. The visible region of the spectrum has been expanded.16

Visible radiation initiates vital processes in living organisms, such as photosynthesis; ultraviolet radiation plays a key role in maintaining a low concentration of ozone in the upper atmosphere, which in turn absorbs most of the more harmful short wavelength radiation preventing it from reaching the surface of the planet. Absorption of light can lead to changes in energy and electronic structure without altering the original molecular species, but it can also lead to photochemical changes, which result in whole new entities. The foundations for understanding photochemistry consist of two main principles. The Grotthuss-Draper law17 states that in order for the photochemical reaction to occur, light must be absorbed by a compound and the Stark- Einstein law states18 that for each photon of light absorbed by a chemical system, only one molecule is activated. Once light is absorbed, new electronic excited states are populated. Such excitation results in a change in molecular orbital occupancy, an increase in the energy and changes in local bonding as well as charge distribution. The reorganization of the electrons occurs much more quickly than any subsequent movements, which led to formulation of the Franck- Condon principle stating that electronic transitions occur much faster than nuclei can respond.19 Because of the fast timescales there is little or no geometry change in the molecular system, and transitions occur vertically on a potential energy diagram, as shown in Fig.1.2.

Fig.1.2 Potential energy diagram for the ground state (purple) and an excited state (navy) of a diatomic molecule. The letter r represents the interatomic distance, A- absorption and F- fluorescence. The numbers indicate vibrational states. Adapted from reference. 20

After absorption of light a number of events may take place. A scheme of photophysical processes that can follow, for compounds having more than two atoms, is shown in Jablonski diagram (Fig.1.3).

Fig.1.3 Jablonski diagram for a hypothetical organic molecule illustrating the absorption of light and the photophysical processes that can follow the light absorption. Straight arrows show radiative transitions; the wavy arrows show the non-radiative transitions. Letters S represent singlet states, and letters T- triplet state. Adapted from21.

These processes can be separated into two main types- the radiative and non-radiative transitions. Photoluminescence due to a transition from the lowest vibrational level of the excited electron state (S1) to any vibrational level of ground state (S0) is called fluorescence. The non-radiative transition of the electron from S1 to metastable triplet state (T1) can result in emission connected to the T1→ S0 transition, shifted towards longer wavelengths compared to fluorescence, which is called phosphorescence. The relative positions of typical spectra are shown in Fig.1.4. The excitation of the molecule causes changes in electronic configuration, so the nuclei must adjust to a new configuration, which instantaneously creates molecular vibrations. The excitation leads to a transition to a non-equilibrium state (Franck–Condon state) and the approach to thermal equilibrium is very fast, on order of picoseconds, in liquid solutions at room temperature, due to a number of collisions with the molecules of solvent. According to Kasha’s rule in the electronic excited state, molecules quickly relax to the lowest vibrational level.22 It states that the molecule will only emit light from its lowest energy excited state. The Franck-Condon principle and Kasha’s rule combined are the reason for the mirror symmetry of the absorption and fluorescence spectra. During the excitation/emission cycle the molecule will experience a loss of vibrational energy, and 23

the emission will always occur at lower energy, which is called the Stokes shift.23 The magnitude of the Stokes shift is characteristic for each of the fluorophores.

Fig.1.4 Relative positions of different spectra of absorption, fluorescence and phosphorescence. The shift in fluorescence spectra relative to the absorption is called the Stokes shift and it is the difference between the peak excitation and the peak emission wavelengths. Letters S represent the singlet states, and letters T represent the triplet states, the arrows indicate from and to which state the transition occurs. Adapted from21.

The nonradiative transitions between different excited states of the same multiplicity (S2→S1) are called internal conversion, and nonradiative transitions between states of different multiplicity (S1→T1 or T1→S0) are called internal crossings. These types of nonradiative transitions occur when multidimensional hypersurfaces of potential energies of specific states approach each other around a certain point or even cross each other. Both internal conversions, S1→S0 (T2→T1) and S1→T1 (T1→S0), take place without changing the atomic configuration. The approximate timescales for different transitions are given in the Table 1.1.

Table 1.1 The approximate timescales for different transitions. The non-radiative decay in the last row, might take place by intermolecular energy transfer to a different molecule, in a process of quenching (when the focus is on the initially excited species) or sensitization (if a subject of interest is the newly created states).24

Assuming that sufficient energy is provided, electrons can be excited from the ground state and promoted to higher, excited levels and exist in them for a brief moment before relaxing back to previously occupied and energetically more stable state, which will be accompanied by releasing energy in the form of photon. If there is not enough energy to incentivise the electrons, no transition will happen. Transitions are not observed between all pairs of energy levels. Some transitions have very low probability of taking place and are therefore called ‘forbidden’. The ones that do occur efficiently are ‘allowed’ by a set of Selection Rules. The first of them is known as the Spin Rule:25

ΔS = 0 Eq.1.4 stating that allowed transition must involve the promotion of electrons without any change in their spin meaning that transitions can only occur between states of the same spin and therefore the same spin multiplicity. The second one of the rules is called the Orbital Rule or Laporte Rule:

Δl = ± 1 Eq.1.5 This states that if a molecule has a centre of symmetry, transitions within a given set of p or d orbitals are forbidden.

1.1.2 Photochemical reactions

Different to a photophysical process is a photochemical reaction, in which absorption of energy in the form of light leads to the formation of new products, the properties of which are different than the properties of the original molecule. The resulting species may dissociate, incorporate, isomerise and transfer hydrogen atoms, protons or electrons. An example of a photochemical reaction is chemiluminescence, which is found in many ocean creatures. Fireflies use bioluminescence to produce a chemical substance called luciferin, which undergoes conversion into an intermediate compound and then rapidly degrades into oxyluciferin and carbon dioxide accompanied by the emission of photon. Another example is the ability to perform photosynthesis by plants which can be affected by a process of photosensitization occurring due to powerfully oxidative molecular oxygen that can oxidise a nearby molecule changing its structure and colour.

This process is known as photo-bleaching. Some organisms have found a way to protect themselves from damaging processes occurring from excited states by forcing highly efficient internal crossings and utilizing the absorbed energy as heat. This method of photoprotection is used by all known photosynthetic organisms and also the eyes of animals. One of the processes when absorbed light causes changes in shape of the molecule is called photoisomerization. The change occurs due to electron distribution in the excited state and in the ground state being different; the molecule attempts to adopt the new conformation by rotating until the shape of its nuclei matches the distribution of the electrons. Light might force a molecule to rearrange its structure and form a new species, an example of which is conversion of 7-dehydrocholesterol to vitamin D in the skin. Due to changes in electron configuration of the excited molecules, also their chemical properties change with respect to the ones in the ground state and an electron transfer (ET) between two molecules in the ground state might take place. To achieve this, the energy of the highest occupied molecular orbital (HOMO) of the electron donor must be higher than the energy of the lowest unoccupied molecular orbital (LUMO) of the electron acceptor.26 The ET is also possible in cases where the ground state HOMO of the donor molecule is energetically lower than the LUMO of the acceptor, when one of them is photochemically excited. The endothermicity of the transfer in the ground state is compensated by the excitation energy. ET in biological systems is usually fast and often coupled to other biochemically significant reactions (such as substrate transformation and proton transfer). Photochemical methods offer a convenient systems approach to the study of these processes because of their time resolution and chemical selectivity. When the absorption of light leads to dissociation of a molecule into two fragments the process of photodissociation occurs. The absorbed energy takes the molecule into an excited state, where one of the bonds, cleaves homolytically and results in formation of two fragments. These highly reactive species can also be created when ET between two organic molecules27 or hydrogen abstraction take place and are known as radicals.28

1.2 Radical Pair Chemistry

Since the 1970s it has been known that certain chemical reactions can be affected by external MFs. The key species are radical pairs (RP), which are pairs of

transient radicals created simultaneously from non-radical precursors; the two electron spins of the radicals are correlated. Experimental and theoretical studies of the radical pair mechanism (RPM) have provided data on the magnetic properties, kinetics and dynamics of radicals and their reaction. This field has come to be called spin chemistry.29

1.2.1 Radicals

Radicals are molecules with unpaired electrons, which are known to be highly reactive and unstable, usually due to being electron deficient.30 Radicals can be generated by different processes initiated either thermally or photochemically, as listed below. Homolysis31 A-B → [A• + B•] Electron Transfer31 A-B → [A•+ + B• -] Hydrogen Atom Transfer31 A-H + B → [A• + H-B•] Radical Transfer32 A• + B → A + B• Radical Addition32 A• + B → AB•

A radical pair (RP) is a reaction intermediate, usually of a short lifetime, which consists of two radicals. They are known as geminate RPs, which means that they are ‘born together’.

1.2.1 Properties of spin

Electrons were long known to possess properties of charge, but it was soon discovered that they possess one more important property. In 1921 Stern and Gerlach performed an experiment using a beam of silver atoms passing through inhomogeneous magnetic field (MF) which would then be detected on the photoplate. The atoms of silver allowed studying the magnetic properties of a single electron, because they have a single outer electron which moves in the Coulomb potential. No interaction with the external MF was expected, due to the electron having a zero orbital angular momentum (orbital quantum number l, is equal to 0).33 While propagating through the field the magnetic dipole moment would experience a force proportional to the field gradient since the two ‘poles’ will be affected by different fields. What they expected to observe

was continuous smear on the plate, but what actually happened was that beams were being separated into two distinct parts. This indicated that magnetic moment of the electron can only have two possible orientations; following the pattern of quantized

1 angular momentum, an angular momentum quantum number is required to be ⁄2. The arising question was how an electron with a zero angular momentum could achieve a magnetic moment and it was further studied and determined that electrons possess an additional property- intrinsic spin angular momentum, or simply spin, which meant that it will also have associated MF known as the magnetic dipole moment of the electron. The magnetic moments for an electron and proton are defined as34:

μel= −푔푒 · 휇B · 푚s Eq.1.6 푒·ℏ μpr= Eq.1.7 2푚p

where e represents the charge, 휇B is the Bohr magneton, mp stands for mass of the proton, the ge is the g-factor and ms is the electron spin quantum number. The magnetic dipole moment of the electron results in their sensitivity to externally applied MFs. For the electron, the magnetic moment is opposite to the spin vector, because it has a negative charge, but for a proton, the magnetic moment lies in the same direction as the spin vector. The electron’s spin can only have two possible values- + ½ (↑) or – ½ (↓). Due to the Pauli Exclusion Principle, each orbital can contain only one electron of each spin; if every occupied orbital holds a pair of electrons with opposing spin, the molecule is in a singlet state (S), which is the pattern for the ground state of most molecules. When the molecule is excited, for example by absorption of a photon, one electron is promoted to a previously unoccupied orbital, and, if its spin does not change, the two (now unpaired) electrons still have opposing spin (↑↓) and the molecule is still in a singlet state. If the unpaired electrons have parallel spins (↑↑), the molecule is in a triplet state (T). A change in intrinsic electron spin is forbidden, so conversion of a molecule from singlet to triplet or vice versa is slow compared with other molecular processes. The unpaired electron on the radical can also have only two possible spins- ± ½. A RP consists of two radicals, both of which have odd number of electrons. Consequently spins of RP can be aligned antiparallel, in which case it will be in an S- state or parallel, when the RP will be in a T-state. Furthermore, since chemical reactions are usually spin-conserving1 the multiplicity of the RP remains the same as the

multiplicity of its precursor. The overall spin of the RP can be determined as shown below:

1 1 (ms= + ⁄2 ) R1↑ ↓R2 (ms= - ⁄2) the multiplicity of 2S+1= 1 one SINGLET 1 1 (ms= + ⁄2 ) R1↑ ↑R2 (ms= + ⁄2) the multiplicity of 2S+1= 3 three TRIPLETS

The multiplicity of the state indicates the number of eigenstates the RP can exist in.35 The Pauli Exclusion Principle states that no two electrons can occupy the same energy eigenstate,4 therefore, the two electrons cannot have the same spin-state and the z-components of each radical have to be aligned antiparallel. If radicals that encounter are in a triplet state they are either unreactive or they react to form a triplet product meaning that the recombination takes place mostly via the singlet state. Many atomic nuclei have spin, which is consequence of the spins of their protons and neutrons, and therefore the associated magnetic moments that interact with the unpaired electron. This is known as hyperfine interactions (HFI)1 and it gives a rise to a process known as spin-state mixing.36

1.2.3 Spin- state mixing

Radicals in both S and T states are highly reactive and non-stationary meaning that once created, they are likely to convert into each other, known as spin-state mixing. Although the interconversion between these states is very fast it is energetically unfavourable in some regions. Before the radicals are able to undergo spin-state mixing in zero applied MF they have to diffuse apart in order to overcome the energy difference between them as shown in Fig.1.5.

Fig.1.5 Energy diagram for the approach of two hydrogen atoms. The red line represents the triplet state and the green one- the singlet. 2J(r), the energy difference between the two configurations represents the electron exchange interaction. Adapted from.8

One might note from Fig.1.5 that if the two radicals are too close to each other, the energy gap between them is simply too high and no conversion can take place. The radicals must therefore diffuse apart in order for the exchange interaction to become small enough for the process of spin-state mixing can take place. In order for the radicals to diffuse away, a certain amount of time is required. The frequency of spin-state mixing can be estimated with a use of, shown in Eq.8, semiclassical description of spin motion in radicals:37

퐻1−퐻2 ω= 푔 · 훽 Eq.1.8 ℏ where g is the g-value of the free electron, β stands for the Bohr magneton and H1 and H2 represent the average hyperfine couplings for each of the unpaired spins. It can be noted from this equation that large hyperfine couplings result in higher frequency of the mixing. For most organic radicals these values are not much different from those of a II free electron, but the values for the Cbl / adenosyl RP are much higher (15.9 mT and

2.71 mT respectively). In the absence of external MF the process of mixing is facilitated by the existence of HFI and both electron and nuclear magnetic spins precess about their 37 combined local MF (Blocal) with a Larmor precession frequency:

𝑔·휇 ·퐵 ω= 퐵 푙표푐푎푙 Eq.1.9 ℏ

This precession can be represented as a vector model shown in Fig.1.6. The figure represents the singlet state (S), and three triplet states (T-1, T0, T+1) in a strong external MF. If the hyperfine couplings (HFC) within each radical of the RP are different, the electron spins will precess with different frequencies, leading to interconversion between the S and T0 states.

Fig.1.6 Vector representation of RP spin states in an applied external MF. The green circles represent the electrons and the arrows- spin vectors, each of which is in constant precession about the MF axis at the Larmor frequency of the respective radicals. Adapted from.8

Since spin-state mixing involves interaction of internal magnetic fields, it is expected to be sensitive to application of an external MF. The extent of the conversion can be monitored through the concentration of the radicals or reaction products over time. If the lifetime of the RP is too short, the radicals will not have enough time diffuse from each other and the energy gap will be too big to overcome. Consequently no spin- state mixing will take place. The mixing of the states may be stopped by one more process; after the RP is created it will start to relax towards the equilibrium, where all correlations are gone and a MF cannot affect it. Although relaxation in non-viscous solutions occurs on timescales slower than lifetimes of most organic RPs, there are situations, when the relaxation is more rapid and the spin-state mixing might be affected.

1.2.4 Spin Hamiltonian of a Radical Pair

In order to describe the energetics and populations of the RP spins states a spin Hamiltonian is used. A suitable Hamiltonian for a RP consisting of radical 1 and 2 is shown below: 37

퐻̂= 퐻̂z(B) +퐻̂ihf +퐻̂ex(r) Eq.1.10

which can be further expanded into:

1 ̂퐻 = ∑2 ( 휇 g ·푆̂ B + Σa 퐼̂ 푆̂ +Σa 퐼̂ – J( + 푆̂ 푆̂ ) Eq.1.11 RP 푖=1 퐵 · i i· ij ij i k k 2 1 · 2

The first term, 퐻̂z(B), describes the electronic Zeeman energy, which is the interaction of the two electrons of the RP with the MF, B; 푆̂ is the spin operator for each radical and g is the g-factor of each radical, a chemical shift of the unpaired electrons.

The second term, 퐻̂ihf, describes the hyperfine interaction between each radical and the nuclei on this radical, where 푆̂ is the spin operator, 퐼̂ is the nuclear spin operator and aij and ak are the hyperfine coupling constants. The final term, 퐻̂ex(r), is the exchange interaction (J) between the unpaired electrons. The spin-state mixing occurs mostly at RP separations where the exchange interaction J, is negligible and can be omitted from calculations. 8

1.2.5 Radical Pair Mechanism

Although it has been known for many years that migratory birds and other animals use Earth’s MF in order to navigate 38, the process of detecting it and passing the information to brain remains unclear. The MF generated by the Earth is very low

(25 – 65 μT) and for many years scientists believed that it had no significant effect on chemical or biochemical reactions. The magnetic energy of most molecules is much smaller than thermal energy kBT (where kB is the Boltzmann’s constant and T is temperature). It is considered that unless energy at least equal to that is supplied, no chemical reaction will take place.39 The situation, however, changed in the 1970s, when a series of MFE studies40,41,42,43 were reported. It was demonstrated that if a chemical system has first been brought into a non-equilibrium state, even tiny interactions can play an important part. Biological processes, such as the Radical Pair Mechanism (RPM) rarely run at equilibrium.30 According to the RPM an externally applied MF can influence chemical reactions by affecting the electron spin state of a weakly coupled RP.44 Once two radicals begin to diffuse away from each other, the exchange interaction J(r) becomes negligible and the S-T interconversion can take place due to 32

weak magnetic interactions including Zeeman and the hyperfine interactions (HFI), both are described further in section 1.3.2. Opposite effects on the product yield will be described in section 1.3.3, the Δg mechanism, which becomes appreciable in higher fields. Finally, the S-T conversion can be affected by Low Field Effect (LFE) typically occurring in very low fields of around 1 mT. The response of the reaction yields and the kinetics of the RP to an externally applied MF are highly non-linear and the general trends are shown in Fig.1.7.

Fig.1.7 The effect of increasing MF on a singlet reaction yield for a T-born RP. Adapted from reference.45

If radicals continue diffusing apart they will form free radicals and escape products, and the conversion will no longer occur.46 Consequently no MFE will be observed. Until today there have been a few hundred different studies reported, in which

MFEs have been observed in fields of up to 100 mT. The RPM is now admittedly authentic, but it has not yet been proven to be the origin of magnetoreception in birds. Nevertheless magnetoreceptors based on RPM still remain the only plausible theory 3,47 since it was first proposed in 1978 by Schulten. It is believed that a ‘receptor’ protein called cryptochrome found in bird’s eyes undergoes a light-activated reaction which is affected by magnetism. By detecting the rate of this reaction, birds can not only perceive the magnetic strength but also the alignment of the field. No other photoreceptor molecule has been found to form RP upon the excitation by light and the only one other biomolecule, chlorophyll is known to generate RP in vivo in the photosynthesis process,48 however it is not found in birds.

1.3 Magnetic Field Effect (MFE)

1.3.1 Chemical processes

Spin chemistry focuses on the influence of electron and nuclear spins on chemical reactivity. The number of studies, where a MFE on a chemical reaction were observed is limited. Furthermore, many early studies were often missing a plausible explanation and the data reproduction has often failed.49 One of the first convincing studies was not published until the 1960s, when Johnson and co-workers50 reported that the intensity of fluorescence resulting from mutual annihilation of triplet excitons in anthracene crystals at room temperature increased by 5% in weak MF up to 35 mT and decreased by around 20% in fields higher than 0.5 mT. A similar approach was taken investigating the processes of radiolysis, where due to collision of particles of high energies51 radical ions are created which then react with each other. It was Brocklehurst, who noticed that during chemical reactions electron spin is conserved.52,6 He was able to predict that the radical ions recombination will be sensitive to applied MFs. At the same time, several independent researchers noticed anomalous line shapes in the EPR spectra of radical intermediates and lately also in NMR spectra53 of the radical reaction products, which were then interpreted independently54,55 in terms of the RPM. In the beginning of 1970s the emissive signals in chemically induced dynamic electron polarization (CIDEP) were explained by the triplet mechanism (TM), in which the polarization originates in the triplet as the result of spin selective intersystem crossing from the photoexcited singlet state56, both in UK57 and Canada.58 Not long after that Pederson and Freed published a series of articles, providing a comprehensive description of CIDEP theory.59,60,61 The number of studies in spin chemistry field has been increasing and in 1970s Schulten proposed the RPM and the origin of magnetoreception in birds, which initiated another growth in the field. By the end of the decade research groups investigating spin chemistry were established in different countries including USA, Japan, Russia, UK and many others in Europe.

1.3.2 Hyperfine mechanism and the effect of the Zeeman interaction

Without the application of an external MF the spin-state mixing takes place between all spin states, and so the RP will exist in different proportion in all states at the same time. Once the MF is applied however the degeneracy among the three triplet states is removed by Zeeman energy splitting as shown in Fig.1.8.

Fig.1.8 The Zeeman effect. An external magnetic field lifts the degeneracy between the singlet and triplet sublevels. As the field increases the T+1 and T-1 states become increasingly separated in energy from the S0 and T0. Mixing between S and T ±1 becomes slower and slower until it no longer occurs. The grey area represents the situation, when process of spin state mixing takes place.

T+1 and T-1 have a net magnetisation along the direction of the applied field leading to splitting of these levels. The S state is not a magnetic state and the T0 state has a magnetic vector orthogonal to the direction of an applied field, so their energies are not affected by increasing MF and they stay separated by a value of exchange interaction energy 2J(r). As the field increases so does the separation between T+1 and

T-1. Mixing between those becomes slower and slower until it no longer occurs and the only states that can still interconvert are S and T0. The HFIs facilitate the spin-state mixing, but as the external field increases beyond the level of the effective hyperfine coupling constant, ISC via the HFI becomes smaller because fewer RPs are converted to the triplet state, and the net rate of

recombination may increase (assuming that the spin-correlated radical pair is born from a singlet precursor). This results in a narrower magnetic field window beyond which ISC is disfavoured.

1.3.3 The Δg mechanism

Although at very high MFs the T+1 and T-1 states are very different in energy and the mixing of the states is limited, the Δg mechanism can affect the reactivity by 62 increasing the rate of spin-state mixing between S and T0. A MF of a specific strength will produce a net decrease in RP recombination by converting some of the singlet radical pairs into triplets. It can be noticed from the first term of the spin Hamiltonian for a RP: ̂ 2 ̂ 퐻푧(퐵) = ∑푖=1( 휇퐵 ·gi·푆i·B) Eq.1.12

that the required fields in this case are relatively high. For most of organic radicals the values of g- factors of the two radicals are very similar, and this term can be omitted from calculations, however, when the applied field is large, it is no longer negligible. Hence, the effect of the Δg mechanism may be observed only in high MFs and it is opposite to a MF-induced change through hyperfine mechanism.

Another way that the reactivity of a RP might be affected by MF is the S/T-1 level crossing mechanism. For a relatively strongly coupled RP, in which the two radicals are fixed at a distance of less than 10 Å, the energy difference between the singlet and triplet spin states, ΔEST, can be large as well as the exchange interaction, J.

If 2J >> EHFI (the energy of hyperfine interaction), then hyperfine interactions cannot mix the singlet and triplet states. However there might be a region on the energy surface where the S and T-1 states are degenerate. If the appropriate magnetic field is selected such that the Zeeman interaction energy splits the three triplet spin states by exactly this amount, mixing between the S and T-1 can happen. Hence, a narrow region of MF values will produce an increase in ISC.

1.3.4 The Low Field Effect (LFE)

The LFE typically occurs in very low fields of around 1 mT for hydrocarbon radicals. The effect is in the opposite phase to the ‘normal’ MFE. The MF lifts some state degeneracies and the two states that had quantum coherence with zero amplitude, have now non-zero amplitude which opens new S-T mixing pathways. From the Zeeman interaction for a singlet born RP the yield of geminate recombination products is expected to initially increase with field, but in this case, it decreases, and the other way around if the RP is a triplet born. The LFE is dominant at longer RP lifetime. In these studies effects of higher MF are the main interest and the LFE will not be further discussed.

1.3.5 Influence of MF on biological systems

Devices generating high MFs are used in many industrial processes as well as medicine and more studies have been performed in order to determine the influence that 63 MFs may have on us , but the relevance of MFEs on human health remains uncertain. Although it is well established that MFs can affect biological processes involving photochemical reactions, the MFE on biological activity remains unclear and only few enzyme systems, have been reported to be influenced by application of a MF. An exception to this is a series of studies performed by Buchachenko and Kouznetsov 64,65,66, who reported MFE on the rate of enzymatic synthesis of ATP in vitro. The observed MFEs were clear and large and the proposed mechanism credible.67 More support has been provided by further studies on phosphorylation, in which rates for the magnetic and nonmagnetic isotopes were twice as high when an external MF of 80 mT 65 68 was applied. Crotty and co-workers attempted to replicate these experiments using the same enzyme, but with different techniques. This work was performed by two independent groups in Dublin and Colchester, but neither one of them managed to confirm the existence of an MFE. Although the conditions were kept as similar as possible, the source of enzyme was different, but it was pointed out by the authors that these changes were probably irrelevant for these studies and the data irreproducibility could not be explained.

One of the earliest studied enzymes is ethanolamine ammonia lyase (EAL), adenosylcobalamin (AdoCbl) dependent enzyme responsible for the conversion of ethanolamine to acetaldehyde and ammonia. It was proposed by Harkins and Grissom, who observed MF influence on kinetic parameters V, Vmax/Km and proposed that the magnetically-sensitive step in AdoCbl activation is the reversible C-Co bond homolysis.69 The 5’-deoxyadenosyl radical that results from the homolysis abstracts an atom of hydrogen from the substrate forming substrate radical, which then undergoes a 1.2-rearrangement of the amine group to the product radical followed by abstraction of hydrogen back from the adenosine and dissociating into products. The kinetic parameter

Vm is limited by the release of the product, hence no MFE was observed. The Vmax/Km parameter decreased by up to 25% with increasing applied field reaching the minimum value at 100 mT. The observed MFEs were large, but further studies of Jones and co- workers were unable to reproduce these data, for two separate enzyme reactions in 70 vitro- the conversion of ethanolamine to acetaldehyde by EAL , and the reduction of hydrogen peroxide by HRP.71 There are many reported studies, where MFEs were observed in very weak fields, but often they lack explanation and the replication attempts were unsuccessful, although a study of the influence of MF on seeding growth in the model plant Arabidopsis thaliana72 makes its mark. The MF sensitivity in this case was said to lie in photoinduced RP reaction in cryptochrome photoreceptors, which was reported to be enhanced when external MF of 500 μT was applied. The attempt by Harris et al.73 to replicate these experiments have once more failed. Although the experimental conditions were chosen to match those of the original study, no consistent MF sensitivity was reported, the reason for which is not clear. A variety of flavin-dependent redox enzymes has been studied,74 but no MFE was observed. It was suggested that the reaction mechanism does not involve RP intermediates and that the generations of RP intermediates is not sufficient for a MFE to be apparent. MFEs have been observed in studies of intramolecular ET in flavin adenine 11 dinucleotide (FAD) in low pH . The TA signal in 200 mT observed at 600 nm has increased from that in zero MF. The MFE curve obtained by plotting the increment of TA versus the MF was said to be characteristic of the hyperfine and relaxation mechanism37 and has provided evidence that the RP is generated through intramolecular ET reactions. The time profiles were highly affected by pH and the MFEs became much slower in higher pH and disappeared within few hundreds of ns. 38

In order for any MF- induced changes to appear a number of conditions must be met. The lifetimes of the RP must be long enough for radicals to diffuse apart and the process of spin-state mixing needs to be very fast. Furthermore the reaction has to be spin-selective, where at least one of the reactants form exclusively S or T spin state. Even if all these conditions are met in an enzyme system it would not necessarily result in any change in its biological functions. Despite radicals being involved in many biological processes, MFEs are rarely observed. In 1975 an interesting group of living organisms called magnetotactic bacteria were discovered that are able to synthesise organic chain structures containing magnetite (Fe3O4). The magnetite crystals are known to have a net permanent magnetic moment. It was determined that magnetotactic bacteria synthesize chains of nano-sized magnetic particles that function as a compass needle,75 which enables the microbes to align themselves with the MF generated by the Earth. The magnetic nanoparticles are synthesized by a specific set of proteins within membrane-bound organelles.76 Recently Magnetococcus marinus magnetic bacteria have been suggested as a potential method for drug delivery system to target cancer tissues without harming healthy cells and in oxygen-depleted hypoxic regions in the tumour that are generally resistant to therapies. 76,77 An interesting approach was taken in one of the first studies of the effects upon living organisms of exposure to MF equal or lower than that of the Earth. The research was conducted by NASA in the early 70s and a very different aspect was considered, where Beischer and co-workers expressed their anticipation for biological problems related to very low MFs found in space.78 Their hypothesis was that ‘the presence of MF during the major part of the development of life on Earth has played a certain role in development and that living beings probably cannot be removed from the geomagnetic environment without penalty’. During various experiments groups of mice were subjected to low fields generated by coils and data were compared with mice that were shielded from MF by Mu-metal, but no significant difference was noticed. Similarly, no effect was observed either in high or low fields upon the in vitro activities of three enzymes studied. Today the influence of MF in biochemistry is studied in two main areas- the magnetic compass sense that migratory birds and other animals possess and the effect it may have on human health. The results of some studies have suggested an increase in childhood leukaemia in areas close to large power lines, but data collected until now do

not provide a sufficient evidence. Studies have also looked at whether MF exposure is linked to the risk of other illnesses such as Alzheimer’s disease. Although there have been some results suggesting a link, the overall balance of evidence is towards no effect, even weaker than that for childhood leukaemia. The complexity of the problem of biological MFEs may seem overwhelming and to fully explain it, experiments in various fields including chemistry, physics, biology and medicine must be performed and collaboration between scientists is required. Furthermore there is a need for studies in wide range of MFs, so if and when biological effect is observed a correlation between the strength and biological changes can be made.

1.4 Aims, Objectives and Motivation

As discussed, the spin dynamics of RPs can be significantly affected by the application of external fields. The response to MFs is highly non-linear and three distinct field ranges can be distinguished, in which different RP interactions dominate.8 The aim of this project is to design and construct a novel experimental setup which would enable the investigations of MFEs in biological and chemical systems with a particular interest in the ultrafast spin-state mixing processes in adenosylcobalamin and its dependent enzymes. Adenosylcobalamin is a perfect candidate for the investigation of MFEs with features not usually found in typical organic RPs: competition between ultrafast spin-state interconversion and rapid relaxation, and the possible contribution of the Δg at relatively low MFs (i.e., < 1 T). Such investigations, however, will require specialised apparatus capable of combining ultrafast kinetic measurements and a large range of MF-exposure amplitudes. The project will be divided into three main parts- designing and constructing variable MF generating apparatus and interfacing it with existing ultrafast TA setup, testing the performance of the new instrument with known, magnetically sensitive small molecule and finally investigations of MFEs in a series of cobalamin type compounds where the coherent spin state mixing lends itself to expected MFEs on ultrafast timescale using the newly developed experimental setup. The new setup will hopefully allow for the collection of TA data with better signal to noise ratio than that collected using the currently available setup.

1.5 References

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4. P. Atkins, R. Friedman; 'The Pauli Principle' in Molecular Quantum Mechanics; Oxford University Press, 2005, pp. 225–228.

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6. B. Brocklehurst; Spin correlation in the geminate recombination of radical ions in hydrocarbons. Part 2 - Time resolved single-photon counting study of the magnetic field effect. Faraday Discuss. Chem. Soc.; 1977, vol. 63, pp. 96–103.

7. C.R. Timmel, U. Till, B. Brocklehurst, K.A. McLauchlan, P. J. Hore; Effects of weak magnetic fields on free radical recombination reactions. Mol. Phys.1998, vol. 95, no. 1, pp. 71–89.

8. J. R. Woodward; Radical pairs in solution. Prog. React. Kinet. Mech. 2002, vol. 27, no. 3, pp. 165–207.

9. C.B. Grissom, E. Natrajan; Use of magnetic field effects to study coenzyme B12- dependent reactions. Methods Enzymol. 1997, vol. 281, no. 7, pp. 235–247.

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Chapter 2

Experimental methods

2.1 Transient absorption spectroscopy

Transient absorption (TA) is a technique usually used to observe events that occur on timescales from femtosecond (fs) to seconds (s). To monitor very fast (sub- nanosecond) processes a technique known as a pump-probe TA is used, where two synchronised laser pulses are used. The first of the beams, called the pump, traverses through the sample and initiates the reaction, while the second one, called the probe, which is delayed relative to the pump (although the reverse can also be used, where the pump is delayed relative to the probe), is then used to interrogate changes induced by the first beam.1 The basic TA scheme is shown in Fig.2.1. The laser pulses employed for ultrafast pump-probe TA experiments have a short duration and can therefore resolve processes that may occur in the femtosecond-picosecond regime, such as rapid relaxation to the lowest vibrational level (10-13 s), slower radiative (10-10-10-8 s) and non-radiative transitions (energy that is transferred by internal conversion, 10-13-10-12 s).

Fig.2.1 Basic scheme of transient absorption. The green colour represents the pump pulse, which initiates the reaction in the sample. The blue one shows the probe pulse, which is delayed on a delay stage and propagates through the sample at a certain time after the pump. After propagation through the sample, only the probe pulse is detected.

This type of spectroscopy encompasses a powerful set of tools for probing and characterizing the electronic and structural changes of short-lived excited states of many chemical, physical and biological systems. The TA technique employs time resolution of short pulses without relying upon the speed of the detectors, so the temporal resolution is usually only limited by the temporal resolution of the laser pulse. Furthermore, due to use of lock-in detection signals occurring only at a specific frequency are observed and any random noise is eliminated. Another advantage compared to other optical absorption based methods is that there are no limits due to non-emissive states or weakly-reflecting samples. The absorbance of the sample can be defined according to the Beer-Lambert law: 2,3 퐼푝푟표푏푒 A= -log [ ] = εLc, Eq.2.1 10 퐼0 where ε stands for molar absorption coefficient, c for concentration and L is the length of the distance that light traverses through the sample. Absorbance can also be defined by either amount of transmitted light, T, which passes through the sample, or by the probe intensity before (I0) and after transmitting through the sample (Iprobe).

퐼푝푟표푏푒 A= -log10T = -log10 [ ] Eq.2.2 퐼0

In pump-probe techniques an optical chopper is used to reduce (for example) the

1 kHz pulse frequency of the pump beam to 500 Hz, while the probe beam remains at 1 kHz. By using a lock-in amplifier to detect changes in signal at 500 Hz only the pump induced changes are detected. The fractional change in transmittance, ΔT/T can be defined as:

on off ∆T Iprobe−Iprobe = off Eq.2.3 T Iprobe

Δ푇 When << 1, approximations can be used which lead to a final equation for change in 푇 absorbance, ΔA: 1 Δ푇 ΔA= - ⋅ Eq.2.4 ln10 푇 In TA we observed changes in absorption (ΔA) as a function of time. If the probe beam arrives before the pump, no change in absorbance should be observed

except from species with lifetimes longer than the separation of probe pulses. There are three processes, which contribute to a typical TA spectrum (shown in Fig.2.2):

Fig.2.2 ΔA spectrum (thick solid line), due to ground state bleach (dashed), stimulated emission (dotted) and excited-state absorption (solid) 1 contributions. Adapted from reference.

The ground-state bleach (negative signal) occurs as the number of molecules in the ground state has decreased because they have been promoted to the excited state. Stimulated emission is the process occurring, when a photon from the probe pulse induces emission of another photon from the excited molecule, which then returns to the ground state. The produced photon is emitted in the exact same direction as the probe photon, and both will be detected, as a result the light intensity on the detector increases and the ΔA is negative. The stimulated emission is Stokes shifted with respect to the ground-state bleach. Excited-state absorption results from the optically allowed transitions from the excited (populated) states to higher excited states, this absorption of the probe pulse will results in the observed positive ΔA. In addition to these excited state features, photochemical reactions themselves can produce intermediates or products with different absorption spectra to the initial material which will result in ground state bleach and positive features from these reaction products.

2.1.1 TA Artifacts

The use of an intense pump pulse to excite the sample is often responsible for the existence of unwanted signals, so-called femtosecond artifacts, which arise due to simultaneous interaction of pump and probe photons. They can occur when the pump and probe are overlapped at a zero-delay time (T0), on a timescale comparable to the 3 pump-probe cross-correlation function. These artifacts disfigure the TA signal, and

therefore must be considered while performing data analysis, they can however also provide a useful measurement of the time resolution of the pump-probe setup. The most often encountered example of such signals is stimulated Raman 4 Scattering Amplification (SRA) of the solvent, which is observed when the pump wavelength is set close to the probe wavelength range. Although transient Raman scattering signals carry very interesting information, it is most often a strong and undesirable effect altering femtosecond transient absorption spectroscopy data. The analysis of a SRA signal can provide important information on the pump pulse itself. Apart from laser pulse duration, the pump’s temporal chirp can be determined.5 The chirp of an optical pulse is usually a time dependence of its instantaneous frequency. A beam with ‘spatial chirp’ has its different frequency components separated in space transverse to the propagation direction.6 Application of very short laser pulses (on the order of 100 fs), which result in extraordinary pump power density, encourages two photon absorption (TPA) which appears as a Gaussian-like peak around time-zero and is more pronounced for shorter pump wavelengths, since the TPA signal has been shown to increase with the square of the pump energy. Furthermore, while TPA signals are Gaussian-like below probing wavelengths of 500 nm, above this threshold the effects of cross phase modulation (XPM) begin to dominate and the associated oscillatory behaviour causes the Gaussian nature to deteriorate. Application of a spectrally very broad probe pulse, typically a white light continuum in the 300–1100 nm range, together with temporal chirp in the continuum and high pulse intensities, can lead to efficient XPM. The XPM signal emanates from 7 empty cell windows as well as from solvent contained in the cell. This too gives rise to an unwanted signal around the time-zero point.8 Lower frequencies, hence longer wavelengths, and ‘less chirp’ result in a smaller and broader XPM signal due to group- velocity dispersion (GVD). Therefore compensating for the chirp on the probe white- light continuum can significantly reduce the XPM signal.

2.2 Standard TA setup

2.2.1 Laser System

The Ti: Sapphire amplifier system is a Spectra Physics Solstice Ace, producing 6 mJ of 800 nm pulses at 1 kHz with pulse durations of 100 fs. The Solstice-Ace contains an Empower pump laser and a Mai-Tai seed laser as well as the pulse stretcher, a regenerative amplifier and pulse compressor (shown in Fig.2.3). The Empower is a frequency-doubled, Q-switched, diode pumped Nd:YLF laser which produces ~28 mJ of 527 nm output at 1 kHz. The Mai-Tai SP is a mode-locked Ti:Sapphire oscillator which produces ~0.95 W of power at 800 nm at a repetition rate of 80 MHz.

Fig.2.3 Block diagram of the Solstice Ace assembly. The Empower pump laser is a Q- switched, frequency-doubled laser that provides the optical energy used to amplify the pulses. The mode-locked Ti: Sapphire Mai Tai laser produces the ultrafast seed pulses for amplification.

Fig.2.4 The experimental amplifier laser setup.

As shown in Fig.2.4 the output of the Solstice Ace is split (65:35), with 2.1 W directed into one of the Optical Parametric Amplifiers (OPAs), TOPAS Prime 1, and the

remaining 65% becomes separated once more (50:50) with 1.95 W used to seed the second of the OPAs, TOPAS Prime 2. The wavelengths ranges of the OPAs are extended with two NIRUVis and one NDFG monolithic unit to cover wavelength ranges of 235-2600 nm and 235-11000 nm respectively. The remaining amplifier output

(1.95 W) is directed onto a beam splitter, where it is again split and 95% of the beam is directed onto a beam dump and remaining 5% (~100 mW) is reflected by set of mirrors, separated by another beam splitter, such that 90% of the remaining power is used for the Halcyone setup (an ultrafast fluorescence experiment, not discussed further in this work) and 10% for the Helios setup, where it is used to generate the probe beam. The output from the OPAs can be routed to any of the three experimental setups to be used as pump and/or probe beams.

2.2.2 Helios/Eos experimental setup

The output of TOPAS Prime 1 is directed onto mirror and attenuated by neutral density filter wheel and then directed by a set of mirrors into the Helios/Eos setup, shown in Fig.2.5, where it is used as the pump beam.

Fig.2.5 Helios/Eos experimental setup; the Helios/Eos pump path is represented by the navy line, the Helios probe path by the light blue and the Eos probe beam by the green ones.

The pump beam is routed into the enclosure via an iris; it is then attenuated by a variable neutral density filter wheel and depolarized before passing through the optical chopper, which reduces the frequency of the pump beam by a half. The beam is then routed by mirrors and focussed by a lens onto the sample. In most experiments it is desirable to remove the rotational deactivation component from the excited state decay kinetics, which can be achieved by either setting the polarization of pump and probe at the magic angle (54.7°) or by depolarizing the excitation beam, both techniques cancel out the dipole-dipole interactions between the excited molecules and probe light. The Helios probe beam path consists of ~10 mW of 800 nm light from the amplifier (via an extended beam path to match path lengths with the pump beam from an OPA), this light is routed into the enclosure via an iris and reflected by mirrors onto a retroreflector on the delay stage (twice). The time window of Helios, as determined by the delay stage (in double pass set-up the path length is 25 cm•4= 100 cm, 30 cm=1 ns), is 3.3 ns. The instrument response function is around 0.2 ps. The intrinsic temporal resolution is 7 fs as defined by the smallest step possible on the delay stage. The temporal chirp of the probe pulse (the time range over which the different wavelengths monitored arrive at the detector) is usually on the order of 1 – 2 ps, and can be corrected for after the experiment if a scan of a sample cell containing solvent, similar to that used in the experiment, is performed. The 800 nm probe beam is focussed to generate a white light continuum (WLC). Using a sapphire crystal a WLC spectral range of approximately 400 – 1000 nm can be obtained. The beam then passes through a 750 nm short pass interference or blue glass filter to cut out the comparatively very strong 800 nm light. In the standard Helios set-up a probe reference channel is used. The probe beam is split in two parts at beam splitter, one of which traverses through the sample, and the other one does not, this helps correct for pulse-to-pulse variations in the laser output. The two probe beams are then independently recollimated and focused through lenses onto the centre of the fibre-coupled spectrometer inputs. The relative intensities of the two beams can be adjusted with the beam splitter. The spectral resolution of the spectrometers is 1.5 nm. The detectors used are CMOS sensors with a sensitivity range of 200 – 1000 nm and a maximum spectral acquisition rate of 9500 spectra/s (1 spectrum every 0.1 ms). The Eos system uses the same pump beam as the Helios system, although without the use of the optical chopper, while the probe beam is generated by a fibre based laser with a spectral range of 350 – 2400 nm. The Eos probe beam operates at the

repetition rate of 2 kHz and is never chopped leading to twice as many pulses per second monitored compared to the Helios setup. The time window is limited to 50% of the pump laser repetition rate (i.e. 500 μs for a 1 kHz laser). The temporal resolution is 500 ps. The time delay between pump and a probe pulse is controlled electronically by the Eos software. Ultrafast Systems provide LabVIEW based software for instrument control and data acquisition. The software allows for experiment automation, such as pre-set optics delay step sizes, averaging time for each transient spectrum and time window. Alternatively the user can adjust the above parameters during the data collection process. Random delay line stepping is available for the above regimes.

2.3 Sample preparation

2.3.1 FAD sample preparation

TA measurements of FAD in low pH were performed using the pre-existing Eos and Helios setups. FAD was purchased from Sigma Aldrich and used without further purification. Samples were prepared daily by dissolving FAD in buffer solution (4 mM

Na2HPO4∙10H2O/ 98 mM citric acid, pH= 2.3) of distilled water. None of the buffers were degassed and storing samples in anaerobic conditions was not required. During experiments a capillary flow cell (described in detail in section 3.3) was used to prevent photobleaching processes and to provide fresh portion of sample for every measurement. Absorption spectra of samples before and after measurements were taken in order to confirm that samples were not photodamaged.

2.3.2 AdoCbl sample preparation

TA measurements of AdoCbl were performed using both pre-existing Helios setup and the newly developed optical setup. AdoCbl was purchased from Sigma Aldrich and used without further purification. Samples were prepared daily by dissolving the AdoCbl in previously degassed HEPES buffer and 50% volume of glycerol of various pH values. Special measures were taken in order to keep the sample anaerobic; degassed buffers were stored in the glove box, where each sample was

prepared. Furthermore, the setup was situated within a closed box and purged with nitrogen. During experiments a capillary flow cell (described in detail in section 3.3) was used to prevent photobleaching processes and to provide fresh portion of sample for every measurement. Absorption spectra of samples before and after measurements were taken in order to confirm that samples were not damaged.

2.4 Calculations of the experimental error

In ideal conditions lasers produce a concentrated stream of single frequency photons, but various components of the laser introduce a certain amount of constant noise resulting in slight fluctuations and day-to-day variations. As a result, the amplitude of signals as well as the quality of the data collected on different days is unlikely to be the same. To illustrate such variations, FAD data were collected using the Eos setup over a time range of 400 μs, without the application of external field and then analysed. Initially, the data were analysed by fitting the ΔA values averaged in the region 580-600 nm using a single exponential fit. This wavelength range is said to monitor the species expected to be MF-sensitive. An example of a raw data for a scan performed without the application of external field at the beginning and the end of the day is shown in Fig.2.6.

Fig.2.6 A decay observed in the averaged range of 580-600 nm following the excitation of FAD at pH 2.3 with a laser pulse centred at 375 nm without the application of external field a) at the beginning and b) the end of the day. The data was fit with a single exponential decay function and the output values are shown in tables.

The rate values obtained from the analysis of scans performed on different days is shown in Fig.2.7.

Fig.2.7 Rates for the decay observed in the averaged range of 580-600 nm obtained for the fitting of TA time profiles. Scans without the application of field were performed on different days; different methods for calculating the experimental error were used.

Various points in Fig.2.7 represent different methods of calculating the error: a) Grey points- rate value and error obtained from fitting individual scans b) Pink- rate value and error obtained after ΔA values from all scans were averaged and analysed using a single exponential fit c) Blue- rate value and error for individual scans were compared- an average of these rates and the propagated standard deviation were then calculated. As can be seen from this graph, the variations in the laser operation, or slight difference in sample conditions may affect the resulting derived kinetic parameters. Changes induced by the application of MF are expected to be relatively small and it is therefore important to perform experiments more than once in order to account for these fluctuations. To fully characterize the number and nature of components present in the spectrum, if a multidimensional dataset is available (wavelength, time, change in absorbance), global analysis is usually recommended.

In order to perform global analysis of the data obtained for the investigation of MFEs on intramolecular electron transfer in FAD described in Chapters 3 and 4, the computer software Glotaran was used. In general, global analysis itself fits the data to spectral components, which correspond to exponentially decaying kinetics. In the simplest kinetic models a number of components decay independently resulting in decay associated difference spectra (DADS).9 In a sequential model, resulting in evolution associated difference spectra (EADS) the first component arises due to the excitation source and subsequently decays into the second one, which then converts into the third one, and so on until the final component decays back to the ground state. Any back reactions are not taken into account in this kind of scheme. Assuming that the model represents the correct reactions in a chemical system, the EADS will correspond to the true spectra for each species. In section 4.3.3.1 the error values are not given because whilst the fitting procedure does output error values, these are standard error, not stanard deviation, and due to the large number of data points used for the fitting these errors are vanishingly small. In order to improve our method of calculating the experimental errors we selected a different analysis method. In order to account for the day-to-day variations in the laser operation, we decided to perform measurement in each applied field more than once. The FAD measurements were performed using the Eos setup, which meant that to make measurements without the application of field, the magnet barrels (described in 3.1.2) had to be fully taken out of the holder. Removing the magnets is not only possibly dangerous, but also time consuming. We therefore decided to make a measurement in

MF 0 mT three times a day - as the first and the last measurements of the day and an additional one performed around the middle of the experimental day. The individual scans were then analysed and the average rate value was calculated, as well as the standard deviation. Measurements in each field were then performed at least three times each, on different days. The order of the measurements was randomly selected, so for

example, measurement in MF = 30 mT was performed on Monday morning, Tuesday afternoon and at the end of the day on Wednesday. Once again the individual scans were then analysed and the relative rate on a specific day calculated. The relative rate for each field was slightly different on different days, so the average value and standard deviation of the individual relative rates were then calculated; a fraction of the data spreadsheet is shown in Fig.2.8.

Fig.2.8 Measurements without the application of external field were performed three times a day; the day average was then calculated as well as standard deviation. Measurements in each field were performed at least three times on different days. Relative rates, their average and standard deviation were then calculated.

As can be seen from Fig.2.8, the rate values for data collected on different days are not the same, for example rates for experiments when an external field of 60 mT was applied varies from 1.80-1.94 μs-1. If only one value was to be selected for plotting the MF-dependence, data would not be fully reliable and it would be easy to accidentally manipulate the data in order to achieve expected or desirable plot. For the datasets collected on the new experimental setup (investigations of MFEs in AdoCbl) measurements with and without the application of external field could be performed easily and quickly, so every measurement in a specific value of applied MF was followed by a measurement, when no external field was present. Each dataset was then individually analysed and the relative rate was calculated. Each pair of measurements was once again repeated three times and the average relative rates as well as standard deviation were then calculated.

2.5 References

1. R. Berera, R. van Grondelle, J. T. M. Kennis; Ultrafast transient absorption spectroscopy: Principles and application to photosynthetic systems. Photosynth. Res; 2009, vol. 101, no. 2-3, pp. 105–118 .

2. C .Rulliere, T. Amand, X. Marie; 'Spectroscopic Methods for Analysis of Sample' in Femtosecond Laser Pulses; Advanced Texts in Physics. Springer, 2005, pp. 223–281.

3. M. Lorenc, M. Ziolek, R. Naskrecki, J. Karolczak, J. Kubicki, A. Maciejewski; Artifacts in femtosecond transient absorption spectroscopy. Appl. Phys. Lasers Opt.; 2002, vol. 74, no. 1, pp. 19–27.

4. N. P. Ernsting, S. A. Kovalenko, T. Senyushkina, J. Saam, V. Farztdinov; Wave- Packet-Assisted Decomposition of Femtosecond Transient Ultraviolet−Visible Absorption Spectra: Application to Excited-State Intramolecular Proton Transfer in Solution. J. Phys. Chem.; 2001, vol. 105, no. 14, pp. 3443–3453.

5. B. Meier, A. Penzkofer; Picosecond pulse generation in a benzene raman generator amplifier system. Appl. Phys. B Photophysics Laser Chem.; 1991, vol. 53, no. 2, pp. 65 - 70 .

6. X. Gu, S. Akturk, R. Trebino; Spatial chirp in ultrafast optics. Opt. Commun.; 2004, vol. 242, no. 4 - 6, pp. 599–604.

7. S. Kovalenko, A. Dobryakov, J. Ruthmann, N. P. Ernsting; Femtosecond spectroscopy of condensed phases with chirped supercontinuum probing. Phys. Rev. A - At. Mol. Opt. Phys.; 1999, vol. 59, no. 3, pp. 2369–2384.

8. G. P. Agrawal, P. L. Baldeck, R. R. Alfano; Temporal and spectral effects of cross-phase modulation on copropagating ultrashort pulses in optical fibers. Phys. Rev.; 1989, vol. 40, no. 9, pp. 5063–5072.

9. J. J. Snellenburg, S. P. Leptenok, R. Seger, K. M. Mullen, I. H. M. van S. Glotaran: A Java -Based Graphical User Interface for the R Package TIMP. J. Stat. Softw.; 2012, vol. 49, no. 3, pp. 1–22.

Chapter 3. Development of MF generating apparatus

Magnetism has been fascinating mankind for thousands of years. Although magnetic attraction was observed in ancient times, the understanding of this phenomenon has taken a relatively long time. In the past century there has been a rapid increase in both knowledge and available technology. The history of magnetism began when the Greeks and Chinese learnt about the properties of magnetite, which is a natural magnet. The name ‘magnet’ itself is said to originate from the Greek region called Magnesia, where the magnetite deposits were first discovered.1 The scientific reason for magnetism remained unknown until the 20th century, when people started exploring the world inside the atom.2 The compass, a device consisting of a small needle that points to the north, was invented in China as early as the 12th century and from there it arrived to Europe, when merchants from Venice realized that it could be used to navigate without having to rely on the sky and the position of the stars. In 1600 an English physicist William Gilbert published the book ‘On the Magnet and Magnetic Bodies, and on That Great Magnet The Earth’,3 in which he hypothesized that the Earth is a big magnet creating around itself a MF which is responsible for the rotation of the compass needle.4 Gilbert’s theory introduced not only the terrestrial magnetism, but also previously unknown ‘distance action’. He was the first one to state that physical contact is not always necessary for bodies to interact with each other, because they are able to interact through the field. Decades later, the same idea inspired Isaac Newton to create the Law of Universal Gravitation. Another important event in the history of the MF was marked by a discovery of the Danish scholar Hans Christian Øersted. In April 1820 during one of his lectures he noticed that the needle of a compass placed near an electrical conductor deflects only when the electricity flow is present.5 He was the first one to conclude that the motion of electrical charges creates the MF. Further studies of Michael Faraday6 proved that not only the current generates a MF, but also the field can cause the current to flow in the conductor. Due to these discoveries a relationship between the electric and MF was

established, but the theory combining both was not known until the 1864, when James C. Maxwell described them in the form of mathematical relations known today as the Maxwell's equations.7 As proven by Faraday and stated in Faraday’s Law of Induction8 every electric charge in motion will generate a MF. Fields of higher values can be obtained by placing a ferromagnetic metal core inside a coil.9 If a direct current is applied, a homogenous, constant electromagnetic field in one direction will be generated, which will disappear once the current is switched off. Electromagnetism has found applications in electric motors, generator, computers, microphones and more new applications are being discovered every day. A promising application of electromagnetism is associated with new form of more energy efficient and environmentally friendly transportation, where a phenomenon of magnetic levitation allows vehicles to travel with speed previously unobtainable. In 2014 Japan revealed their state-of-the-art MAGLEV train setting a world record of just over 600 km/h, which remains the fastest train ever constructed. Magnetism is a force of attraction or repulsion acting at a distance; a magnet is an object exhibiting a strong MF that attracts other magnetic materials. MF is a dipole field, meaning that all magnets have two poles, the north (N) and the south (S). Two magnets will attract each other if their poles are opposite and repel if they are the same. Even though iron was the first discovered naturally occurring magnet, it is not the only material that will become magnetized when placed in MF and others include nickel and cobalt. Some materials have the ability to turn themselves into temporary magnets in order to resist magnetization and will weakly repel MF they have been inserted in. These materials were discovered in 1778 by Brungmans,10 who observed that bismuth and antimony were repelled by MF and called them diamagnetics. In 1845 Faraday6 showed that every material will respond to MF either in diamagnetic (repelling) or paramagnetic (attracting) way. Atoms of paramagnetic materials are aligned randomly, so their magnetic momenta cancel each other out resulting in no magnetic properties, although, once the material is placed within MF, the magnetic momenta will align themselves with its direction and create a local non-zero MF. When the field is removed, the magnetic arrangement is removed as well as magnetic properties. The structure of ferromagnetic11 materials contains so called domains, within which the alignment of the magnetic momenta is the same. When an external MF is applied these domains align themselves along the direction of the field, and remain unchanged even if the external field is removed remaining permanently magnetized. Examples of such

materials are iron, cobalt, nickel or gadolinium. By the end of the century neodymium12 was discovered and the production of neodymium magnets began. Today neodymium magnets still remain the strongest permanent magnets that exist.13 MFEs are a helpful tool in investigations of the chemical composition of matter. Particles possess magnetic momenta, so externally applied MF will cause them to spin. The frequency of this motion depends on the intensity of the field and is different for each chemical compound. This technique is known as magnetic resonance and it has found its use in various disciplines such as drug research, medical diagnosis and even drug detectors found in the airports worldwide. MF measurements are often exploited in space research and it is due to studying the magnetosphere of Saturn that the Cassini spacecraft14 discovered water in liquid state on one of its moons, Enceladus. It has been found that disturbance in the Earth’s MF often appears in places, where petroleum or natural gas are sourced.15,16,17,18 The Earth’s field originates from magnetized rocks within the planet’s crust, which are not found in places with large deposits of oil or gas, therefore the field measured is much lower. Investigations of field anomalies around the world allow the prediction of the location of areas, where such deposits may appear. Due to the use of devices generating MFs in industrial processes, energy production and medicine the number of studies focusing on the biological effects and the potential health risks associated with the exposure to MFs has been increasing.19 For the investigations of MFEs on biochemical processes described in this thesis, a wide range of MF strength values was required. The MF generated by the disc magnets covers the range between 30 - 950 mT depending on the disc thickness and the distance between them.

3.1 Neodymium disc magnets

In order to achieve strong, uniform MFs in the sample volume, a pair of neodymium disc magnets N52 shown in Fig.3.1 (also known as NdFeB or NIB), were employed. The N-rating refers to the Maximum Energy Product (MEP)20 of the material that the magnet is made from and it provides information about the maximum strength the material can be magnetized to.

Fig.3.1 An example of N52 neodymium disc magnets. All dimensions are given in mm.

Neodymium is the most commonly used type of rare-earth magnets, and it is a permanent magnet made from an alloy of neodymium, iron and boron to form the 21 Nd2Fe14B tetragonal crystalline structure. Neodymium magnets are the strongest of the rare earth magnets and the strongest permanent magnets available. This type of magnets is very sensitive to high temperatures, which result in loss of a fraction of its magnetic strength. Two important temperatures can be distinguished- the Maximum Operating Temperature (MOT), below which no strength loss will occur, and the Curie temperature,22 above which the magnet will lose all of its strength. These temperature 23 values for ND52 type of magnets are 60°C and 312 - 380°C respectively.

3.1.1 Magnet and sample holder- version 1.

In order to vary the MF strength easily, a magnet and sample holder has been designed and built. The initial version of the holder was designed using the computer programme CorelDRAW; the drawings of this version of the holder are shown below.

Fig.3.2 Magnet holder- version 1. The red arms hold the magnets and the MF is changed by varying the gap between the two arms. The green part represents the sample holder located in the middle of the gap.

The holder was built of plastic and brass in order to prevent magnets from being attracted to it. The discs are held by the magnet holder in such way that the opposite poles face one another, so the magnets are attracting each other, while separated by a small gap. Change of the magnetic strength is caused by moving the magnets towards and away from each other. In this version the magnet holder was built of small pieces and once the holder was ready, it was noted that it would not be able to hold the powerful magnets, which would allow us to reach MF close to 1 T. Inserting magnets into it would be dangerous and might cause harm. Measurements of MF using this holder were not performed and a new magnet holder was designed and built.

3.1.2 Magnet and sample holder- version 2.

To ensure that the magnets could be safely inserted and removed the new holder was built so that each magnet was held in a hollow screw made from a single piece of brass. The screws were then inserted directly into the holder. The drawings of the new holder are shown in Fig.3.3

Fig.3.3 Magnet holder- version 2. The red colour shows the arms and base of the holder, the green represents the screws made of brass which hold the magnets. The screws are empty inside and the magnets are inserted into it. The magnetic field is changed by screwing the brass holders in or out the holder to vary the gap between the two disc magnets. All measurements are given in mm.

Multiple magnets could be placed within each brass screw, and the field, measured at the centre of the holder at the sample position, was much stronger than achieved with a single magnet. By varying the thickness, grade, diameter and distance between the magnets the field strength can be varied. Furthermore, in order to perform experiments in low MF, the orientation of the discs can be changed, by inserting them in such way, that the same poles are repelling each other. Measurements of both configurations are shown in the next section.

3.1.2.1 Comparison of MF simulations and measurements

Measurements of MF depending on the thickness and gap between the magnets as well as the orientation of the two poles were performed. The sensor of the

Gaussmeter was located in the centre of the sample holder in the gap between the two magnets. Combined results are shown in Fig.3.4.

Fig.3.4 Comparison of computer simulations (green) and measurements (red) of MF between N52 neodymium disc magnets. The diameter of discs is 2 inches and the thickness is a) 0.5’’, b) 1.0’’, c) 1.5’’, d) 1.0’’ magnets are repelling each other.

The MF simulations were made performed using the Gap Calculator software provided by magnets suppliers K&J Magnetics.24 The Gap Calculator derives its answers from a series of finite element analysis studies conducted for specific configurations of magnets and is checked by comparing a number of specific sizes and shapes. The calculator makes a number of assumptions that can be different for specific setup, so it is only an estimation of MF obtainable by a specific set of magnets.

The MF simulations suggested that a range between 80-650 mT was obtainable

for 0.5 inch thick magnets (the gap varied from 2 - 60 mm). The values obtained from measurements were 100-600 mT (the gap range was the same), giving an average difference between calculated and measured values of ~20 mT. For 1 inch thick magnets

(with the gap varied from 2-60 mm) the simulations suggested a range of 200-980 mT, and the values obtained from measurements were in the range of 215-810 mT, which gave an average difference of ~110 mT. Although for 1.5 inch thick magnets computer simulations suggested a MF range between 250- 1200 mT would be achievable, during the joining the thee 0.5’’ magnets one of them was crushed and only half of it could be used; so rather than three 0.5’’ magnets on either side of the sample, one side had three, and the other two and a half, the resulting highest measured field was 950 mT.

3.2 Sample cell

In order to maximize the MF that can be experienced by the sample placed between the disc magnets and holder shown in Fig.3.2 a new sample cell was designed. To obtain higher values of the field at the sample position, the gap between the magnets needed to be smaller than 15 mm, as on distances shorter than this the MF increases significantly. Therefore, a new flow cell was built of a rectangular glass capillary, the width of which is only 2 mm. The drawings of the capillary flow cell are shown in Fig.3.5.

Fig.3.5 Newly designed and built capillary flow cell. The capillary cell is 2 mm wide and the tubing has an external diameter of 1.3 mm, internal diameter of 0.8 mm.

The newly built flow cell not only allows for the field to be maximized, but also reduces the amount of sample required compared to other flow cells. The minimum sample volume required is 0.7 ml.

3.3 MF generating apparatus test system

MFEs on flavin-based systems have previously been investigated.25,26,27 In order to test the new MF- generating setup described in section 3.1.2, TA measurements equivalent to those previously published were conducted to investigate the influence of external fields of 200 mT on the photochemistry of flavin adenine dinucleotide (FAD) at low pH.

3.3.1 Reproduction of previously reported MFE on intramolecular ET in FAD

Murakami and co-workers studied the mechanism and the intermediates generated during the intramolecular ET reactions in FAD.28 The presence of two components was reported, the first one with a spectral maximum at 680 nm and a lifetime of 0.7 μs and a second one with a spectral maximum at 510 nm and a longer lifetime of about 50 μs. As can be seen from Fig.3.6 in the presence of an externally applied MF of 200 mT, the signal at 580 nm, which is said to monitor both flavin radical and the triplet state, increased indicating that the RP is generated from the triplet state precursor, assuming that the back ET takes place only from the singlet RP.29

Fig.3.6 Time profiles of TA observed at λ= 600 nm with (upper B= 0.2 T) and without (lower B= 0 T) MF. Taken from reference.28

These measurements were then reproduced using the experimental setup Eos over a timeframe of 10 μs. All samples were prepared by dissolving FAD (purchased from Sigma Aldrich, used without further purification) in buffered aqueous solution, the

pH of which was adjusted to 2.3. An example of a MFE on the decay kinetics of data averaged over the wavelength range of 580-600 nm, where the MF sensitive species is observed, is shown in Fig.3.7.

Fig.3.7 An example of MFE on decay kinetics at 580- 600 nm with and without the application of external MF of 200 mT and in pH= 2.3. Data were then fitted using single exponential function.

In order to further investigate the influence of MF on the kinetics of intramolecular ET in FAD, TA measurements were then performed in a wide range of fields up to 1 T and are described fully in Chapter 4. As can be noted from Fig.3.7 in the presence of an externally applied MF of 200 mT, the signal increased and the previously reported MFE was successfully reproduced. The investigations of MFEs in this system were then performed in a wide range of field up to 950 mT.

3.4 References

1. W. Lowrie; Fundamentals of geophysics. Cambridge University Press, 2007.

2. C. Woodford; Magnetism. Available: http://www.explainthatstuff.com/magnetism.html. [Accessed March - 2017].

3. W. Gilbert, A. Dowling; De Magnete, Magneticisque Corporibus, et de Magno Magnete Tellure (On the Magnet and Magnetic Bodies, and on That Great Magnet the Earth). 1600.

4. How Magnets Work. Available: http://www.howmagnetswork.com/types.html. [Accessed March - 2017].

5. R. De Andrade Martins; Resistance to the Discovery of Electromagnetism: Ørsted and the Symmetry of the Magnetic Field. Volta and the history of electricity Available: http://www.ghtc.usp.br/server/pdf/RAM-Oersted1.PDF. [Accessed March - 2017].

6. M. Faraday; The Correspondence of Michael Faraday: Vol. 5: 1855-1860 Institution of Engineering and Technology, 2008.

7. N. Forbes, B. Mahon; Faraday, Maxwell, and the Electromagnetic Field: How Two Men Revolutionized Physics. Prometheus Books, 2014.

8. F. Ulaby, U. Ravaioli; Fundamentals of applied electromagnetics 5th Edition. Pearson Education Limited, 2007.

9. C. D. Mee, E. D. Daniel; Magnetic recording handbook: Technology and Applications. McGraw - Hill, 1998.

10. Jackson, R.; John Tyndall and the Early History of Diamagnetism. Ann. Sci.; 2015, vol. 72, pp. 435 - 489.

11. S. Chikazumi; Physics of ferromagnetism. Oxford University Press, 2009.

12. C. A. von Welsbach; Die Zerlegung des Didyms in seine Elemente. Monatshefte für Chemie und verwandte Teile anderer Wissenschaften; 1885, vol. 6, no. 1, pp. 477–491.

13. C. K. Gupta, N. K Krishnamurthy; Extractive metallurgy of rare earths. International Materials Reviews. CRC Press, 1992.

14. Martinez, C. NASA’s Cassini Discovers Potential Liquid Water on Enceladus. Available: http://www.nasa.gov/mission_pages/cassini/media/cassini- 20060309.html. [Accessed April - 2017].

15. H. Lyatsky; Magnetic and Gravity Methods in Mineral Exploration: the Value of Well-Rounded Geophysical Skills. CSEG Recorder; 2010, vol. 35, no. 8, pp. 1– 9.

16. J.P. Busby, R.J. Peart, C.A. Green, R.D. Ogilvy, J. P. Williamson; A Search for Direct Hydrocarbon Indicators in the Formby Area. Geophys. Prospect.; 1991, vol. 39, no. 5, pp. 691–710.

17. A. L. Piskarev, M. Y. Tchernyshev; Magnetic and gravity anomaly patterns related to hydrocarbon fields in northern West Siberia. Geophysics; 1997, vol. 62, no. 3, pp. 831–841.

18. O. Menshov, R. Kuderavets, S. Vyzha, I. Chobotk, T. Pastuchenko; Magnetic mapping and soil magnetometry of hydrocarbon prospective areas in western Ukraine. Stud. Geophys. Geod.; 2015, vol. 59, no. 4, pp. 614–627.

19. Zanella, S. Biological effects of magnetic fields. CISE SpA, Segrate (Milan), Italy, 1997.

20. J. Park, Y. Hong, J. Lee, W. Lee, S. Kim, C. Choi; Electronic Structure and Maximum Energy Product of MnBi. Metals, 2014,. vol. 4, no. 3, pp. 455–464.

21. J. Fraden; Handbook of Modern Sensors: Physics, Designs, and Applications. Springer, 2010.

22. H. Luth, H. Ibach; Solid-state physics: an introduction to principles of materials science. Springer, 2009.

23. Magnetization Direction for Neodymium Magnets. Available: https://www.kjmagnetics.com/magdir.asp. [Accessed April - 2017].

24. Magnetic Strength Calculator. Available: https://www.kjmagnetics.com/calculator.asp. [Accessed April - 2017].

25. M. Horiuchi, K. Maeda, T. Arai; Magnetic field effect on electron transfer reactions of flavin derivatives associated with micelles. Appl. Magn. Reson.; 2003, vol. 23, no. 3-4, pp. 309–318.

26. M. Horiuchu, K. Maeda, T. Arai; Dynamic process of the photo-chemically produced flavin radicals in a neutral micelle studied by a magnetic field effect. Chem. Phys. Lett.; 2004, vol. 394, no. 4 -6, pp. 344–348.

27. T. Miura, K. Maeda, T. Arai; Effect of coulomb interaction on the dynamics of the radical pair in the system of flavin mononucleotide and hen egg-white lysozyme (HEWL) studied by a magnetic field effect. J. Phys. Chem. B; 2003, vol. 107, no. 26, pp. 6474–6478.

28. M. Murakami, K. Mawda, T. Arai; Structure and kinetics of the intermediate biradicals generated from intramolecular electron transfer reaction of FAD studied by an action spectrum of the magnetic field effect. Chem. Phys. Lett; 2002, vol. 362, no. 1-2, pp. 123–129.

29. E. W. Evans, C. A. dodson, K. Maeda, T. Biskup, C. J. Wedge, C. R. Timmel; Magnetic field effects in flavoproteins and related systems. Interface Focus; 2013, vol. 3, no. 5, pp. 1 - 17.

Chapter 4 Investigations of MFEs on flavin adenine

dinucleotide (FAD) photochemistry

4.1 Photochemistry of FAD

Flavin adenine dinucleotide (FAD), the structure of which is shown in Fig.4.1, is one of the most important cofactors found in the blue-light activated proteins of the photolyase group, which use blue or near-UV light to repair DNA defects that have been caused by the exposure to light.1 FAD is also found in the retina in the eyes of migratory birds.2,3,4

Fig.4.1 The structure of FAD.

Redox properties of flavins are the reason for a huge diversity in their function and their ability to exist in oxidized, one-electron and two-electron reduced states, as shown in Fig.4.2, means that they can take part in a wide variety of redox processes.5

Fig.4.2 Different redox states of FAD under various physiological conditions. Adapted from reference.6

FAD has an ability to undergo various oxidation-reduction reactions and is able to accept either one or two electrons.7,8 Reduction is achieved by hydrogen atom addition to a specific atom of nitrogen of the isoalloxazine ring. FAD can exist in different states: fully oxidized, one-electron reduced radical semiquinone and two- electron fully reduced hydroquinone.9 The pKa values for the semi- and hydroquinone 10 are 8.3 and 6.7 respectively and in physiological conditions, both of them can be found in either neutral or anionic form. Although the primary mechanism of migratory birds detection of the MF generated by the Earth remains unclear, the evidence points to a photochemical reaction in cryptochromes. It has been established that both cryptochrome and photolyase protein possess all the physical properties necessary to respond to MFs and to convert the physical signal into the cell signalling mechanism.11,12 The photoexcitation of FAD can trigger three consecutive intraprotein ET along a conserved triad of tryptophan (Trp) residues to produce a (FAD•− Trp•+) RP,13,14,15 which is MF-sensitive in vitro and possibly in vivo.16,17

There has been extensive research focused on the excited state properties of FAD and flavin mononucleotide (FMN), which lacks the adenine moiety in its structure. Studies have shown that in aqueous solution, oxidized FAD exists in two distinct conformations: closed, where the flavin and adenine moieties are in the close proximity, and open, where the two moieties are separated.18 A schematic representation of various structures of FAD depending on the pH is shown in Fig.4.3.

Fig.4.3 Schematic representation of various FAD structures in different pH. Adapted from reference.19

The photochemical cycle of flavins (Fig.4.4) is well understood. Photoexcitation of flavins results in populating the singlet excited state followed either by fluorescence or the rapid ISC to the triplet state on the timescale of a few nanoseconds. In a similar reaction, photoexcited FAD forms an intramolecular triplet spin correlated RP in which the adenine moiety donates an electron to the isoalloxazine ring.20,21,22 The resulting RP then undergoes a process of coherent spin state mixing, in which triplet-born RP converts into a singlet state. The recombination of the RP to the ground state takes place only from the singlet state.

Fig.4.4 Photochemical reaction scheme for intramolecular RP formation between flavin (F) and an electron donor (D). The double headed arrow represents the process of coherent spin state mixing. Adapted from reference.11

In the pH range 4-8 and at room temperature around 80% of ground-state FAD is in the closed conformation, while 20% remains in the open one. The open conformer of FAD starts appearing in significant concentrations below pH =3 and the relative population of closed form is significantly smaller. The lifetime of the excited state open 19,23 conformation of FAD is reported to be between 3-9 ns, while the lifetime of the 24 closed one is much shorter with the values reported to be between 1-20 ps. Various spectroscopic techniques have been applied to investigate the behaviour of both 25,26,23,24,6,27 28,29 conformations as well as the pH dependence of flavin fluorescence. It was found that fluorescence appears mostly from the neutral form, while the anionic form is weakly fluorescent and the cationic form is practically non-fluorescent.28,29 The pH dependent dynamic behaviour of FAD and FMN were extensively studied using transient absorption spectroscopy with femtosecond resolution by Li and Glusac.30 Their results suggested that at low and high pH FAD adopts an open conformation and behaves similarly to FMN and that the conformational changes of FAD take place at pH~3, due to adenine protonation, and pH~10, due to flavin deprotonation. In a femtosecond transient absorption and fluorescence study of fully oxidized FAD in neutral pH by Kao et al., two separate components were observed, the first one with a lifetime of 4.5 ps and the second one of a 30-40 ps. The short-lived component was assigned to the excited-state decay of the closed conformation of FAD with the RP formation, and the long-lived one to a lifetime of the RP decaying by charge recombination.6

The existence of the two conformations has been further investigated by Sengupta and co-workers, who performed fluorescence lifetime measurements of FAD and FMN in order to investigate the pH dependent conformational dynamics of FAD on femto and nanosecond time scale. The group reported that below pH =3.0 the open conformer is favoured due to protonation of either adenine or flavin moiety resulting in reduced fluorescence quantum yields and lifetimes. In the pH range between 5- 10 the population of the closed conformer dominates over the open one. They suggested the existence of an additional third conformation, a partially closed one in which both moieties do not ‘stack’, but only interact with each other. In this pH range three separate components were observed with the lifetimes of ~10 ps, ~3.5- 5 ns and ~2.2 ns, which can be assigned to closed, open and partially closed conformers accordingly. In pH ≥10.0 the open conformer dominates due to repulsion between the deprotonated flavin ring and the phosphate groups. In biological systems FAD plays an important role in electron transportation as a system, where an electron donor (adenine moiety) and acceptor (flavin) are linked by a covalent bond.21 The photo-induced intramolecular ET, the basic scheme of which is shown in Fig.4.5, leading to a formation of a RP was studied by CIDNP by Kaptein et al.31

Fig.4.5 Basic scheme of the photoinduced ET in FAD. The green lines represent the flavin (acceptor) and the blue lines the adenine moiety (donor). Adapted from reference. 32

The authors reported a pH dependence similar to that observed in the fluorescence measurements with a significant maximum at pH= 2.4 and a decrease at both low and high pH. The absence of any signals at higher pH was explained by a large exchange interaction in the closed conformation.11 Experiments performed at low MF demonstrated that the polarization in this field region arises from the spin state mixing of the formed RP.

Studies of the excited state dynamics of both FAD and FADH in aqueous 33 solution and pH= 8.0 were reported by Brazard et al using femtosecond TA spectroscopy. The group investigated the formation of the RP intermediate in the excited state decay of the closed conformer of FAD. It was previously reported that FMN at submillimolar concentrations undergoes dimerization,34,35,36,37 hence the experiments were performed using both diluted and highly concentrated samples in order to determine the influence of concentrations on FAD excited state dynamics. The absorption spectra of FAD at different concentrations were taken and although the spectral features remained the same, their amplitude decreased with higher concentrations, which is characteristic for molecular aggregates or biopolymers.38 In the case of FAD it was said to perhaps be due to some screening of the chromophores arranged in stacked aggregates. The data were analysed using the simplest model for the aggregation, in which the dimer is formed out of two monomers. The fraction of the dimer in typically used concentrations of samples used in TA experiments is said to be not negligible and the aggregation of FAD was demonstrated. By changing the concentration of the sample it was shown that the excited state decay was dependent on the fraction of the dimer present. Three excited state lifetimes were observed and assigned to three separate species- the closed and open conformer of the monomer and the dimer with the lifetimes of ~5.2- 5.5 ps, ~2.6- 3.0 ns and ~22-31 ps respectively. It was shown that the intramolecular ET from the adenine to the isoalloxazine ring in the stacked conformer of the monomer occurs with a time constant of 5.4 ps and is followed by a rapid charge recombination within 390 fs.

4.2 Reported MFE in flavins

MFEs on flavin-based systems have previously been investigated with transient absorption spectroscopy.39,40,41 Murakami and co-workers studied the mechanism and the intermediates generated during the intramolecular ET reactions.21 The presence of two components was reported, the first with a spectral maximum at 680 nm and a lifetime of 0.7 μs and a second one with a spectral maximum at 510 nm and a longer lifetime of about 50 μs. The short-lived component was assigned to the absorption of the cation form of the flavin triplet excited state from the similarity to that of FMN.42 The assignment of the second component was more difficult and since the proportion of it

increased with higher laser power it was said to reflect an intermediate generated from a bimolecular reaction.43 Measurements in the pH region 2.0-3.6 have shown that the spectral shape of the short-lived component did not change, but its lifetime decreased in higher pH, as shown in Fig.4.6.

Fig.4.6 pH dependence of the time profile of TA observed in MF= 0 mT at 650 nm following the excitation of FAD with a laser pulse centred at 375 nm. Taken from reference.21

44 Although, the signal from the RP (λmax= 550 nm) could not be clearly observed due to broad T-T absorption in the range 500 – 730 nm, the absorption at 580 nm is said to monitor both flavin radical and the triplet state. As can be seen from Fig.4.67 in the presence of an externally applied MF of 200 mT the signal increased indicating that the RP is generated from the triplet state precursor, assuming that the back ET takes place only from the singlet RP.11

Fig.4.7 Time profiles of TA observed at λ= 600 nm with (upper B= 0.2 T) and without (lower B= 0 T) MF following the excitation of FAD with a laser pulse centred at 375 nm. Taken from reference.21

The MFE- action spectrum obtained by a calculating the difference between the ΔA values with and without the application of an external MF suggested that the dynamics of the FAD excited state and RPs themselves are modulated by pH. The MFE

curves were characteristic of the hyperfine and relaxation mechanisms,45 as shown in Fig.4.8.

Fig.4.8 MFE- action spectra on the TA at pH= 2.3 (filled circle), 2.9 (open circle), 3.3 (filled square) and 4.1 (open square). Solid lines represent simulated spectra by linear combination of the template spectra. Taken from reference.21

The experimental data were then successfully reproduced by simulations. A strong MFE has been observed in the T-state of flavin in low pH and it is assumed that the flavin T- excited state is regenerated from the RP. Both transient species absorb 46 light at 532 nm with almost equal extinction coefficients. In the pH region of 2- 4 the time profiles of observed MFE at λ= 550 and 600 nm show similar shapes, which leads to conclusion that the interconversion reactions are so fast that the system reaches a quasi-equilibrium condition on the observed time scale.21 It has been shown that for the contribution of the T-T absorption the critical value of pH is about 2.3, which is the pKa value of flavin neutral radical to cation radical. Recently the MFE on the RP generated upon the photoexcitation of FAD in aqueous solution at low pH was observed with high sensitivity and spatial resolution using a newly developed transient optical absorption detection (TOAD) imaging microscope.46 The LFE on FAD was observed for the first time and MFE experiments on cryptochrome protein in solution are said to be underway. The MFEs in flavins and indole rings were investigated using detergent micelles and the results have been related to the variations in the hydrophobic properties of various flavins.39 The presence of micelles allows for the diffusion control and provide environment more similar to that in biological systems.47 The results have shown that in hydrophobic flavins the triplet excited state is rapidly quenched by ET, but the anionic radical is then only slowly protonated. Measurements of hydrophilic flavin have

confirmed that although the triplet state quenching is slower, the ET takes place before the rapid protonation. MFE studies of FMN and hen egg-white lysozyme (HEWL) have shown an 41 increase in the radical yield by 13% in a field of 250 mT. The MFE observed in an analogous system of FMN and tryptophan is much smaller with the increase in radical yield of only 2%, which suggests that that the lifetime of the RP in flavin/protein system is longer, due to slower diffusion. The MFE in FMN/HEWL system was shown to decrease in higher salt concentrations, which was explained by the authors as due to stronger Coulombic interaction between the negatively charged phosphate group of FMN and positively charged HEWL at lower salt concentrations. It was concluded that the dynamics of the ground state as well as the RP are affected by the attractive Coulombic interaction.

4.3 Investigations of MFEs in FAD

In order to investigate the influence of the pH on the excited state dynamics of

FAD, experiments on the picosecond timescale over a timeframe of 3 ns using Helios experimental setup and over a timeframe of 400 μs using the Eos experimental setup were performed. Measurements were performed in pH 2.3, 6.0 and 8.0. In all experiments the excitation light of 375 nm and the WLC in the range 430- 700 nm as the probe beam were used. The newly designed and constructed apparatus, as described in section 3.1.2, was used to generate the MF. All samples were prepared by dissolving the FAD (purchased from Sigma Aldrich and used without further purification) in buffer solution

(4 mM Na2HPO4∙10H2O/ 98 mM citric acid) of various pH. The extinction coefficient -1 -1 48 for FAD at 450 nm is 11,300 M cm - the concentration calculated using this value and the Beer-Lambert law was 0.2 mM.

4.3.1 Results and discussion

UV/Vis spectra of samples were taken before and after each measurement to ensure no sample degradation occurred, examples are shown in Fig.4.9 Flavins absorb light in the visible region of the spectrum due to conjugation of the isoalloxazine ring.

The fully oxidized FAD has two absorption bands centred at 450 and 375 nm corresponding S0 → S1 and S0 → S2 transitions.

Fig.4.9 Normalized UV/Vis spectra of FAD in pH 2.3 taken before (red line) and after (green line) measurements.

4.3.1.1 Excited state dynamics of FAD at a range of pH

Experiments at various pH on a 3 ns timescale were performed in order to determine the most suitable fitting model as well as the number and nature of species observed in the spectra. All measurements were performed using the Helios experimental setup. In order to investigate the influence of pH on the excited state dynamics of FAD, experiments without the application of MF in the pH range 2.3- 8.0 were performed. Fig.4.10 shows an overview of the TA spectra of fully oxidized FAD after excitation with a laser pulse centred at 375 nm for pump- probe time delays ranging from 0.3 ps to 3 ns. Data for the time delays below 0.3 ps are not shown, due to spectral contamination by a cross-phase modulation artifact generated during the overlap of the two beams. At all-time delays two positive bands can be observed- a fairly small one around 510 nm and a broad structure beginning around 625 nm and extending beyond the range of WLC used in these experiments. The region between 450 and 490 nm is dominated by a ground-state bleach, while the range of 540- 625 nm by the stimulated emission, where it produces a negative peak.

Fig.4.10 Time-resolved visible spectroscopy data for FAD in a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) recorded between 0.1 ps and 3 ns after photoexcitation with a laser pulse centred at ~375 nm at pH 2.3, 6.0 and 8.0.

49 The computer software Glotaran was then used to perform global analysis of the data obtained. First, Singular Value Decomposition (SVD) was used to estimate how many different species were observed in the spectrum and how many parameters would have to be specified in order to describe the data. SVD is a mathematical operation, which separates the contributions to the data into linearly independent vectors scaled by a number. The global analysis itself fits the data to spectral components, which correspond to exponentially decaying kinetics. In the simplest kinetic models a number of components decay independently resulting in decay associated spectra (DAS).49 In a sequential model, resulting in evolution associated spectra (EAS) the first component arises due to the excitation source and subsequently decays into the second one, which then converts into the third one, and so on until the final component decays back to the ground state. Any back reactions are not taken into

account in this kind of scheme. Assuming that the model represents the correct reactions in a chemical system, the EAS will correspond to the true spectra for each species.

The pH-dependence data over the timeframe of 3 ns was globally analysed using two separate components and an additional one with a lifetime fixed as ‘infinite’ for pH=2.3 and three separate components and an additional one with a fixed ‘infinite’ lifetime for pH values higher than that. The comparison of EAS of different components in various pH is shown in Fig.4.11.

Fig.4.11 EAS and SVD of the residual matrix resulting from global analysis of time-resolved visible spectroscopy data for FAD in a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) of various pH values recorded between 0.3 ps and 3 ns after photoexcitation with a laser pulse centred at ~375 nm; a) 3 and b) c) 4- component fitting shows no significant structure in the residuals, especially in the first principal time component (black lines), which implies a good fit has been obtained.

The best global fit EAS obtained with 3 (low pH) and 4 (high pH) components and the lifetimes of the exponentials for all pH values are given in the Table 4.1. Errors are not given because whilts the fitting procedure does output error values, these are standard error, not standard deviation, and due to the large number of data points used for the fitting these errors are vanishingly small. Table 4.1 The lifetime values of all components in various pH values obtained with sum of three and four exponential decays.

τ1 τ2 τ3 τ4 pH 2.3 2 ps - 1.2 ns Infinite pH 6.0 0.3 ps 6.5 ps 1.3 ns Infinite pH 8.0 0.3 ps 5.1 ps 1.0 ns Infinite

The short lived component in all pH values is in agreement with previous studies24,6 and can be assigned to early solvation dynamics- the relaxation of solvent molecules around the excited state of FAD. The value for the lowest pH is significantly higher than for the other two,which may be due to it being close to the time resolution of the setup or due to the open structure of FAD resulting in the chromophore being more solvent exposed. The EAS of the the other components are positive in the absorption domains and negative in the range of bleaching and stimulated emission. The second one of the components with a lifetime of 5-7 ps represents the excited-state decay of closed conformation of FAD, and is observed only in high pH, whilst the lifetime of about 1 ns represents the excited-state decay of the open conformer, which is found in all pH values. The last of the components with the lifetime value fixed as infinite (i.e. substantially longer than the 3 ns time window) may be assigned to other, long-lived intermediates, such as triplet species. An additional component with a lifetime of ~30 ps was previously reported and identified as the deactivation of the excited states of FAD dimers, or more precisely, the aggregates; however, the concentration of samples used in these experiments was not sufficient for the aggregates to appear. The lifetime of fully oxidized FMN, which lacks the adenine moiety, is 23,50 longer (about 4.7 ns ) which implies that the quenching role of the adenine is still active even in the open conformation, as the distance between the two moieties may not be separated far enough and the interactions are not fully cancelled. The measurements on an ultrafast timescale allowed for a correct analysis model to be determined and for the identification of species resulting from the photoexcitation

of fully oxidized FAD. The experiments on a slower, microsecond, timescale were then performed in order to identify and characterize the longer- lived components that may be affected by the application of the external MF. The experiments were performed using experimental setup Eos without the application of the external MF in the pH range 2.3- 8.0; examples of the TA spectra of FAD in different pH are shown in Fig.4.11. The pH-dependence data over the timeframe of 400 μs were then globally analysed using three separate components in a sequential model. The comparison of EAS of different components in various pH is shown in Fig.4.12.

Fig.4.12 Time-resolved visible spectroscopy data for FAD in buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) of various pH recorded between 0.5 ns and 2 μs after photoexcitation with a laser pulse centred at ~375 nm.

Fig.4.13 EAS and SVD of the residual matrix resulting from global analysis of time-resolved visible spectroscopy data for FAD a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) of various pH values recorded between 5 ns and 400 μs after photoexcitation with a laser pulse centred at ~375 nm. A 3- component fitting shows no significant structure in the residuals, especially in the first principal time component, which implies a good fit has been obtained. The EADS3 is not clearly noticeable at higher pH due to very low intensity.

In order to compare spectra of individual components in various pH, separate EAS spectra were normalized to the ground state bleach feature at ~450 nm and compared, as shown in Fig.4.14.

Fig.4.14 Comparison of EAS spectra resulting from global analysis of time- resolved visible spectroscopy data for FAD a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) in various pH values and lifetime values for separate components.

In the pH-dependence experiments three separate species can be observed. The first of the components, observed in the whole pH range, has a lifetime between 5-9 ns and neither its spectral shape nor lifetime showed any significant pH dependence. This component reflects the excited singlet state of the open conformer. The lifetime values and difference spectra obtained are in agreement with previous studies.19,20 The lifetime of this component seems to be only slightly shorter in high pH, which may be due to ET taking place faster in the stack conformer of FAD. The spectral shape of the second species varies across the investigated pH range.

It can be seen from Fig.4.14 that above pH = 4.0 there is an additional peak around 720 nm. Furthermore the lifetime of this component is also ~10 times longer, suggesting that it is not the same species in the whole pH range. The lifetime value obtained is in agreement with previous studies,21,20 becomes shorter at higher pH and it may be 88

assigned to a combination of a triplet excited state of the chromophore, achieved through the cation form of flavin and the RP (or protonated triplet state and the radical pair- PTS/RP).46 Low pH is required in order to stop the two moieties from stacking and to facilitate rapid forward and backward ET. The two intermediates are in equilibrium, and as mentioned before, absorb in the same wavelength region with almost the same extinction coefficient. Deconvolution of these two signals is said to be too complicated, due to adenine interaction in the open conformation of FAD. It was previously suggested that the critical pH value for the appearance of the contribution of the T - 6 absorption is about 2.3, which is identical to the pKa value of flavin neutral radical to cation radical, meaning that the formation of the cation form of flavin radical plays an important role in shifting the equilibrium to the triplet state. The differences in the spectral shape of the second component in various pH suggest that the cation form of the flavin is not generated in pH above 4.0. Therefore, if any MFEs are expected to be observed, it is most likely that this component will be affected. The third component may be assigned to a long-lived intermediate, the lifetime of which varied significantly in the whole pH range and this species was previously 20 described as a laser power - dependent intermediate. In pH > 4 about 80% of FAD is in the closed conformation, while below this point the majority of FAD is in the open conformation. In neutral pH both forms are said to interact with each other, in so called ‘butterfly motion’, during which a number of intermediates are said to exist. Computer simulations have demonstrated that these can have very long lifetimes.51,52 Above pH 4, a ground state bleach, but no positive features, are observed suggesting either the presence of some excited state or that the remaining bleach results from the flavin photobleaching. The reduced flavin has hardly any absorption in this region, so the loss in populations of the oxidized ground state should be observed. It is possible that the photobleaching takes place in the whole pH range, but in low pH there is also a very long lived triplet state. The observed long-lived intermediate has previously been 53 - observed by Brazard et al. for the spectrum of FADH , who assigned it to a combination of the neutral radical product and solvated electron. A similar component 54 was then observed for FADH2, when the pulse energy exceeded 500 nJ, however, the band disappeared, when the energy was reduced and it was said to be induced by multiphoton absorption.5 In experiments described in this thesis, the power of the excitation pulse was attenuated to 0.4 mW and, since the repetition rate of the pump beam is 500

Hz, the calculated power of each excitation pulse was equal to 800 nJ, suggesting that

multiphoton electron ejection are likely to take place in our system. In order to confirm this, power dependence measurements should be performed, however, it could not have been done due to time constraints.

4.3.1.2 MFE investigations up to 950 m T

In order to investigate the influence of MF on the kinetics of intramolecular ET in FAD, experiments in pH= 2.3 were performed in a wide range of fields. As identified in the pH-dependence measurements, the magnetically sensitive species has a lifetime below 1 μs, so a timeframe of 10 μs was sufficient for field dependence experiments. The lifetime of the longest component was determined to be between 2- 200 μs, and of very low intensity, meaning that although it can be properly characterized in all datasets in 400 μs window, the 10 μs scan is simply not long enough to characterize this component properly. All MF-dependence data were therefore analysed using a sequential model with two individual components and an additional one with a lifetime fixed as infinite. An example EAS of different components in various fields, normalized to the ground state bleach feature at 450 nm, are shown in Fig.4.15.

Fig.4.15 Comparison of EAS spectra of individual components resulting from global analysis of time-resolved visible spectroscopy data for FAD a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) various MF values. All data were normalized to the ground state bleach at 450 nm.

The calculated lifetime values for EAS1 and EAS2 were then plotted against the applied MF, as shown below in Fig.4.16.

Fig.4.16 Comparison of the a) EAS1 and b) EAS2 lifetime values resulting from global analysis of time-resolved visible spectroscopy data for FAD a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) in various MF values against the applied MF. The blue line represents the lifetime value for measurements performed without the externally applied MF.

Table 4.2 Relative rate values for the decay of the second component in applied MF up to 950 mT resulting from global analysis of time-resolved visible spectroscopy data for FAD a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) in various MF values.

Measurement MF k2 τ2 k0mT average % -1 -1 krel St.Dev. Date (mT) (μs ) (μs) (μs ) krel Error 02/05/2017 2.32 0.43 2.37 0.97 04/05/2017 5 2.13 0.47 2.27 0.94 0.96 0.02 1.99 05/05/2017 2.28 0.44 2.40 0.95 04/05/2017 1.85 0.54 2.26 0.82 30 0.79 0.03 4.01 05/05/2017 1.78 0.56 2.40 0.77 02/05/2017 1.92 0.52 2.37 0.81 04/05/2017 50 1.73 0.57 2.26 0.76 0.75 0.05 7.15 05/05/2017 1.69 0.59 2.40 0.70 04/01/2017 1.80 0.55 2.29 0.79 12/05/2016 60 1.94 0.51 2.24 0.80 0.82 0.04 4.85 25/02/2016 1.72 0.57 2.23 0.86 02/05/2017 1.86 0.54 2.37 0.78 04/05/2017 80 1.84 0.54 2.26 0.81 0.79 0.02 2.12 05/05/2017 1.88 0.53 2.40 0.78 02/05/2017 1.81 0.55 2.37 0.76 04/05/2017 90 1.68 0.59 2.27 0.74 0.77 0.03 3.27 05/05/2017 1.90 0.52 2.40 0.79 04/01/2016 1.81 0.55 2.29 0.79 05/01/2016 120 1.82 0.54 2.31 0.78 0.78 0.01 1.48 06/01/2016 1.76 0.56 2.29 0.77 06/01/2017 1.80 0.55 2.29 0.78 17/01/2017 150 1.76 0.56 2.33 0.75 0.76 0.02 2.28 21/12/2016 1.41 0.70 1.85 0.76 06/01/2017 1.61 0.61 2.29 0.70 23/02/2017 200 1.57 0.63 2.23 0.70 0.67 0.05 7.52 23/06/2017 1.37 0.72 2.22 0.61 13/05/2016 1.80 0.55 2.23 0.80 17/01/2017 250 1.73 0.57 2.33 0.74 0.75 0.04 5.77 20/12/2016 1.38 0.72 1.91 0.72 04/01/2017 1.79 0.55 2.29 0.78 05/01/2017 300 1.76 0.56 2.31 0.76 0.77 0.01 1.52 06/01/2017 1.78 0.56 2.29 0.77 20/12/2016 1.36 0.73 1.91 0.71 21/12/2016 350 1.40 0.71 1.85 0.75 0.72 0.02 3.62 25/02/2016 1.56 0.63 2.19 0.71 13/05/2016 1.80 0.55 2.23 0.80 20/12/2016 400 1.30 0.76 1.91 0.68 0.74 0.08 11.80

20/12/2016 1.37 0.73 1.92 0.72 17/01/2017 450 1.68 0.60 2.34 0.72 0.71 0.005 0.67 23/06/2016 1.58 0.63 2.23 0.71 92

17/01/2017 1.81 0.54 2.33 0.77 17/01/2017 500 1.77 0.56 2.33 0.75 0.76 0.01 1.92 21/12/2016 1.38 0.72 1.85 0.75 04/01/2017 1.72 0.58 2.29 0.75 06/01/2017 550 1.77 0.56 2.29 0.77 0.73 0.04 5.79 23/02/2016 1.54 0.64 2.22 0.69 13/05/2016 1.79 0.56 2.24 0.80 15/06/2016 600 1.65 0.60 2.24 0.74 0.76 0.04 5.37 20/12/2016 1.39 0.72 1.92 0.73 21/12/2017 650 1.36 0.73 1.85 0.73 0.73 - - 13/05/2016 1.76 0.57 2.24 0.79 15/06/2016 700 1.61 0.62 2.24 0.72 0.74 0.05 6.16 23/02/2016 1.56 0.64 2.23 0.70 04/01/2017 1.67 0.60 2.29 0.73 05/01/2027 740 1.73 0.58 2.31 0.75 0.75 0.02 2.51 15/06/2016 1.72 0.58 2.24 0.77 24/01/2017 1.68 0.59 2.24 0.74 800 0.74 0.0025 0.33

27/01/2017 1.67 0.58 2.23 0.75

24/01/2017 1.67 0.58 2.24 0.74 850 0.75 0.01 1.41 27/01/2017 1.69 0.59 2.23 0.75 24/01/2017 1.70 0.58 2.24 25/01/2017 900 1.59 0.62 2.19 0.75 0.74 0.02 2.32 27/01/2017 1.69 0.59 2.23 20/01/2017 1.73 0.57 2.23 0.80 950 0.79 0.02 3.10 24/01/2017 1.71 0.58 2.24 0.77

The component with the shortest lifetime, assigned to the relaxation of the singlet excited state, did not show any significant MF dependence, but the field dependence can be clearly observed in the longer lifetime component assigned to be some combination of a triplet excited state and the RP. In order to calculate an experimental error rather than fitting error, which is calculated during the global analysis, measurement in each field was performed three times on different days. The individual relative rates were then calculated by dividing values in a specific field by the value when no field was applied. The final relative rate values and the errors were then

calculated as the average of the individual measurements and their standard deviation. The final relative rates values and the respective errors are shown in Fig.4.17 and the table of all values obtained from the data analysis in Table 4.2.

Fig.4.17 Relative rate constants for the decay of the second component against the applied MF in fields a) up to 100 mT and b) up to 950 mT resulting from global analysis of time-resolved visible spectroscopy data for FAD a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) in various MF values.

When no external field is applied, most of the RP population initially resides in the three T-states, all of which can interconvert with the S-state. The rate of this spin-

state mixing depends on the strength of the HFCs of the two radicals. If any other magnetic interactions become stronger than the hyperfine one, the process of mixing may be interrupted. As the external field is applied, the T±1 states become more and more energetically separated due to Zeeman effect, the mixing between them and the S- state becomes slower and slower until it no longer occurs. This will be observed on the relative rates graphs as a plateau. The Zeeman Effect in FAD is expected to be statured in comparatively low fields (up to 20-30 mT) and previous simulations have shown that the value of Bsat is 20 around 25 mT . As can be seen from Fig.4.16a the saturation takes place, as expected, within 30 mT. Once the T± become fully separated, the interconversion between the S and T0 states is driven only by the HFI in the region between ~35-900 mT. In the higher fields (~950 mT) one can notice a slight increase in the rate constant for the decay due to the Δg mechanism- as the field increases, so does the rate of the spin state mixing. The process of spin-state mixing and its effect on the overall reaction depends on the amount of time, that the RP can undergo the mixing, while still being able to reencounter and react. It is important for biological systems that the two radicals do not become free radicals, therefore the diffusion control plays an important part in the observations of MFEs. For most non-viscous solutions, the rate of the escape product formation is fairly rapid and increasing the viscosity allows for the lifetime of the RP to be prolonged. In case of FAD, the two radicals are not able to become fully separated and independent as the RP is the result of intramolecular reaction. The differences in amplitudes of the signals in various MF (Fig.4.15) show that the conditions in different measurements vary. The difference in the spectra may result from fluctuations of light intensities or overlap of the pump and probe beams, which are shifting as the laser operation time passes or simply, the position of the sample itself. The overlap of the two beams required adjusting between measurements and so, the values of absorption at specific wavelength and time point should not be subtracted from each other and the MFE-action spectra were not created. It is apparent that the current method of such investigations is not perfect. It is clear that a new technique, allowing for the measurements with and without the field can be performed more efficiently, would make these types of measurements easier and the data obtained more reliable.

4.4 Summary

The MF generating apparatus has been designed and constructed and numerous tests and experiments have been performed. TA measurements on a test system, FAD in low pH in different values of MF, have been performed with the existing TA setup. The previously reported MFE in 200 mT was successfully reproduced and the investigations have been extended in the range of fields up to 950 mT. Both saturation of the Zeeman Effect and the Δg mechanism were observed in these studies demonstrating the suitability of the newly designed MF generating kit, as it allows for the field to be easily changed in a broad range, therefore enabling for the influence of different mechanisms to be observed. In traditional experiments investigating MFEs, measurements in the externally applied MF are followed by the measurements when no field is present. The data are then analysed and finally compared. It is therefore important for the conditions between the separate measurements to remain as similar as possible. In order to minimize the changes resulting from moving the magnets, between the field on and field off measurements, we have designed a new technique, where an additional path was created to enable measurements in field on and off without the need to move the MF-generating apparatus and the data to be acquired with a significantly improved signal to noise ratio. The newly developed setup will be described in the next chapter.

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Chapter 5

Development of Femtosecond – Nanosecond Transien t Absorption Instrument for the Investigations of Magnetic Field Effects

The idea of a traditional pump-probe spectroscopy is rather simple- molecules within the sample are excited by a short laser pulse and another pulse arriving sometime after the first one is then used to investigate changes of the absorption of the sample. By varying the arrival time of the second pulse with respect to the first one, the entire time- dependence of the absorption change can be measured. Small changes in absorption can be detected because the pump beam is modulated to be at least half the repetition rate of the probe beam, allowing the pump on / pump off difference to be detected by observing the signal produced at the specific frequency of the pump beam. Pump-probe spectroscopy has become a prominent method for time-resolved studies leading to development and advancements in many scientific fields. The high temporal resolution of pump-probe measurements, due to the very short laser pulses employed, has opened doors to new experiments investigating the dynamics of ultrafast processes.1 In MFE investigations, measurements with and without the application of the external MF are performed, analysed and finally compared. Although in TA techniques the signal to noise ratio is usually very good, a critical factor is the quality of the pump and probe beams overlap, which usually requires correcting between experiments due to small changes in beam profile. The quality of the data may therefore be different for separate data sets and it is important not to change the position of the sample between the measurements, which may be difficult in some cases. The newly designed MF generating apparatus allows us to change the field easily and quickly, but the sample may move when the distance between the magnets is changed. In order to minimize small differences between the measurements, we have developed Selene- a novel TA setup, where an additional, third path was created allowing for these measurements to be performed more efficiently and reproducibly.

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5.1 Experimental setup development

In designing a MFE-specific TA set-up we had two main aims: to improve the signal to noise ratio of the data collected, and to be able to measure data with and without an applied MF with as few physical changes as possible. For the first aim, in order to improve signal to noise ratio, a lock-in amplifier and biased Si detectors were employed as this type of signal detection allows for much smaller signals to be observed compared to the Helios setup. Furthermore, instead of using a WLC as the probe beam, which has limited light intensity, we used the output of an OPA, which not only allowed us to selectively probe a finite wavelength range (i.e. the spectral bandwidth of the OPA output), but also produced considerably more power than was available from the WLC. The modulation of the pump beam is provided by the optical chopper in the Helios set-up which allows only every second pulse to pass, reducing the repetition rate of the beam from 1 kHz to 500 Hz. The employed lock-in amplifier will then record changes occurring only at this frequency and ignore signals at all others, thus eliminating most of the background noise. For the second aim of measuring transient absorption signals with and without a MF with as little external input as possible, a third path was created. The first of the paths includes a sample shielded by a mu-metal enclosure ensuring no influence of external MF, the second one not only includes a sample, but also an applied MF (neodymium disc magnets described in section 3.1), and the reference beam being the third path. Although MFs cannot be blocked and there is no such thing as a magnetic insulator, they can be re-directed around objects. If the object is surrounded by a shielding material, which can ‘conduct’ the magnetic flux more efficiently than the object inside, the field will flow along the material. The effectiveness of the shielding depends on many factors, such as the nature of the MF, its strength and the thickness of the shielding. For the Selene setup four layers of mu-metal sheets were used in a semi- cylindrical shape, as shown in Fig.5.1.

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Fig.5.1 Semi- cylindrical mu-metal shielding (grey) with gaps for access of the laser beam to the sample cell. The green lines represent the MF lines, the red line shows the laser beam path.

After splitting off the reference beam, the remaining probe beam was split into two traversing through either one of the two sample beam paths, the choice of which is provided by the presence of an externally controlled shutter. Creating a second ‘sample’ path meant that an additional sample would be required. In order to minimise the amount of sample needed and to minimise any variations between the two cells, we designed and built a double capillary flow cell (Fig.5.2), in which the sample is flowed in parallel from a single reservoir.

Fig.5.2 Newly designed and built double capillary flow cell. The capillary cell is 2 mm diameter square profile quartz tubing and the flow tubing has an external diameter of 1.3 mm, internal diameter of 0.8 mm.

Due to space limitations Selene enclosure had to be located on an additional breadboard above the existing Helios/Eos setup. This allowed the use of certain Helios components, such as the optical delay stage and optical chopper, to be used in Selene.

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The newly designed optical setup is shown in Fig.5.3 and the photos of it in Fig.5.4.

Fig.5.3 Ultrafast transient absorption laser instrumentation development- ‘Selene’, a breadboard located above the existing Helios setup. Both pump and probe beams have been directed onto it by periscopes (P1, P2). The pump beam is represented by the purple lines, the probe beam by the pink lines and the reference by the green one.

Fig.5.4 Ultrafast transient absorption laser instrumentation development- ‘Selene’, a breadboard located above the existing Helios setup.

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After passing through the depolariser and optical chopper of the Helios setup the pump beam, generated by OPA1 is directed through a periscope to the breadboard above, where it passes through a focussing lens and is then is immediately split by a reflective ND filter into two separate beams. The first of the pump beams is directed onto the translation stage for path length matching, reflected by mirrors and onto the sample which is shielded from the applied MF. The second of the pump beams passes through the ND filter, is reflected onto a sample surrounded by the MF. After passing through samples, both pump beams are blocked. The probe beam is generated by OPA2 and after passing through the delay stage in the Helios setup it is routed through a periscope to the breadboard above. A fraction of the probe beam is split to be used as a reference beam and the remaining light as the two probe beams. The reference beam is reflected and focused onto the detector (Det-B, Thorlabs Det100-A). After passing through a focussing lens the remaining probe beam is split once more and the two beams are directed onto the respective samples- surrounded either by MF or the mu-metal shielding. The shutter positioned after the two probe beams are split allows for only one probe beam to pass at a time, which is controlled electronically from outside the enclosure. After passing through the samples the probe beams are reflected onto the detector (Det-A, Thorlabs Det100-A). The detectors have a relatively slow time resolution (43 ns rise) and measure only the amount of light detected, there is no wavelength separation. The detectors are connected to the lock-in amplifier (Stanford Research 830), which records the difference between Det-A and Det-B at the specific frequency of the pump beam. This newly developed setup allows for the MF to be changed easily and quickly within wide range of fields up to nearly 1 T and for the measurements with and without the applications of the field to be performed without significant changes in the position of the sample. The use of a lock-in amplifier and a single probe wavelength rather than a broad WLC spectrum with CMOS array detectors significantly improved signal to noise ratio. Both CMOS (Helios) and Det100-A (Selene) detectors were tested using the new optical setup, so that the levels of noise could be compared and the intensity of the probe beam changed in order to order to determine the required power for further experiments. The comparison of the signals obtained using both types of detectors is shown in Fig.5.5 and 5.6.

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Fig.5.5 Decay observed on a FAD in a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) sample in the shielded path using a probe pulse centred at 450 nm after photoexcitation with a laser pulse centred at 375 nm using a) Helios and b) Selene detectors. Powers quoted are probe intensities measured before the sample position. When Helios detectors were in use, different probe wavelength ranges were averaged in order to determine the best method for data analysis. The indistinct transient of the Selene 50 μW dataset is due to poor pump probe overlap.

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Fig.5.6 Decay observed on a FAD in a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) sample in the MF path using a probe pulse centred at 450 nm after photoexcitation with a laser pulse centred at 375 nm using a) Helios and b) Selene detectors. Powers quoted are probe intensities measured before the sample position. When Helios detectors were in use, different probe wavelength ranges were averaged in order to determine the best method for data analysis.

As can be noted from Figures 5.5 and 5.6, the combination of lock-in amplifier and DET100-A detectors has significantly improved the signal to noise ratio compared to the Helios detection hardwear, with background noise on the level of only ~5% of the maximum signal. Changes induced by the application of MF are expected to be

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relatively small, so reducing the noise levels in the data is necessary in order for those to be observed. The amplitude and the position of the signals on the two different paths is the same, although the vertical offset is slightly different. This may be explained by the variations of the background noise- it is possible that the pump scatter on the two paths is not the same, but this will have no effect on the kinetics itself. The time resolution of the Selene setup was then determined by performing scans on both paths using buffer solution as a sample. The data acquired were then fit with a Gaussian function; examples of such scans are shown in Fig.5.7.

Fig.5.7 Signal observed at 450 nm after the photoexcitation of a FAD in a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) with a laser pulse centered at 375 nm on a a) shielded and b) MF path. The data were then fit with a Gaussian function and the FWHM was determined.

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The full width at half maximum (FWHM) and therefore time resolution of the Selene setup was determined to be around 0.33 ps. The same method can be used to determine the time resolution of the Helios setup, however, in this case the acquired data must be fit with a multiple peaks function; example of such scan is shown in Fig.5.8. The time resolution in this case is determined by the FWHM value of the middle peak only2 and it is therefore equal to around 0.2 ps.

Fig.5.8 Signal observed after the photoexcitation of FAD in a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) with a laser pulse centered at 375 nm using Helios setup and detectors. The data was then fit with a multiple peaks function and the FWHM was determined.

The time resolutions of the existing and the newly developed setups are comparable. The slight increase in the Selene setup is not unexpected given the increased number of optics the beam travels through, and the different probe source.

5.2 Software development

The Helios setup uses custom built control software and electronic cards installed in the PC, in this case the system receives a 1 kHz trigger signal from the laser and the software then control the chopper and its internal lock-in collects the transient absorption data collected from the spectrographs and CMOS array detectors. The

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Helios software also moves the delay stage and performs data processing in order to convert the incoming signals to change in absorbance. The new setup required stand-alone software to perform these functions, for which systems engineering software LabVIEW was used. All LabVIEW programs are called virtual instruments (VIs), as the appearance and operation usually imitates physical instruments. LabVIEW contains a number of tools and options for the data acquisition and analysis. Every VI consists of two separate windows- the front panel, which is the user interface containing the controls and indicators, and the block diagram, which includes terminals, functions and structures that are responsible for the data transfer. After an individual VI is written, it can be saved as a subVI, added to the main pallet and easily inserted into another VI. If a VI is broken or there are any errors within the code, the ‘Run’ button appears broken and the VI is non-executable. In such cases, a list of errors will be generated and possible solutions suggested. When developing a program for instrument control, users can save time by utilizing the LabVIEW Instrument Driver Finder, which will automatically identify the connected instruments and install the required drivers, libraries, programme examples and templates. The delay stage (ILS220, Newport) receives commands from a separate control box (SMC100, Newport), which in turn is instructed by commands sent from the experiment control software running on a PC. Different types of delay stage are commercially available and will have different lengths and different minimum steps, which define how long the time window can be observed, and with what time resolution. Both of these parameters are usually defined within the software as millimetres and can be easily converted to time (using the speed of light, c ≈ 2.99 x 10-8 m·s-1). In the newly designed software the computer communicates with the delay stage control box via a USB cable and with the lock-in amplifier (SR830) via a RS232 cable interface and a USB converter. The lock-in amplifier receives an input reference frequency of 500 Hz from the optical chopper which remains under the control of the Helios software, operating at the laser reference frequency divided by two. The lock-in amplifier also receives input signals from the two detectors (DET100A, Thorlabs), reads their signal values and finally sends the calculated difference between them at the reference frequency of 500 Hz to the control software. The software then records this signal as a function of the delay stage position.

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The first challenge of the new software design was to successfully communicate with both the delay stage and the lock-in amplifier. The initial software focused simply on testing the communication with both instruments- the software sends a command requiring the name of the two instruments; the front panel and block diagram of this part of the code are shown in Fig.5.9.

Fig.5.9 Front panel and block diagram of the communication test for the Selene software. The initial software focuses only on testing the communication with both instruments- the software sends a command requiring the name of both instruments.

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The standard settings for the lock-in amplifier were then specified in the code, as shown in Fig.5.10, in such way that once turned on and connected to a computer, various options (time constant, sensitivity etc.) are set automatically.

Fig.5.10 Default settings block diagram for the newly developed Selene software. Once the software it turned on and both instruments are connected to the computer, various options (time constant, sensitivity etc.) are set automatically.

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The initial working version of the software simply required choice of a range of movements, over which the delay stage is told to move and a number of positions, at each of which it should stop for a fixed amount of time (set as double the value of the lock-in dwell time). After reaching the specified position, the delay stage sends information to the lock-in, which then reads the voltage of the transmitted light on both detectors (sample and the reference) for a certain dwell (integration) time and produces the difference between them. Due to the design of the lock-in amplifier only the difference in signals, not the signal’s absolute value is recorded. In order to convert the measured values of the voltage of the transmitted light to change in absorption, the voltage on the sample beam needs to be measured and entered manually in the software. The software will then convert the values of the change in transmittance into change in absorbance and save all values as an ASCII text file. The block diagram of the ‘Save’ Sub-VI and an example of Selene data file is shown in Fig. 5.11.

Fig.5.11 Block diagram of the Selene Save Sub-VI. All data will be given a unique name including the date and time of the measurement. Data from both channels are saved.

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Further software development was required in order to improve data collection and more options were added including number of scans and various movement ranges. The T0, where pump and probe beams are overlapped perfectly in time, varies daily and it is therefore important to correctly choose the movement range and step size. The number of ranges can be changed at any point, as this part of code was saved as an individual Sub-VI and can be easily inserted into the code. Both regular and randomly arranged stepping is available; after the ranges have been specified, the software will generate the positions for the delay stage to stop at. The calculated position will then be inserted in ascending order to an array for the regular stepping, or will be first shuffled, if the random stepping was selected. The amount of time the delay stage waits at a specific position can be determined by the user as well, although it needs to be manually adjusted on the amplifier to be correlated. If the amplifier has not been properly phased prior to the measurement, the pump-induced decay on the lock-in may appear on either one of the two channels (CH1 and CH2) or may be observed partially on both of them. The Selene software was designed to record and plot both channels at the same time, in case any errors arise and signal is not observed on the expected channel. All data are automatically saved in a default folder unless specified differently and each measurement will be given a unique name including the measurement date, time and the scan number to ensure that no data are accidentally overwritten or unsaved. All parameters for the delay stage, such as velocity or movements limits are visible on the front screen and can be changed within the software code only. Additionally the stage’s current position can be read from the rubric. The front panel of the finished software is shown in Fig.5.12.

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Fig.5.12 Front panel of the newly designed software. Once the computer is connected to both instruments and the software is turned on, the initial setting on the lock-in amplifier will be set automatically. If the ID of the instrument does not show, the communication with it has not been established. Various ranges of movements are available, as well as regular stepping order.

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5.3 Further instrument development

Traditional TA experiments include two paths- the sample path and the reference. Small changes in absorption can be detected because the pump beam is modulated to be at least half the repetition rate of the probe beam, allowing the pump on

/ pump off difference to be detected by observing the signal produced at the specific frequency of the pump beam. The relative position of the pulses used in traditional TA setup is shown in Fig.5.13.

Fig.5.13 Pump and probe beam pulses in traditional TA. The pump beam is modulated to be at least half the repetition rate of the probe beam, allowing the pump on /pump off difference to be detected by observing the signal produced at the specific frequency of the pump beam.

In Selene only one of the probe beams is allowed to pass through the sample, whilst the other one is fully blocked by the shutter. For more direct measurements of the MFE, we decided to try replacing the shutter with an optical chopper operating at a specific frequency and instead of fully blocking one of the paths, we alternated which path reached the detector. The chopper still allows one of the beams to pass at a time, but after passing through the samples, the two beams will be recombined and directed onto the detectors as one. The pump beam will no longer be chopped. The relative positions of the pulses in this setup are shown in Fig.5.14.

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Fig.5.14 In the proposed new scheme the probe beam is modulated to be half the repetition rate of the beam generated by the OPA2. After passing through the samples the two beams are recombined and directed onto the detectors as one.

The basic scheme for this new configuration is shown in Fig.5.15. This is still a pump-probe technique, with ‘sample’ and ‘reference’ paths, but in this design the ‘sample’ beam actually consists of two probe beams combined into one, with the change at the reference frequency being the difference between MF and no MF instead of the more usual pump on, pump off scenario. The detectors are connected to the lock- in amplifier, which records only signals at a specific frequency, in this case at 500 Hz, eliminating any background noise. Therefore the observed absorption changes should be those directly induced by the applied MF.

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Fig.5.15 The basic scheme for the modified Selene development. The probe beam generated by the OPA2 is split, half of which is used as the reference and is directed onto the detector (Det-B). The remaining portion becomes split once more. The two probe beams then pass through the optical chopper operating at the frequency of 500 Hz. After passing through the samples the two probe beams are recombined on the beam splitter.

After being split into the two the probe beams pass through an optical chopper, which operates at the frequency of 500 Hz. The chopper allows for only one of the beams to pass at a time, as shown in Fig.5.16, thus modulating the probe beam. After passing through the samples, both beams are recombined by a beam splitter (BS) and then coupled as one beam onto the detector (Det-A).

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Fig.5.16 The Selene chopper head- red and green lines represent beams passing through samples with and without applied MF respectively. Only one of the beams is allowed to pass at any time, while the other one is blocked by the blades of the chopper, as shown in a) green beam is blocked, red beam is allowed to pass b) green beam is allowed to pass while red beam is blocked.

In theory, if the applied MF has no effect on any species resulting from the photoexcitation, the observed spectrum should be a flat line. This design would not only allow for more direct measurements of field induced changes, but also significantly reduce the time required for the process of data analysis, as only the species susceptible to external fields would give a rise any signals. Furthermore, the MF-generating apparatus can be replaced so changes induced by temperature, pressure etc. could also be investigated. The new development was tested using FAD in pH 2.3 buffered solution. Samples were adjusted to a concentration such that the absorbance at the pump wavelength was ~0.5 using a 2 mm path length quartz cuvette. In all experiments the excitation light of 375 nm and the 450 nm probe beam were used. The TA traces observed when only one of the two probe paths was monitored are shown in Fig.5.17.

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Fig.5.17 TA traces observed at 450 nm following the excitation of FAD in buffered solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) at pH 2.3 with a laser pulse centered at 375 nm. The navy line represents the decay observed on the path surrounded by an external MF; the grey line represents the decay observed on the shielded path.

As can be seen from the example data in Fig.5.17 the beam paths and overlap of pump and probe can be adjusted so that the T0 observed on the two paths is very similar, within 1 ps, meaning that the two beams can be correctly recombined and the amplitude of the peaks is also very similar for the two paths, which implies that the intensities of the probe beams are well balanced. In order to create a difference between the two paths that could result in the observations of absorption changes, we monitored the recombined probe beam, at 1 kHz, and blocked the pump beam on one of the paths at a time. In theory, if the pump- probe overlap and T0 are the same, an identical decay should be observed on each path. The data obtained from this test are shown in Fig.5.18.

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Fig. 5.18 TA traces observed at 450 nm following the excitation of FAD in a buffer solution (4 mM Na2HPO4∙10H2O/ 98 mM citric acid) at pH 2.3 with a laser pulse centred at 375 nm a) when pump beam is allowed to pass through the sample surrounded by the MF and b) when the pump beam is allowed to pass through the sample surrounded by mu-metal shielding.

The results shown in Figure 5.18 were unfortunately not reproducible; over several weeks of testing the new setup only appeared to give the expected data twice. Many parameters were checked: the basic alignment, the length of the various beam paths, the position of T0, the frequency of the recombined beams, the stability of the pump and probe beams, the chopper phase, the alignment of the two beams through the chopper wheel, the chopper itself was changed, as was the chopper blade. Although

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during the next weeks of measurements occasionally a decay-like feature could be distinguished, the randomness of its appearance made it difficult to determine the cause of the problems. It became apparent that the lack of reproducibility was due to variations of the two probe beams. As the laser operates, its power and precise position varies making it impossible for the two paths to be identical at all times. The difference between the two probe beams is at 500 Hz, so the lock-in would detect it. The MF- induced changes are expected to be relatively small, so unless the variation in the two probe beam paths is even smaller no changes due to the MF would be observed. With regret this advanced setup was set aside and the original version of Selene was used for further measurements.

5.4 Optical setup test system- adenosylcobalamin

5.4.1 Photochemistry of vitamin B12

Enzymatic systems have long been of interest to chemists due to the highly productive and selective reactions in their pathways. Since the discovery of vitamin B12 as a treatment for pernicious anaemia (1920s), interest in B12-dependent enzymes and the isolated cobalamin coenzymes has been growing due to a presence of an unusually labile carbon-cobalt bond. Biologically active derivatives of vitamin B12 are organometallic complexes, which play an important role in normal functioning of the human brain, nervous system as well as the process of blood formation. B12 derivatives also act as cofactors to many thermally-driven metabolic enzymes. The behaviour and properties of vitamin B12 derivatives depends on the nature of the upper axial ligand. The chemical structures of selected ones are shown in Fig.5.19.

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Fig.5.19 Chemical structure of selected B12 derivatives with variable upper axial ligands (R). Methyl- and adenosylcobalamin are the only ones biologically active. Adapted from reference.3

The commercially available form of vitamin B12 is cyancobalamin (CNCbl), and although this form is stable, it is not biologically active and must be converted in vivo to either 5′-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) or methylcobalamin (MeCbl).4,5,6,7,8 CNCbl is commonly used in the treatment of pernicious anaemia as well as many other haematological diseases and its structure was first characterized by Dorothy Hodgkin, who due to her work on X-ray crystallography became only the third woman to win the Nobel Prize in Chemistry.

The photochemistry of vitamin B12 derivatives has been widely studied, but the main focus of investigations has centred on its function as a cofactor for thermally driven enzymes. The enzymes crucial to human and animal metabolism, such as Me- Cbl-dependent methyltransferases, are well characterized.9,10,11 Although methyltransferases catalyse the transfer of methyl groups via a SN2 mechanism, most of the AdoCbl-dependent enzymes catalyse the reactions by utilizing highly reactive radicals. Upon substrate binding, the Co-C bond to the upper axial ligand breaks

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homolytically, resulting in the formation of a cob(II)alamin/5’-deoxyadenosyl RP, as shown in Fig.5.20a, followed by an abstraction of a hydrogen atom from the substrate by the latter and a radical rearrangement. The question arising is how AdoCbl- dependent enzymes achieve the ~1012 enhancement in the rate of thermal Co-C bond 12,13,14,15 homolysis. The mechanism of B12-dependent dehalogenases involving bacterial organohalide respiration remains not yet fully understood, but it was recently proposed to proceed through the formation of a halide-cobalamin structure and either heterolytic or homolytic cleavage of the Co-halide bond.16,17 The homolysis of the Co-C bond can also result from the photoexcitation of alkylcobalamins (Fig.5.20b), such as MeCbl and AdoCbl.

Fig.5.20 a) A scheme of the Co-C bond homolysis upon substrate binding to AdoCbl- dependent mutases and eliminases resulting in the formation of a cob(II)alamin/5’-deoxyadenosyl RP, followed by a hydrogen atom abstraction from the substrate and the radical rearrangement. b) A scheme for the RP formation upon the photoexcitation of alkylcolbalamins. Adapted from reference.3

4,6,5,18,7 The structural studies of the B12 derivatives started in the 1950s and 1960s and were soon followed by the investigations of their photosensitivity.19,20,21,22 Although the early studies correctly identified a number of photoproducts and their quantum yields, the photochemical dynamics remained unknown until the 1970s,23,24 when various TA techniques were first employed. The RP generated upon the substrate binding to an enzyme is the same as the RP resulting from the photoexcitation,19 which enabled investigations of the influence of the cofactor environment on the formation of the RP and the dynamics of such reactions. The quantum yield of the solvent separated RPs in anaerobic conditions has been reported to be about 0.20-0.2425,26 and is determined by the competition between

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the geminate recombination and the formation of the escape products. The bond

cleavage in aqueous solutions takes place almost fully within 100 ps, and is independent of the excitation wavelength.25,27 The proposed AdoCbl photolysis mechanism in water is shown in Fig.5.21.

Fig.5.21 The photolysis of AdoCbl. Adapted from reference. 25

The TA spectral evolution following the photolysis of free AdoCbl (Fig.5.21) has been previously investigated by Sension and co-workers.25,27,28,35 According to this scheme, the excitation of the ground state AdoCbl is followed by an internal conversion on sub-ps timescale to the first excited state (AdoCbl*), which the proceeds via -1 (AdoCbl**) species with a rate constant of kex1= 0.69 ± 0.05 ps and then to an -1 intermediate state (AdoCbl***) with kex2= 0.071 ± 0.001 ps . The intermediate state has been reported to have a UV-Vis spectral shape similar to cob(II)alamin and may therefore represent an excited state, in which the Co-C has not yet been fully broken, but simply weakened and it is said to exist up to 110 ps after the excitation. The

(AdoClb***) can then either relax to the ground state (kR1) or undergo a further homolysis (kH) of the already weakened Co-C bond and generate a geminate, S-born RP, which can then undergo a spin-state mixing, a process which is known to be sensitive to externally applied MFs. The lifetime of the RP has been reported to be solvent dependent and the probability of the geminate recombination increases in solution of a higher viscosity28 with the escape product formation rate reduced from 0.57 ± 0.06 ns-1 in water to 0.11 ± 0.03 ns-1 in ethylene glycol. A similar cage effect was observed after photolysis of AdoCbl bound to its dependent enzymes29, 30 and

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UV/Vis TA experiments have demonstrated that the photolysis of ethanolamine ammonia lyase (EAL) is dependent on the viscosity of the solvent surrounding the protein.30 The nature of the lower axial group has been demonstrated to have a significant influence on the quantum yield of the photolysis.31,26,32 At low pH, the lower axial atom of nitrogen becomes protonated and the cobalt ion is now coordinated by the molecules of water, which leads to a yield of the close RPs reduced from near unity to only 0.12.31,33,26 The lack of the lower axial group in so called base-off AdoCbl results in changes in the electronic structure and opens a new channel for the rapid non-radiative decay that competes with the bond cleavage.31 The transition from base-on to base-off can be achieved by lowering the pH of the sample and it is also blue-shifted with the change of the dominant absorption peak from 525 to around 460 nm, as can be seen from Fig.5.22.

Fig.5.22 Absorption spectra of adenosylcobalamin in a 50/50 volume mixture of HEPES buffer and glycerol in pH 2.2 (grey) and in pH 7.5 (navy). The dashed lines represent the dominant absorption peak.

Although no evidence for the presence of geminate recombination was observed for base-off form, the quantum yield of solvent separated RPs has been demonstrated to decrease to 0.045,26,32 which implies that the recombination does in fact take place, but 126

on much slower timescale. This may be explained by the RP being born in a T-state, which would first have to undergo a conversion to a S-state in order to recombine.31 The steady state spectrum of base-off cob(II)alamin was measured by photolysis of AdoCbl in pH 2.0 and with the addition of the radical scavenger TEMPO.34,35 The excitation at

400 nm was reported to be followed by a strong bleach on the order of few picoseconds. Data were then fit to a sum of three exponential decay components with the rate values as follows: 2, ~ 0.35 and ~0.016 ps-1. The presence of an additional component with a very short lifetime has been suggested, but it could not be fully characterized due to the contributions of cross-phase modulation and the chirp of the WLC. The timescale for the separation due to diffusion of the adenosyl radical at neutral pH was previously 36 determined to be 1.6 ns, however, further studies by Sension and co-workers suggested that bond homolysis occurs on a timescale much faster, within a few tens of picoseconds, and it was hypothesized that the quantum yield may be affected by a geminate recombination of the RP on longer timescales. Although no evidence for geminate recombination was reported,37 the authors did not exclude its existence, as the signals were said to be of very small amplitude. The presented data provided new insight into the electronic structure and the reactivity of alkylcolbalamins. The quantum yields of base-off AdoCbl photolysis is dominated by the competition between the bond homolysis and fast internal conversion back to the ground state. The photolysis of these types of alkylcolbalamins leads to a rapid cascade via the excited state to produce a, presumably the S1, excited state, the lifetime of which is said to be between 18 and 60 ps. The population in this state is divided between the homolysis and the nonradiative decay recovering to the ground state, with the rate estimated to be about 40-50 ns-1, 38,39,40 which is significantly different to the rate of ground state recovery in base-on cobalamins on a nanosecond timescale. By contrast the photolysis of base-on AdoCbl at neutral pH is determined by the competition between the primary geminate recombination of the RP and the formation of free radicals.28,35 The rate constant for the internal conversion is no bigger -1 31 than 0.85 ns . The apparent lack of the geminate recombination in base-off form suggests that there is a change in the recombination of the RP as well. The MFEs were investigated using a broad band white light as the excitation source by Jones and co-workers,41 who deduced that the RP is formed from a singlet precursor independent of the excitation wavelength. The lack of geminate recombination in base-off cobalamins is consistent

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with the formation of a T-born RP, since only the RPs in S state can undergo the recombination. Further measurements and calculations of the photolysis of base-off and base-on cobalamins, may provide information on the influence of the axial ligand on the electronic structure. A cage effect similar to that observed in the presence of ethylene glycol was demonstrated after the photolysis of AdoCbl bound to dependent enzymes.29,42,43 The TA experiments have exposed the effect of the viscosity of the solvent surrounding the protein43 on the rate of the formation of escape products following the photolysis of AdoCbl bound to its dependent enzyme ethanolamine ammonia lyase (EAL), which was later explained using the time resolved IR detection as the results of the vibrational coupling between the AdoCbl and the local protein environments.44 Similar vibrational signals from EAL residues were then demonstrated by stopped-flow Fourier-transform infrared spectroscopy (FTIR) indicating the relevance of this type of coupling to enzyme catalysis. The origin of this coupling was proposed to be a mobile glutamate residue responsible for binding the substrate in EAL.45,46 The experiments investigating the reaction dynamics of the geminate recombination following the photolysis of AdoCbl have been performed mostly under anaerobic conditions. After the separation of the RP in anaerobic conditions the main photoproducts are the cob(II)alamin radical and 5′-deoxy-5′,8-cycloadenosine.19,47 while photolysis in aerobic conditions results in the formation of aquacobalamin 20,47,48 (OH2Cbl) or OHCbl and adenosine 5’-aldehyde. The photolysis of MeCbl results in the formation of a similar, cob(II)alamin/methyl RP, however, the MeCbl-dependent enzymes do not proceed through radical intermediates, as the Co-C bond breaks heterolytically leading to a change in the oxidation state from Co(III) to Co(I).9,49 The photolysis of MeCbl has previously been demonstrated to be wavelength dependent.48,25 It has been suggested that the geminate recombination following the photolysis of MeCbl in water is limited27,25,50,51 and affected mainly by the competition between internal conversion (from higher S1 state to S0 ground state) and bond homolysis.31 Despite the similarities in the photochemistry of AdoCbl and MeCbl, the reaction dynamics are quite different. It is apparent that the nature of the upper axial position of cobalamins has a big influence not only the electronic structure, but also on the reactivity. The effect of the upper ligand has been investigated in the photolysis of various alkylcobalamins.50,52 The radical diffusion is said to be highly affected by

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various factors such as viscosity,35 solvent properties, mass and volume3 and even the geometry of the radicals.53 In 5’-deoxyadenosyl radical, due to the location of the spin density, the unpaired electron is pointed towards the atom of cobalt, which leads to an enhanced recombination. In case of MeCbl, the excitation energy provides the newly formed methyl radical with additional translational energy leading to the lack of geminate recombination.3 Furthermore, the methyl radical is not only small sized allowing for the rapid diffusion to take place, but also planar, meaning that the unpaired spin is pointed away from the central atom, which increases the probability of chemical quenching making the recombination unlikely.53,54

5.4.2 Results and discussion

UV/Vis absorption spectra of samples at pH 2.2 and 7.5 were taken before and after each measurement to ensure no sample degradation occurred. The Selene enclosure was purged with nitrogen in order to minimize the amount of oxygen present, as the cob(II)alamin radical reacts with molecular oxygen to form hydroxocobalamin (OHCbl),55 photoexcitation of which does not result in a significant dissociation of the upper axial OH.56 Examples of these spectra are shown in Fig.5.23.

Fig.5.23 Normalized UV/Vis spectra of AdoCbl in 50/50 volume mixture of HEPES buffer and glycerol at a) pH 7.5 and b) pH 2.2 taken before (navy blue) the measurement, after the measurement when the Selene enclosure was purged (light blue) and after the measurements, when the enclosure was not purged (grey).

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5.4.2.1 Photolysis of AdoCbl using the experimental setup Selene

In order to test the performance and suitability of the Selene experimental setup, the AdoCbl photolysis experiments were performed at room temperature. The acquired data were then fit to a sum of three exponential decay components. Examples of typical

TA traces obtained with a probe centred at 525 nm, following the excitation of free

AdoCbl with a laser pulse centred at 375 nm are shown in Fig.5.24.

Fig.5.24 TA traces and regular residuals plots obtained on different days using a probe beam centred at 525 nm following the excitation of free adenosylcobalamin in 50/50 volume mixture of HEPES buffer and glycerol solution with a laser pulse centered at 375 nm.

A three - component fitting shows no significant structure in the residuals, which implies a good fit has been obtained. The rate constants calculated for the three exponential decay components shown in the graphs above are in agreement with previous studies and the variation of the values are acceptable.28 The observed rates of k1 and k2, equivalent to τ1 and τ2, represent the formation of the excited state and the intermediate. The future of the formed RP has previously been demonstrated to be

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dependent on the nature of the solvent with increasing probability of the geminate 28 recombination in solutions of higher viscosity. The calculated rate constant k3 represent the combination of kE and kR, the latter being the rate of the RP recombination. According to the RPM an externally applied MF can influence chemical reactions by affecting the electron spin state of a weakly coupled RP.57,58,59,60 The MFEs on photolysis of AdoCbl were first reported in 1993,61 although the range of MF used was limited. In order to fully investigate the MFEs as well as spin dynamics in B12, the experiments were extended to studies in external fields up to 900 mT at both neutral and acidic pH. These experiments will be described in the next chapter.

5.5 Summary

The aim of this project was to design and construct variable MF generating instrumentation which would enable the investigation of ultrafast spin-state mixing in

B12 species using MFEs in TA. We have demonstrated the newly designed and built Selene setup can be used for the investigation of MFEs in biological and chemical systems. The signal to noise ratio has been significantly improved compared to that achievable with the Helios setup. Creating an additional path and using the double capillary flow cell significantly minimized the variations between the scans. Further development of the experimental setup required the use of both paths simultaneously whilst chopping the probe beam would allow for the MF-induced changes to be observed more directly. Unfortunately, the expected decay was observed only on a small number of occasions, which is most likely caused by the variations in the laser power as well as its positions overwhelming the pump (or MF) induced change. With regret this advanced setup was set aside and the original version of Selene was used for further measurements. In order to test the suitability of the Selene setup for further MFEs experiments, the photolysis of free AdoCbl measurements were then performed. The rate constants calculated for the three components are in agreement with previous studies and the variations of the values are acceptable.28 The investigations of MFEs in AdoCbl were then performed in a wide range of external MFs up to 950 mT.

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38. T .A. Stich, N. R. Buan, J. C. Escalante-Semerena, T. C. Brunold; Spectroscopic and Computational Studies of the ATP: Corrinoid Adenosyltransferase (CobA) from Salmonella enterica : Insights into the Mechanism of Adenosylcobalamin Biosynthesis. 2005; vol. 127, no. 5, pp. 8710-8719.

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40. T. A. Stich, N. R. Buan, T. C. Brunold; Spectroscopic and Computational Studies of Co2+ Corrinoids: Spectral and Electronic Properties of the Biologically Relevant Base-On and Base-Off Forms of Co2+ Cobalamin. J. Am. Chem. Soc. 2004; vol. 126, no. 31, pp. 9735-9749.

41. A. R. Jones, J. R. Woodward, N. S. Scrutton; Continuous wave photolysis magnetic field effect investigations with free and protein-bound alkylcobalamins. J. Am. Chem. Soc. 2009; vol. 131, no. 47, pp. 17246-17253.

42. R. J. Sension, D .A. Harris, A. Stickrath, A. G. Cole, C. C. Fox, E. N. G. Marsh; Time-Resolved Measurements of the Photolysis and Recombination of Adenosylcobalamin Bound to Glutamate Mutase. J.Phys.Chem.B. 2005; vol. 109, no. 31, pp. 18146-18152.

43. A. R. Jones, S. J. O. Hardman, S. Hay, N. S. Scrutton; Is there a dynamic protein contribution to the substrate trigger in coenzyme B12-dependent ethanolamine ammonia lyase? Angew. Chemie Int. Ed. 2011; vol. 50, no. 46, pp. 10843-10846.

44. A. R. Jones, H. J. Russell, G. M. Greetham, M. Towrie, S. Hay, N. S. Scrutton; Ultrafast infrared spectral fingerprints of vitamin B12 and related cobalomins. J. Phys. Chem. A. 2012; vol. 116, no. 23, pp. 5586-5594.

45. Z. Chen, M. A. Zie, H .J. Russell, S. Tair, S. Hay, A. R. Jones, N .S. Scrutton; Dynamic , Electrostatic Model for the Generation and Control of High-Energy Radical Intermediates by a Coenzyme B12 -Dependent Enzyme. Chem. Bio. Chem. Commun. 2013, vol. 14, no. 1, pp. 1529-1533.

46. K. Mori, T. Oiwa, S. Kawaguchi, K. Kondo, Y. Takahashi, T. Toraya; Catalytic Roles of Substrate-Binding Residues in Coenzyme B12 -Dependent Ethanolamine Ammonia-Lyase. Biochemistry. 2014; vol. 53, no. 16, pp. 2661-2671.

47. H. P. C. Hogenkamp; A Cyclic Nucleoside Derived from Coenzyme B12. J. Biol. Chem. 1963; vol. 238, no. 1, pp. 477-481.

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48. T. Taylor, J. Gill, M. Leslie, A. Hantu: Aerobic Photolysis of Alkylcobalamins: Quantum Yields and Light-Action Spectra. Arch. Biochem. Biophys. 1973; vol. 156, no. 4, pp. 521-533.

49. R. G. Matthews, M. Koutmos, S. Datta; Cobalamin-dependent and cobamide- dependent methyltransferases. Curr. Opin. Struct. Biol. 2008; vol. 18, no. 6, pp. 658-666.

50. A. G. Cole, L. M. Yoder, J. J. Shiang; Time-Resolved Spectroscopic Studies of B12 Coenzymes: A Comparison of the Primary Photolysis Mechanism in Methyl-, Ethyl-, n -Propyl-, and 5‘-Deoxyadenosylcobalamin. J. Am. Chem. Soc. 2002; vol. 124, no. 3, pp. 434-441.

51. R. J. Sension, D. A. Harris, A. Stickrath, A. G. Cole, C. C. Fox, E. N. G. Marsh; Time-resolved measurements of the photolysis and recombination of adenosylcobalamin bound to glutamate mutase. J. Phys. Chem. B. 2005; vol. 109, no. 38, pp. 18146-18152.

52. D. A. Harris, A. B. Stickrath, E. C. Carroll, R. J. Sension; Influence of Environment on the Electronic Structure of Cob(III)alamins : Time-Resolved Absorption Studies of the S1 State Spectrum and Dynamics. J. Am. Chem. Soc. 2007; vol. 129, no. 3, pp. 7578-7585.

53. W. B. Lott, A. M. Chagovetz, C. B. Grissom; Alkyl Radical Geometry Controls Geminate Cage Recombination in Alkylcobalamin. J. Am. Chem. Soc. 1995; vol. 117, no. 49, pp. 12194-12201.

54. J .R. Woodward: Radical pairs in solution. Prog. React. Kinet. Mech. 2002; vol. 27, no. 3, pp. 165-207.

55. P. A. Schwartz, P. A. Frey; 5’-Peroxyadenosine and 5'-peroxyadenosylcobalamin as intermediates in the aerobic photolysis of adenosylcobalamin. Biochemistry. 2007; vol. 46, no. 24, pp. 7284-7292.

56. A. S. Rury, T. E. Wiley, R. J. Sension; Energy Cascades, Excited State Dynamics, and Photochemistry in Cob(III)alamins and Ferric Porphyrins. J. Am. Chem. Soc. 2015; vol. 48, no. 3, pp. 860-867.

57. Y. Zhang, G. P. Berman, S. Kais; The radical pair mechanism and the avian chemical compass: Quantum coherence and entanglement. Int. J. Quantum Chem. 2015; vol. 115, no. 19, pp. 1327-1341.

58. J. R. Woodward; Radical pairs in solution. Prog. React. Kinet. Mech. 2002; vol. 27, no. 3, pp. 165-207.

59. U. E. Steiner, T. Ulrich; Magnetic Field Effects in Chemical Kinetics and Related Phenomena. Chem Rev. 1989; vol., 89, no. 1, pp. 51-147.

60. A. R. Jones; Magnetic field effects in proteins. Mol. Phys. 2016; vol. 114, no. 11, pp. 1691-1702.

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61. A. Chagovetz, C. B. Grissom; Magnetic field effects in adenosylcob(III)alamin photolysis: relevance to B12 enzymes. J Am Chem Soc. 1993; vol. 115; no. 25, pp. 12152-12157.

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Chapter 6 MFEs on the Ultrafast Spin Dynamics of

AdoCbl in MFs up to 900 mT

6.1. Reported MFE studies

The rate and reaction yields of thermochemical, photochemical and biological reactions containing RP intermediates can be altered by the application of an external MF,1 which influences the extent of ISC between the singlet and triplet electron spin states. The rate of this interconversion may be either increased or decreased, meaning that the competition between RP recombination and separation can be affected. The spin conservation rules state that the RP multiplicity will be the same as that of its precursor, meaning that the singlet excited state precursor will generate a S-born RP and a triplet excited state a T-born one. If the hyperfine interaction constants of the resulting radicals are not identical and their lifetime is long enough for them to diffuse away in order to overcome the exchange interaction, the spin state will evolve and the S and T spin states will undergo a process of coherent interconversion, before relaxing incoherently to an equilibrium state. When no external field is applied, the spin state mixing is facilitated by the hyperfine interaction and its frequency can be described with the Eq. 1.8. As can be noted from this equation, the efficiency of the mixing depends on the difference between the HFI constants. Although for most organic radicals the differences of the HFI constants are fairly small, they are particularly large for the AdoCbl (2.71 mT for 5’-deoxyadenosyl radical and 15.9 mT for cob(II)alamin),3 suggesting that the state mixing takes place on the ultrafast timescale. Using equation 6.1, the process of spin state mixing for AdoCbl is therefore estimated to take place with a period of approximately 430 ps. The recombination of the RP can usually only take place from the S-state due to the Pauli exclusion principle. Both radicals possess an unpaired electron, which, being a moving charge, has an associated local magnetic field. The process of spin-state interconversion may therefore be affected by the application of external fields.4,5,6 Furthermore, the g-value for the 5’-deoxyadenosyl radical is typically close to the value of a free electron (ge~2.0023), while cob(II)alamin generated in the absence of an

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enzyme7 has a g-value around 2.16 and the value of CblII bound in the active site of an enzyme may be as high as 2.30.8 This would also result in an enhanced Δg mechanism, and thus the increase of the net rate of ISC in the presence of higher fields. Spin-lattice relaxation is the mechanism, by which the component of the magnetization vector along the direction of the static magnetic field and its surroundings, often referred to as ‘lattice’, establish a thermodynamic equilibrium.9 Relaxation results from the fluctuating MFs in the sample.10 The sources of such fields are numerous, for example the presence of paramagnetic substances in the solution or ever- present dipole-dipole coupling, which can be described by the equation11:

A E = (1 – 3cos2θ) Eq. 6.2 푟3 where r is the distance between the two spins and θ is the angle between the external field and the vector joining the two spins. A is a constant depending on the magnetic moment of the two spins and their orientation (up or down).11 When molecules move and rotate in solution, the θ angle changes rapidly, which leads to a fluctuating MF.11 If the molecule is of a large size, its rotation is slow as well as changes of the θ angle leading to relaxation, however, not all fluctuating fields are efficient enough to do so.5,10 The frequency of such fluctuation needs to be fairly large, but not too large- the relaxation is efficient only when the frequency of the motion matches the transition frequency. Large molecules tumble slowly, so the θ angle does not change significantly, while small molecules rotate too rapidly and as a result, the relaxation is inefficient in both cases. Another factor that may cause relaxation is the internal motion within the molecule itself. Different parts of molecules can have different motion frequencies, due to rotation around single bonds, and various sizes of elements in its structure- for example, motion of the methyl group is too rapid to accommodate efficient conditions for relaxations, but the motion of a heavy element is much slower resulting in an enhanced relaxation. The relaxation processes in AdoCbl are therefore expected to be ultrafast, due to the presence of a heavy transition metal atom of Co in its structure. If the RP relaxes too quickly, the spin-state mixing will not be able to occur. The relaxation processes have more time to affect RPs, the lifetimes of which are longer and are more damaging to those.10 The MFEs on the photolysis of AdoCbl performed in 199312 reported an increase of the observed rate of the RP recombination in viscous solutions when a field

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of up to few hundreds of mT was applied. As the T±1 spin state levels become separated due to Zeeman effect, the interconversion between them becomes increasingly less efficient, allowing only for the S↔T0 interconversion to take place. The probability of recombination increases due to the increase in S-state population. The original plots for the CblII quantum yields are shown in Fig.6.1. Shortly after, the same group reported a similar MF-dependence on the steady- state turnover parameter Vmax/Km for the B12-dependent enzyme, ethanolamine ammonia lyase (EAL),13 reporting the first observation of the field effect on a wildtype protein with its natural substrate. The Vmax/Km parameter was reported to decrease by 25 % when a static field of ~100 mT was applied. The origin of the field effect was proposed to be caused by a MF-induced change in intersystem crossing rates between the singlet and triplet spin states in the cob(ll)alamin:5'-deoxyadenosyl spin-correlated radical pair.13

II Fig.6.1 MF dependency of the Cbl quantum yield for the cw photolysis of 200 μM AdoCbl at 20°C. A) 514 nm, 50 mM Hepes/ 75% glycerol. B) 514 nm, 50 mM Hepes. 20% Ficoll-400. A’) 248 nm, 50 mM Hepes/ 75% glycerol. B’) 248 nm, 50 mM Hepes / 20% Ficoll-400. All plots taken from reference.12

The MFEs were then revisited by Jones and co-workers.14 A continuous wave (CW) photolysis study investigating MFEs in both free and enzyme-bound AdoCbl and MeCbl was performed.14 Experiments on free AdoCbl were conducted under anaerobic

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conditions and in the presence of the TEMPO radical, to selectively scavenge the alkyl radical and allow an accumulation of the CoII radical.15,16,17,18,19 The resulting traces observed at 525 nm with and without the externally applied MF of 190 mT and MFE plot are shown in Fig.6.2.

Fig.6.2 a) Overlaid and difference traces acquired at 525 nm for the anaerobic cw-photolysis (140 µmol s-1 m-2) of 26 µM free AdoCbl in 20mM Hepes / 67 % w/w glycerol, pH 7.5, and 1 mM TEMPO. b) MF-dependence of the relative, observed rate coefficient from the initial downward phase representing CoII accumulation. The data in a) were fit with a triple exponential fitting function, and only k1obs showed MF sensitivity. Taken from reference.20

A large field-dependence can be observed on the rate of the initial downward phase, which represents the accumulation of the CoII radical. Calculations of relative rate coefficient for each investigated value of MF were performed in order to generate a MFE plot (Fig.6.2b), from which a saturating MF dependence is clear. The observed increase of MFE amplitude in solutions of higher viscosities was explained as due to longer cage lifetime and a potential for enhanced spin-state mixing resulting in higher probability of the RP recombination.14 In MFE investigations on EAL-bound AdoCbl, neither the addition of TEMPO radical nor anaerobic conditions are required, due to the protective effect of the protein.

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As the proportion of AdoCbl bound to the protein increases, so did the absorbance at 525 nm, which implied that the reaction between the CoII radical and molecular oxygen to 5′-peroxyadenosylcobalamin is replaced by an absorption decrease, due to CoII becoming protected by the active site.14 The kinetics trace and a single exponential fit from the EAL-bound to AdoCbl data is shown in Fig.6.3.

Fig.6.3 (a) The aerobic cw-photolysis of 10 µM AdoCbl in the presence and absence of a 190 mT MF titrating EAL apoenzyme (∼0-20 µM active sites). The relative extinction coefficients at 525 nm of the various cobalamin species implicated are listed: OHCbl, hydroxycobalamin; PerAdoCbl, 5′-peroxyadenosylcobalamin; AdoCbl, 5′-adenosylcobalamin; Cbl(II), cob(II)alamin. (b) Data (O) and single exponential fit (red line) representing the aerobic cw-photolysis of the EAL holoenzyme (∼26 µM active sites, 10 µM AdoCbl), acquired at 525 nm, 0 mT, and 25 °C. (c) Overlaid traces acquired at 0 and 190 mT, and corresponding difference trace (blue line); conditions as for (b). A clear magnetically induced change in photolysis rate is evident. Taken from reference.14

The change in absorbance is said to be caused by the loss of the AdoCblIII αβ band on homolysis of the Co-C bond. Despite the close-pair recombination being highly favored,21,22 a population of adenosyl radicals diffuse away from the CoII and possibly

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removed. The accumulation of the CoII species observed, is the result of the repeated photolysis of the bond and continuous removal of the alkyl radicals. The change caused by the application of a 190 mT MF shown in Fig.6.3 c) revealed that the MFE is most likely kinetic in origin, with single exponential fits of both data sets giving a relative rate of an 18% reduction. Competing pathways of reactions have been suggested due to presence of a slight quantum yield effect, the origins of which are not yet fully understood. The data were in agreement with the theory of protein protective effects, confirming that the MFEs are a result of the protein- bound AdoCbl reaction dynamics. The relative rates for all applied fields were then calculated and plotted against the field values, as shown in Fig.6.4.

Fig.6.4 Magnetic field dependence (O) of the aerobic cw-photolysis relative rate of the AdoCbl bound to EAL. The data points are the mean of 5-7 acquisitions, and the error bars represent the standard deviation. The observed relative rate coefficient, extracted from the data decreases with increasing field strength until saturation at a relative rate of ∼0.82. A Lorentzian line shape (red line) predicting the MF dependence was produced from the calculated B1/2 value. The predicted and experimental dependencies are in good agreement. Taken from reference.14

The field dependence shown in Fig.6.4 was said to be in excellent agreement with that predicted by the Lorentzian line shape, produced from the calculated B1/2 value using the method described previously.23 The CW-photolysis of free MeCbl (Fig.6.5) has been shown to be reduced in comparison to AdoCbl, because of the presence of relatively small methyl radical which makes the cage escape from the active-site cage more probable. In this case the results

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showed faster quenching kinetics and much reduced amplitude in comparison to AdoCbl, which suggest that the quenching processes are more rapid and abundant, but when MeCbl was bound to EAL, no MFE was observed.

Fig.6.5 a) The MF-dependence of the anaerobic cw-photolysis (140 µmol s-1m-2) of free MeCbl (40 µM) in 20 mM Hepes / 67 % glycerol, pH 7.5 and 1 mM TEMPO, in the presence (O) and absence (O) of a < 400 nm cut-off filter. b) Overlaid traces acquired at 525 nm for the anaerobic cw-photolysis (140 µmol s-1 m-2) of MeCbl (10 µM) in 20mM Hepes / 67 % w/w glycerol, pH 7.5 and 1 mM TEMPO, in the presence and absence of a < 400 nm cut-off filter. Taken from reference.14

Furthermore, the photolysis of MeCbl has previously been shown to be wavelength-dependent. When an excitation wavelength of 400 nm is used, about 25% of the initial photoproducts undergo a direct homolysis, while the remaining 75% form a metastable intermediate state; when an excitation wavelength of 520 nm is used, only the metastable state is observed.21,24 Regardless of the wavelength, the metastable state can only decay to the ground state or form free radicals. The aerobic cw-photolysis of EAL-bound MeCbl have shown at least a partial protective effect of the enzyme against the side reaction with molecular oxygen14 and no MFE was reported. The data for MeCbl suggested a higher number of faster RP quenching processes and, although the traces at 525 nm were fairly well fit when a double exponential function was used, the physical origin of those is not fully known. The reported MFEs in AdoCbl dependent EAL was the first observations of field effect on a wildtype protein influenced by an externally applied MF.14,25,26 Evidence that physiological process may be affected by the application of MFs has given a rise to concerns about humans’ health and the risks associated with both environmental and occupational exposure.27 Despite the observations of MFE on the

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photolysis of free AdoCbl, the lack of observed MFEs, when the cofactor is bound to the EAL apoenzyme in the presence of a substrate has given a rise to questions about the previously reported existence of such field sensitivity. It was also concluded that the stopped-flow method was12 not sensitive to the pre-steady-state kinetics of geminate recombination, the step they claimed to be magnetically-sensitive. It was further explained that the difference in magnetic-sensitivity may be caused by limited geminate

recombination in the enzyme - catalysed reaction. The observed MFEs on AdoCbl dynamics are consistent with the formation of a S-born RP.25,28 and it has been further supported by the polarisation pattern of the time- resolved EPR data.29,30 In case of MeCbl, however, the MFEs suggest that the RP is generated from a precursor in a S-state, which is inconsistent with the initial time- dependent density functional theory (TD-DFT) calculations performed that proposed the bond photolysis being mediated by a T-state and would lead a T-born RP.31,14 The potential energy surface of the S1 state was constructed as a function Co-C and Co-N bond distance, and two possible dissociation pathways were demonstrated, which can be associated with different excitation wavelengths, providing a theoretical basis for the wavelength dependent photolysis of MeCbl. Although the chemically induced dynamic electron polarisation (CIDEP) signals from EPR measurements suggest that the photolysis of MeCbl proceeds via an excited T-state, the chemically induced dynamics nuclear polarisation (CIDNP) signals propose that it proceeds via the S-state.32 These differences may be explained as the CIDEP signals being dominated by the triplet mechanism polarisation, while the CIDNP and MFEs data by a different reaction channel proceeding through the S-born RPs; this would be consistent with the wavelength-dependence.21 It is possible that the MeCbl photolysis results in the formation of both S and T-born RPs. The photolysis of AdoCbl results in a formation of a RP, in which the HFI constant are very different, which implies that the process of spin-state mixing is very efficient and fast. Furthermore, due to difference in the g-factor for the two radicals, the possibility of the Δg mechanism playing part on the RP recombination is much higher than for most organic radicals. Finally, one would expect fast relaxation owing to the II presence of a Co radical. Coenzyme B12 is therefore a perfect candidate for the investigation of MFEs with features not usually found in typical organic RPs: competition between ultrafast spin-state interconversion and rapid relaxation, and the contribution of the Δg at relatively low MFs (i.e., < 1 T). Such investigations, however,

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will need specialised apparatus capable of combining ultrafast kinetic measurements and a large range of MF-exposure amplitudes.

6.2 TA measurements of free AdoCbl in external MFs up to 900 mT

The ultrafast TA experiments were performed at room temperature using the Selene setup described in section 5.1. For all experiments an excitation wavelength of

375 nm and probe wavelength of 525 nm were used and the absorption changes were monitored at time delays between 0.2 ps and ~3 ns after excitation. In the original MFE studies conducted by Harkins and Grissom the C-Co bond homolysis was monitored at II 1 471 nm (the evolution of Cbl ), however, the largest change in absorbance III accompanies the decay of the AdoCbl species at 525 nm, which results in a significantly improved signal to noise ratio. The changes observed at both wavelengths are known to represent the same kinetics.33 All data were fit to sum of three exponential decay components. The results from experiments at both neutral and low pH are presented.

6.2.1 Results and discussion

6.2.1.1 MFE studies on photolysis of free AdoCbl up to 200 mT

The spin-state of the generated adenosyl/CblII RP can coherently interconvert between the singlet and the triplet sublevels, as shown in Fig.6.6.

Fig.6.6 The reaction and spin dynamics of the separated adenosyl/CblII RP following the photolysis of AdoCbl. During the cw-photolysis, the adenosyl radicals are irreversibly quenched yielding an accumulated CblII signal. Adapted from the reference.34

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Previous cw-photolysis MFE studies with B12 have been conducted under anaerobic conditions, up to 200 mT and in the presence of TEMPO, in order to selectively scavenge the alkyl allowing the accumulation of the CblII radical.15,35 In experiments described in this thesis, ultrafast TA spectroscopy was used to probe changes to the microscopic rate constant, which we hypothesise contribute to the previously reported cw-MFE over the field range.

An example of a typical TA trace obtained at 525 nm following the excitation of free AdoCbl with a laser pulse centred at 375 nm is shown in Fig.6.7.

Fig.6.7 An example of MFE on the decay observed at 525 nm following the excitation of AdoCbl with a laser pulse centred at 375 nm observed on a) the shielded path and b) the MF path. A sum of three exponentials fitting shows no significant structure in the residuals, which implies a good fit has been obtained.

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The lifetime values obtained from a fit to sum of three exponential decay components were then plotted against the applied field, as shown in Fig.6.8, in order to determine, whether any of them were field dependent.

Fig.6.8 Comparison of lifetime values and the fitting errors for the a) first b) second and c) third component in external MF of up to 200 mT. The dashed line represents the lifetime value for measurements performed without the externally applied MF.

The component with the shortest lifetime of 1-3 ps and the second one, with the lifetime between 40-80 ps represent the formation of the excited state and the intermediate respectively. Neither of those show any field dependence, but the lifetime of the third component was more clearly affected by the application of the field. Although the rate for the measurement with externally applied field of 70 mT seems to be not affected by the MF, this data set was of a relative poor quality and the lifetime values of all three components are subject to a large fitting error. The third component can be assigned to the RP and its rate k3, represents the competition between the geminate recombination and the formation of free radicals.

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The experimental error was calculated as described in section 2.4. A comparison of previously published MFE in MFs of up to 200 mT observed using cw photolysis on a picosecond timescale33 and experiments performed using Selene fitted to the expected Lorentzian shape is shown in Fig.6.9.

Fig.6.9 Comparison of the MFE on photolysis of AdoCbl observed in the a) previous and b) current study. The red line represents the predicted Lorentzian shape of the MF-dependence.

Previously reported studies have revealed a MF-dependence on the geminate recombination in the opposite sense to those described in this thesis due to the inverse relationship between the two, in both viscous solvent and buffered water. In conditions of continuous illumination, the adenosyl radicals are irreversibly quenched to yield an accumulated CblII signal leading to a decrease in the apparent rate of this accumulation.34 In TA spectroscopy the kinetics of the recombination are measured, which leads to an increase in the observed rate. The accumulation of CblII and the recombination kinetics show a mutually inverse MFE.

The B1/2 value, at which half of the saturation takes place, can be calculated using the equation below.

2 2 2 (푎1 + 푎2 ) B1/2 = Eq.6.1 (푎1+ 푎2) Where

2 ai = √∑푗 퐼푖푗 ( 퐼푖푗 + 1)푎푖푗 Eq.6.2

The ai in the equations stands for the average hyperfine couplings of the II 29 adenosyl and Cbl radicals and they have previously been calculated to be 2.71 mT 36,37 and 15.9 mT. The calculated B1/2 is therefore 28 mT, which is quite different than the value observed in Fig.6.8, where the B1/2 is closer to ~100 mT. This is caused most

149

likely by the fairly high error, or other mechanisms, such as diffusion and relaxation, which are not taken into account in theoretical calculations. The observed field dependence is however in agreement with the results previously published,33 which implies that Selene setup is a suitable experimental setup for investigations of MFEs in biochemical systems. In order for the process of spin-state mixing to take place, the RP needs to have enough time to separate and overcome the exchange interaction. As discussed in details in Chapter 1, the spin-state interconversion at a very short distance is energetically highly unfavourable, due to the electron exchange interaction being too large. Since the T-born pairs are non-reactive and there are three of these states and only one S-state, 75% of encounters are non-reactive.38 Furthermore, these numbers may be affected by any other mechanisms able to induce the spin-state mixing. The exchange interaction has to drop to values similar to those of the hyperfine coupling constants within the lifetime of the RP. The observations of ultrafast MF- dependence on photolysis of free AdoCbl implies that the diffusion of the two radicals is fast enough in order for them to overcome the exchange interaction. Furthermore, the observed MFEs imply that the S-T interconversion take place on a faster timescale than the relaxation processes and it is not affected by them. The MFEs on the photolysis of AdoCbl were then investigated in a wide range of fields up to 900 mT.

6.2.1.2 MFEs in AdoCbl at neutral pH and external fields of up to 900 mT

The MFE on ultrafast timescale and external fields of up to 200 mT has been observed, which reflects a large difference in the hyperfine constants. The g values in AdoCbl are also much bigger than in most organic RPs, which implies the potential for the Δg effect to be observed in the field range obtainable with the MF-generating apparatus. MFEs on the photolysis of AdoCbl in external fields up to 900 mT were investigated and the data obtained was once again fit to sum of three exponential decay components. The lifetime values for all components were then plotted against the applied field, as shown in Fig.6.10, in order to determine, whether any of them is field dependent.

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Fig.6.10 Comparison of lifetime values of the a) first, b) second and c) third component against the applied MF. The dashed line represents the lifetime value for measurements performed without the externally applied MF.

As determined by experiments in lower fields, neither the component with the shortest lifetime of 1-3 ps nor the second one, with the lifetime between 40-80 ps have shown any significant field dependence. The lifetime of the third component, assigned to the decay of the RP was clearly affected by the application of the field. The decay rate of this component- k3, is the combination of the geminate recombination and the formation of free radicals. Once again the experimental errors were calculated as described in section 2.4 and the resulting values are shown in Fig.6.11. The table with all values obtained from the analysis of the field dependence data can be seen in the Table 6.1

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Table 6.1 Relative rate values for the decay of the third component in applied MF up to 900 mT

Measurement MF k3 τ3 k0mT k average % Date -1 -1 rel St.Dev. (mT) (ps ) (ps) (ps ) krel Error 23/08/2017 0.0018 544.6644 0.0017 1.1107 24/08/2017 25 0.0016 617.2997 0.0016 1.0147 1.0905 0.0681 6.24 21/08/2017 0.0017 597.5617 0.0015 1.1462 23/08/2017 0.0019 537.3859 0.0014 1.3289 24/08/2017 50 0.0021 483.0005 0.0014 1.4786 1.3496 0.1200 8.89

23/08/2017 0.0018 562.8109 0.0016 1.0880 24/08/2017 70 0.0016 612.3218 0.0015 1.0631 1.0848 0.0203 1.87 21/08/2017 0.0018 546.0270 0.0017 1.1034 14/08/2017 0.0024 416.66 0.0013 1.7850 15/08/2017 100 0.0027 370.37 0.0017 1.6197 1.6360 0.1416 8.65 16/08/2017 0.0022 454.55 0.0015 1.5033 17/08/2017 0.0029 344.83 0.0017 1.6889 21/08/2017 200 0.0019 522.7403 0.0013 1.4999 1.6370 0.1198 7.32 22/08/2017 0.0020 508.4665 0.0011 1.7221 17/08/2017 0.0020 507.0125 0.0013 1.5371 18/08/2017 300 0.0034 291.4988 0.0018 1.8760 1.7302 0.1743 10.08 1.7775 11/08/2017 0.0023 434.7192 0.0016 1.4496 14/08/2017 400 0.0026 388.7136 0.0015 1.6706 1.5969 0.1276 7.99 15/08/2017 0.0026 391.1206 0.0016 1.6250 11/08/2017 0.0022 450.0205 0.0013 1.7691 09/08/2017 500 0.0017 588.2126 0.0012 1.4257 1.5546 0.1870 12.03 18/08/2017 0.0030 329.4301 0.0021 1.4690 17/08/2017 0.0020 497.0647 0.0015 1.3658 18/08/2017 600 0.0017 581.2335 0.0012 1.4925 1.3894 0.0936 6.73 1.3099 22/08/2017 0.0022 461.1378 0.0018 1.1811 18/08/2017 650 0.0022 458.7698 0.0016 1.3244 1.2356 0.0776 6.28 21/08/2017 0.0016 612.4382 0.0014 1.2012 10/08/2017 0.0014 693.5963 0.0013 1.0938 11/08/2017 700 0.0014 716.6778 0.0012 1.1394 1.0652 0.0919 8.63 0.9623 10/08/2017 0.0015 664.0084 0.0016 0.9481 18/08/2017 800 0.0017 595.5090 0.0014 1.1840 1.0367 0.1285 12.39 11/08/2017 0.0012 815.2965 0.0013 0.9779 18/08/2017 0.0022 461.2015 0.0019 1.2080 21/08/2017 900 0.0019 525.4770 0.0018 1.0603 1.0718 0.1308 12.21 0.0015 658.4977 0.0016 0.9471

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Fig.6.11 Relative rate values and experimental errors for the decay observed at 525 nm following the photolysis of free AdoCbl with a laser pulse centred at 375 nm against the applied MF. Measurements in each field were repeated three times and were followed by a measurement without the application of the field. The average values of individual measurements as well as their standard deviations were then calculated.

A very large MFE can be clearly observed in the rate representing the decay of the adenosyl radical and when the relative rate coefficient is calculated for each MF investigated, a saturating MF dependence is evident. Increasing the viscosity of the solvent resulting in a longer cage lifetime leads to the potential for a longer separation time and the enhanced spin-state mixing, because there is more time for the process of the spin-state interconversion to as well as higher probability of the RP reencounter.15 By using a solvent containing 50% glycerol (by volume) we have decreased the probability of the formation of the escape products. Two major trends in the rate of RP combination can be observed in Fig.6.11.

Firstly, there is an increase in recombination rate, when exposed to MFs up to about 100 mT, which is consistent with a S-born RP. At these field magnitudes, the T±1 spin-states, the magnetisation direction of which is the same as the applied field, are separated in energy by the Zeeman interaction such that their involvement in interconversion decreases in higher fields. When the field is applied, the population of the S-state increases, as well as the rate of the RP recombination. The second trend observed in

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Fig.6.11 is a decrease in the rate of the geminate recombination at MF above around

600 mT. Because the unpaired electrons on both radicals have different g-factors, the application of higher fields can also enhance ISC, which is called the Δg mechanism. As the Zeeman Effect saturates and is gradually overcome by enhanced S-T0 spin-state mixing due to the Δg mechanism, resulting in the increase of adenosyl radical population and slower recombination rate. One might notice from the first term of spin Hamiltonian for a RP (discussed in Chapter 1, Eq.1.10),

̂ 2 ̂ 퐻푧(퐵) = ∑푖=1( 휇퐵 ·gi·푆i·B) Eq.6.1 that the required fields in this case are relatively high. For most of organic radicals the values of g- factors are very small, and this term can be omitted from calculations, however, when the applied field is large, it is no longer negligible. Table 6.2 shows typical g-values for some organic radicals.

Table 6.2 Isotropic g-values for some typical organic radicals. Adapted from the reference. 38

The effect of the Δg mechanism may therefore be observed only in high MFs and it is opposite to a MF-induced change through hyperfine mechanism. The Δg mechanism may however affect the RP recombination in lower fields assuming that the g-values of the two radicals are significantly different. The g-value for the 5’- deoxyadenosyl radical is typically close to the value of a free electron (g~2.025), while cob(II)alamin generated in the absence of an enzyme7 has a g-value around 2.16 and the value of CblIII bound in the active site of an enzyme may be as high as 2.30,8 which would result in an enhanced Δg mechanism. In case of AdoCbl, the RP is generated from a precursor in an S-state, meaning that in higher fields, its population decreases. It 39 has been previously demonstrated that the yield decrease is proportional to B1/2. If the RP was generated from a precursor in a T-state, the results would be opposite. Theoretical calculation required to fully characterize the photochemistry of cobalamins are said to be very complicated, due to the big size of the tetrapyrrole ring and the number of collateral groups as well as the complexity of the metal centre itself.40

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Therefore, whilst simulations of the full MF dependence may be possible, in this case they fall outside the timeframe of the experiments described in this thesis. The observed MFE curve is in agreement with the theoretically predicted shape calculated using the previously described method.23 In 1993 Sakaguchi and Hayashi combined a nanosecond laser flash photolysis apparatus with a superconducting magnet in order to measure transient optical 41 absorption of radicals in external fields of up to 10 T. The authors investigated the photochemical reaction of benzophenone in a micellar sodium dodecysulfate solution and reported that the decrease of the decay rate of the ketyl radical was saturated around 2 T. The apparent lack of the Δg mechanism contribution in external fields lower than 10 T was explained to be due to a very small Δg (said to be close to zero) values for this system. Further studies of RPs containing heavy atoms in micellar solution as well as in non-viscous solution demonstrated the observations of field induced change due to the Δg mechanism.42,43,44,45 In all studies, the Δg were very small, from 0.000842 to 0.0047344 and required the applied MF to be as high as 10 T in order to facilitate such mechanism. Tanimoto and co-workers46,47,48,49 used a pulsed magnet to generate external field of 14 T and studied the decay of various transient biradicals and RPs and explained their results in terms of relaxation due to hyperfine and g tensor anisotropy. 50 In 1997 Hayashi and co-workers extended the range of MF to 30 T and investigated the photoreduction of benzophenone in micellar SDS and Brij35 solution. As reported, the lifetime of the generated RP increased in higher fields of up to 3.36 T and it remained constant, when the field was continuously increased until it reached 29.6 T. This saturating effect was explained by the RM due to the anisotropic Δg-, HFC-, and dipolar- interactions. Although theoretical calculations performed by Schulten and Epstein estimated that the saturation of MFEs due to the ∆g should take place at extremely large MFs of the order of 103 T for the values of ∆g on the order of 0.01 in non-viscous solutions. Hayashi and co-workers reported the observations of such saturation for the hydrogen abstraction reaction of 4-methoxybenzophenone (MBP) with thiophenol (PhSH) in 2- meth-yl-1-propanol already at 20 T. The g-values for the ketyl and PhS radical are 2.0027 and 2.0082 respectively, meaning that the ∆g= 0.0055. The saturation was said to be due to the isotropic ∆g mechanism and it was interpreted by a complete T0-S spin conversion due to the ∆g mechanism with an extremely fast recombination reaction

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from singlet radical pair. In all studies mentioned the main feature of the observed MFEs was assigned due to the ∆g mechanism, but the interpretation is said to remain unclear.45 Although the studies of the influence of externally applied MFs on chemical reactions have been reported, the effects of high fields on biological systems have not been investigated. The reported MFEs in AdoCbl dependent EAL was the first observations of field effect on a wildtype protein influenced by an externally applied MF14,25,26 and it has given a rise to concerns about human health and the risks associated with both environmental and occupational exposure.27 Later attempts to reproduce these findings by Jones and co-workers have not been successful and have given a rise to questions about the previously reported existence of such field sensitivity. The authors concluded that the stopped-flow method was12 not sensitive to the pre-steady-state kinetics of geminate recombination, the step they claimed to be magnetically-sensitive and suggested that the difference in magnetic-sensitivity between B12 photolysis and B12- dependent enzyme catalysis may be caused by limited geminate recombination in the

enzyme - catalysed reaction. In solution, the newly generated RP has to diffuse apart in order to overcome the exchange interaction allowing for the S-T interconversion to take place. Since both adenosine and CoII radicals are bound to the protein, the classical diffusive processes are less likely to take place;20 such immobilization of the RP within the enzyme has previously been suggested.51 The apparent lack of MFEs has also been explained in terms of changes to the dynamics of the RP upon substrate binding52 and it was reasoned that the sensitivity to external fields is caused by the rapid removal of the adenosyl radical by H-abstraction from the substrate and the consecutive RP separation. The authors concluded that although the active site of the EAL is a suitable environment for the observations of the enhanced MFEs in B12, the magnetic sensitivity is removed if the radicals are removed fast enough.

6.2.1.3 MFEs in AdoCbl at low pH and external fields of up to 900 mT

As discussed in section 5.4 the nature of the lower axial group has a significant influence on the quantum yield of the photolysis.53,15,22 The lack of the lower axial group in so called base-off AdoCbl results in changes in the electronic structure and opens a new channel for the rapid non-radiative decay that competes with the bond

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cleavage,53 resulting in the very low quantum yields of RPs and the apparent lack of geminate recombination.15,22

An example of a typical TA trace obtained at 525 nm following the excitation of free AdoCbl in pH 2.2 with a laser pulse centred at 375 nm is shown in Fig.6.12. The data obtained were fitted initially with the sum of both two and three exponential decays.

Fig.6.12 Transient kinetics observed at 525 nm following the excitation of AdoCbl in 50/50 volume mixture of HEPES buffer and glycerol at pH 2.2 with a laser pulse centred at 375 nm without the application of external MF. Data were then fit to a) sum of two and b) sum of three exponential decays without the application of external fields and c) sum of two and d) sum of three exponential decays in an externally applied field of 75 mT.

One might notice that the standard errors calculated for the sum of three exponential decays are reasonable for the first and the second component, but in case of

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the third one, the error is very high compared to the value itself, also the residuals from the fit are not significantly different when a third component is added. Thus it was concluded that only two exponential components were required to fit the data. As discussed previously, the photolysis quantum yield in base-off AdoCbl is dominated by the competition between bond homolysis and fast internal conversion back to the ground state. It has been previously demonstrated that photolysis of these alkylcolbalamins results in a rapid cascade through the excited state manifold producing an excited state with a lifetime of 18-60 ps.53 The population in this state is divided between the bond homolysis resulting in the formation of the RP and the ground state recovery. The lower axial ligand in base-off AdoCbl is replaced with a molecule of water leading to changes in electronic structure and opening of new, rapid channels for internal conversion, the nature of which have not yet been fully characterized. The absence of any significant geminate recombination has previously been reported and data shown in this thesis seem to be supportive of this claim, as the fit to the data only required two, rather than three exponential decay components. The third decay component, the lifetime of which should be around 500-600 ps could not be distinguished in our experiments. Previous studies have revealed, however, that the quantum yield of solvent separated RPs decreases to 0.045,15,22 which implies that the recombination does in fact take place, but on much slower timescale. This may be explained by the RP being born in a T-state, which would first have to undergo a conversion to a S-state in order to recombine,53 which has been predicted in the time dependent density functional theory (TDDFT) reported Kozlowski and co-workers.31 and is in disagreement with the reported observation of the geminate recombination components in the TA data.54 It was therefore concluded that in case of base-off cobalamins, the RP is produced in the singlet state independent of excitation wavelength.53 Although there is no evidence for the presence of the geminate recombination in this or previous data, the amplitude of the signals is very small, due to absorption spectrum being shifted towards the shorter wavelengths. The existence of this recombination cannot be therefore fully excluded.

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6.3 Summary

We have presented ultrafast TA kinetics of free AdoCbl using the newly developed Selene setup and the investigated MF-dependence on its photolysis. The kinetics of the decay components observed at 525 nm are comparable to those previously reported by Sension and co-workers and are therefore in agreement with the mechanism proposed by them.55,56,57,58 The MFE investigations have been extended and measurements were performed in wide range of fields up to 900 mT. The saturation of the Zeeman effect as well as Δg mechanism were observed, which implies that the newly developed setup allows for the MFE investigation to be performed easily and reliably. The recombination rate decrease due to the Δg mechanism was clearly observed, unfortunately theoretical simulations of this process proved to be beyond the scope of this thesis. The effect of the Δg mechanism in B12 was demonstrated to take place at lower field compared to most RPs and, since it reduces the MFEs due to the Zeeman interaction, it may possibly act as some sort of protective mechanism. To our knowledge, this is the first reported observations of the MFE due to the Δg mechanism in a biologically relevant system. The observed MFE curve is in agreement with the theoretically predicted shape calculated using the previously described method.23

6.4 References

1. T. T. Harkins, C. B. Grissom; Magnetic Field Effects on B12 Ethanolamine Ammonia Lyase: Evidence for a Radical Mechanism. Science, 1994, vol. 263, no. 5149, pp. 958 - 960.

2. R. A. Goldstein, S. G. Boxer; Effects of Nuclear Spin Polarization on Reaction Dynamics in Photosynthetic Bacterial Reaction Centers. Biophys. Journal. 1987, vol. 51, no. 6, pp. 937–946.

3. C. B. Grissom, E. Natarajan; Use of magnetic field effects to study coenzyme B12-dependent reactions. Methods Enzymol. 1997, vol. 281, no. 7, pp. 235–247.

4. J. R. Woodward; 'Carbon centered free radicals and radical cations' in Magnetic Field Effects on Radical Pair in Homogeneous Solution; John Wiley & Sons, 2010, pp. 157–180.

5. U. E. Steiner, T. Ulrich; Magnetic Field Effects in Chemical Kinetics and Related Phenomena. Chem. Rev. 1989, vol. 89, no. 1, pp. 51–147.

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6. A. R. Jones; Magnetic field effects in proteins. Mol. Phys. 2016, vol. 114, no. 11, pp. 1691–1702.

7. L. P. Lee, N. Schrauzer; The Reaction of Vitamin B12 and of Cobaloximes with Carbon Monoxide. Evidence for Self-Reduction of Vitamin B12 in Neutral Solution. J. Am. Chem. Soc. 1968, vol. 90, no. 6541, pp. 5274–5276.

8. J. A. Hamilton, R. Yamada, R. L. Blakely, H. P. Hogenkamp, F. D. Looney, M. E. Winfield; Cobamides and Ribonucleotide Reduction. Cob(II)alamin as a Sensitive Probe for the Active Center of Ribonucleotide Reductase. Biochemistry 1971, vol. 10, no. 2, pp. 347–355.

9. D. W. McRobbie, E. A. Moore, M. J. Graves, M. R. G. MRI. From picture to proton. Cambridge University Press, 2003.

10. S. Worster, D.R. Kattnig, P. J. Hore; Spin relaxation of radicals in cryptochrome and its role in avian magnetoreception Spin relaxation of radicals in cryptochrome and its role in avian magnetoreception. J. Chem. Phys. 2016, vol. 145, no. 3, pp. 1 – 13.

11. E. Breitmaier, W. Voelter; Spin Lattce Relaxation in High Resolution NMR Techniques in Organic Chemistry. Wiley & Sons, 1986.

12. A. M. Chagovets, C. B. Grissom; Magnetic field effects in adenosylcob(III) alamin photolysis: relevance to B12 enzymes. J. Am. Chem. Soc. 1993, vol. 115, no. 25, pp. 12152–12157 .

13. T. T. Harkins, C. B. Grissom; Magnetic Field Effects on B12 Ethanolamine Ammonia Lyase: Evidence for a Radical Mechanism. Science. 1994, vol. 263, no. 5149, pp. 958 – 960.

14. A. R. Jones, J. R. Woodward, N. S. Scrutton; Continuous wave photolysis magnetic field effect investigations with free and protein-bound alkylcobalamins. J. Am. Chem. Soc. 2009, vol. 131, no. 47, pp. 17246–17253.

15. E. Chen, M. R. Chance; Continuous-wave quantum yields of various cobalamins are influenced by competition between geminate recombination and cage escape. Biochemistry. 1993, vol. 32, no. 6, pp. 1480–1487 .

16. Janos, R.; Enzymic Reaction Selectivity by Negative Catalysis or How Do Enzymes Deal with Highly Reactive Intermediates ? Angew. Chemie - Int. Ed. 1990, vol. 29, no. 4, pp. 355–361.

17. W. D. Robertson, K. Warncke; Photolysis of Adenosylcobalamin and Radical Pair Recombination in Ethanolamine Ammonia-Lyase Probed on the Micro- to Millisecond Time Scale by Using Time-Resolved Optical Absorption Spectroscopy. Biochemistry. 2009, vol. 48, no. 1, pp. 140–147.

18. H. P. C. Hogenkamp; The Photolysis of Methylcobalamin. Biochemistry 1966, vol. 5, no. 2, pp. 471 – 422.

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19. P. A. Schwartz, P. A. Frey; 5’-Peroxyadenosine and 5'-peroxyadenosylcobalamin as intermediates in the aerobic photolysis of adenosylcobalamin. Biochemistry 2007, vol. 46, no. 24, pp. 7284 –7292 .

20. A. R. Jones, J. R. Woodward, N. S. Scrutton; Continuous Wave Photolysis Magnetic Field Effect Investigations with Free and Protein-Bound Alkylcobalamins. Supporting Information. J. Am. Chem. Soc. 2009, vol. 131, no. 47, pp. 7286–7292.

21. L. A. Walker, J. J . Shiang, N. A. Anderson, S. H. Pullen, R. J. Sension; Time- Resolved Spectroscopic Studies of B12 Coenzymes: The Photolysis and Geminate Recombination of Adenosylcobalamin. J. Am. Chem. Soc. vol. 120, no. 29, pp. 7286–7292.

22. E. Chen, M. R. Chance; Nanosecond Transient Absorption Spectroscopy of Coenzyme B12. Biochemistry 1990, vol. 265, no. 22, pp. 12987 – 12994.

23. J. R. Woodward, C. B. Vink; Hyperfine coupling dependence of the effects of weak magnetic fields on the recombination reactions of radicals generated from polymerisation photoinitiators. Physi.Chem.Chem.Phys. 2007, vol. 9, no. 47, pp. 6272–6278.

24. J. J. Shiang, L. A. Walker, N. A. Anderson, A. G. Cole, R. J. Sension; Time- Resolved Spectroscopic Studies of B12 Coenzymes: The Photolysis of Methylcobalamin Is Wavelength Dependent. J. Phys. Chem. B. 1999, vol. 103, no. 47, pp. 10532–10539.

25. T. T. Harkins, C. B. Grissom; Magnetic Field Effects on B12 Ethanolamine Ammonia Lyase. Science. 1994, vol. 263, no. 5149, pp. 958 – 960.

26. T. T. Harkins, C. B. Grissom; The Magnetic Field Dependent Step in B12 Ethanolamine Ammonia Lyase is Radical Pair Recombination. J. Am. Chem. Soc. 1995, vol. 117, no. 1, pp. 566–567.

27. A. Ahlbom, E. Cardis, A. Green, M. Linet, D. Savitz, A. Swerdlow; Review of the Epidemiologic Literature on EMF and Health. Environ. Health Perspect. 2001, vol. 109, no. 6, pp. 911–933.

28. T. T. Harkins, C. B. Grissom; The Magnetic Field Dependent Step in B12 Ethanolamine Ammonia Lyase is Radical-Pair Recombination. J. Am. Chem. Soc. 1995, vol. 117, no. 1, pp. 566–567 .

29. A. Bussandri, C. W. Kiarie, H. Van Willigen; Photoinduced bond homolysis of B 12 coenzymes. An FT-EPR study. Res. Chem. Intermed. 2002, vol. 28, no. 7 - 9, pp. 697–710.

30. A. R. Jones; The photochemistry and photobiology of vitamin B12. Photochem. Photobiol. Sci. 2017, vol. 16, no. 6, pp. 820–834.

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31. P. Lodowski, M. Jaworska, T. Andruniow, B. D. Garabato, P. M. Kozlowski; Mechanism of Co − C Bond Photolysis in the Base-On Form of Methylcobalamin. J. Phys. Chem. A. 2014, vol. 118, no. 50, pp. 11718–11734.

32. A. I. Kruppa, M. B. Taraban, T. V. Leshina, E. Natarajan, C. B. Grissom; CIDNP in the Photolysis of Coenzyme B 12 Model Compounds Suggesting That C−Co Bond Homolysis Occurs from the Singlet State. Inorg. Chem. 1997, vol. 36, no. 5, pp. 758–759.

33. A. R. Jones; External Magnetic Fields and Human Health: a Link to Biological Enzyme Reaction Systems. PhD Dissertation. University of Leicester, 2008.

34. A. R. Jones, S. J. O. Hardman, S. Hay, N. S. Scrutton; Is there a dynamic protein contribution to the substrate trigger in coenzyme B12-dependent ethanolamine ammonia lyase? Angew. Chemie - Int. Ed. 2011, vol. 50, no. 46, pp. 10843– 10846.

35. E. Natarajan, C. B. Grissom; The origin of magnetic field dependent recombination in alkylcobalamin radical pairs. Photochem. Photobiol. 1996, vol. 64, no. 2, pp. 286–295.

36. B. M. Babior, T. H. Moss; The mechanism of Action of Ethanolamine Ammonia Lyase, a B12-dependent Enzyme. J. of Biol. Chem. 1974, vol. 249, no. 14, pp. 4537–4544.

37. M. Baumgarten M., W. Lubitz; EPR and ENDOR studies of cobaloxime(II). Chem. Phys. Lett. 1987, vol. 133, no. 2, pp. 102 - 108.

38. J. R. Woodward; Radical pairs in solution. Prog. React. Kinet. Mech. 2002, vol. 27, no. 3, pp. 165–207.

39. H. Hayashi; Introduction to Dynamic Spin Chemistry: Magnetic Field Effects on Chemical and Biochemical Reactions. World Scientific Publishing Company, 2004.

40. A. S. Rury, T. E. Wiley, R. J. Sension; Energy Cascades, Excited State Dynamics and Photochemistry in Cob(III)alamins and Ferric Porphyrins. J. Am. Chem. Soc. 2015, vol. 48, no. 3, pp. 860–867 .

41. Y. Sakaguchi, H. Hayashi; Influence of Large Magnetic Fields on the Dynamic Behavior of a Radical Pair Produced by Photoreduction of Benzophenone in a Micellar Solution. J. Chem. Soc. Japan. 1993, vol. 22, no. 7, pp. 1183–1186 .

42. M. Igarashi, Y. Sakaguchi, H. Hayashi; Effects of large magnetic fields on the dynamic behavior of radical ion pairs in a non-viscous solution at room temperature. Chem. Phys. Lett. 1995, vol. 243, no. 5 - 6, pp. 545–551.

43. M. Wakasa, H. Hayashi, Y. Mikami, T. Takada; Reversion of Magnetic Field Effects Observed in the Reaction of a Triplet-Born Radical Pair Consisting of

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Two Equivalent Sulfur-Centered Radicals. J. Phys. Chem. 1995, vol. 99, no. 35, pp. 13181–13186.

44. Y. Sakaguchi, H. Hayashi; Magnetic field effects on the photodissociation reaction of triphenylphosphine in non-viscous homogeneous solutions. Chem. Phys. Lett. 1995, vol. 245, no. 6, pp. 591–597.

45. M. Wakasa, H. Hayashi; Magnetic Field Effects on the Hydrogen Abstraction Reactions of Triplet Benzophenone with Thiophenol in Nonviscous Homogeneous Solutions. J. Phys. Chem. 1996, vol. 3654, no. 96, pp. 15640– 15643.

46. Y. Fujiwara, M. Mukai, T. Tamura, Y. Tanimoto; A laser flash photolysis study of the effect of intense magnetic fields on the photoreaction of benzophenone in SDS micellar solution. Chem. Phys. Lett. 1993, vol. 213, no. 1 - 2, pp. 89–94.

47. M. Mukai, Y. Fujiwara, Y. Tanimoto; A Laser Flash Photolysis Study of Effects of High Magnetic Fields on the Chain-Linked Biradical Lifetime. J. Phys. Chem. 1993, vol. 97, no. 49, pp. 12660–12662.

48. R. Nakagaki, M. Yamaoka, O. Takahira, K. Hiruta; Magnetic Field and Isotope Effects on Photochemistry of Chain-Linked Compounds Containing Benzophenone and Hydrogen-Donor Moieties. J. Phys. Chem. A. 1997, vol. 101, no. 4, pp. 556–560 .

49. Y. Fujiwara, T. Aoki, T. Haino, Y. Fukaawa, Y. Tanimoto, R. Nakagaki, O. Takahira; High Magnetic Field and Magnetic Isotope Effects on Lifetimes of Triplet Biradicals Consisting of Two Equivalent Benzophenone Ketyls Linked by Methylene Chains. J. Phys. Chem. 1997, vol. 101, no. 37, pp. 6842–6849.

50. M. Wakasa, K. Nishizawa, H. Abe, G. Kido, H. Hayashi; Magnetic Field Effects Due to the ∆g Mechanism upon Chemical Reactions through Radical Pairs under Ultrahigh Fields of up to 30 T. J. Am. Chem. Soc. 1999, vol. 121, no. 39, pp. 9191–9197.

51. B.Brocklehurst, K. A. McLauchlan; Free radical mechanism for the effects of environmental electromagnetic fields on biological systems. Int. J. Radiat. Biol. 1996, vol. 69, no. 1, pp. 3–24.

52. A. R. Jones, S. Hay, J. R. Woodward, N. S. Scrutton; Magnetic field effect studies indicate reduced geminate recombination of the radical pair in substrate- bound adenosylcobalamin-dependent ethanolamine ammonia lyase. J. Am. Chem. Soc. 2007, vol. 129, no. 50, pp. 15718–15727.

53. J. Peng, K. Tang, K. McLoughlin, Y. Yang, D. Forgach, R. J. Sension; Ultrafast excited-state dynamics and photolysis in base-off B12 coenzymes and analogues: Absence of the trans-nitrogenous ligand opens a channel for rapid nonradiative decay. J. Phys. Chem. B. 2010, vol. 114, no. 38, pp. 12398–12405.

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54. A. B. Stickrath, E. C. Carroll, X. Dai, D. A. Harris, A. Rury, B. Smith, K. Tang, J. Wert, R. J. Sension; Solvent-Dependent Cage Dynamics of Small Nonpolar Radicals : Lessons from the Photodissociation and Geminate Recombination of Alkylcobalamins. J. Phys. Chem. A. 2009, vol. 113, no. 30, pp. 8513–8522.

55. L. A. Walker, J. T. Jarrett, N. A. Anderson, S. H. Pullen, R. G. Matthews, R. J. Sension; Time-Resolved Spectroscopic Studies of B12 Coenzymes: The Identification of a Metastable Cob(III)alamin Photoproduct in the Photolysis of Methylcobalamin. J. Am. Chem. Soc. 1998, vol. 120, no. 15, pp. 3597–3603.

56. J. J. Shiang, A. G. Cole, R. J. Sension, K. Hang, Y. Weng, J. S. Trommel, L. G. Marzilli, T. Lian; Ultrafast Excited-State Dynamics in Vitamin B 12 and Related Cob(III)alamins. J. Am. Chem. Soc. 2006, vol. 128, no. 3, pp. 801–808.

57. D. A. Harris, A. B. Strickrath, E. C. Carroll, R. J. Sension; Influence of Environment on the Electronic Structure of Cob(III)alamins: Time-Resolved Absorption Studies of the S1 State Spectrum and Dynamics. J. Am. Chem. Soc. 2007, vol. 129, no. 24, pp. 7578–7585.

58. R. J. Sension, A. G. Cole, A. D. Harris, C. C. Fox, N. W. Woodbury, S. Lin, E. N. G. Marsh; Photolysis and recombination of adenosylcobalamin bound to glutamate mutase. J. Phys. Chem. B. 2005, vol. 109, no. 38, pp. 18146–18152.

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Chapter 7.

Summary and Future Work

7.1 Summary

Although it has been known for many years that migratory birds and other animals use the Earth’s magnetic field (MF) in order to navigate, the process of detecting it and passing the information to the brain remains unclear. From a thermodynamical point of view, weak MF should not be able to affect living organisms at all, as the effects at most magnitudes are negligible when compared to the thermal ‘noise’ within the biological systems, however, the kinetics and reaction yield effects due to the influence of MF on RP reaction dynamics cannot be excluded. To date the only plausible theory explaining the magnetosensitivity of chemical reactions remains the RPM. MF may have a significant effect on some enzymatic reactions and enzyme catalytic cycles containing radical species as reaction intermediates have been identified as an example of potential carriers of biological field sensitivity. It is apparent that there are potentially harmful interactions that we simply know nothing about and the investigations into the effects of MFs on biological systems in light of our essentially continuous exposure to non-ionising radiation from man-made environmental sources are necessary. The spin dynamics of RPs are significantly affected by the application of external fields. The response to MFs is highly non-linear and three distinct field ranges can be distinguished, in which different RP interactions dominate,1 as shown in Fig. 1.7 (Chapter 1).

The application of very low field (typically less than 1 mT) leads to the removal of some of the degeneracies providing an increase in the rate of the S-T mixing,2,3,4 known as the LFE. The LFE is in the opposite phase to the ‘normal’ MFE- for example for a singlet born RP the yield of geminate recombination products will initially

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decrease with field, and not increase as is expected from the Zeeman interaction and the opposite is true for a T-born RP. The LFEs are of particular biological significance, because the magnitude of the MF generated by the Earth is about ~50 μT and sufficient to cause such effects.5 The LFEs have only been observed in radical ion pairs,6,1 where the lifetime of the RP is longer (hundreds of nanoseconds) due to coulombic attraction, in viscous solutions and in micelles.7

As the MF strength increases, the T±1 states become separated in energy due to the Zeeman effect, which leads to a decreased population of a singlet state, as the separated states cannot undergo the conversion to it. The saturation of the Zeeman effect takes place in relatively low fields of up to ~100 mT, where the field effect plateaus as 8 T±1 states become energetically inaccessible. The value of MF, at which the saturation of the Zeeman effect should theoretically take place, can be calculated using Eq.6.1 (Chapter 6.) The lifetime of the RP in this region still has to be fairly long, and it is usually obtained through increasing the viscosity of the solution. Prolonging the lifetime of the RP means that incoherent spin relaxation processes may begin to compete with coherent spin-state interconversion before recombination.9 The singlet population 8 becomes higher as the T±1 are depopulated through spin-lattice relaxation. Spin relaxation times determine how rapidly the non-equilibrium spin population decays. For the ferromagnetic transition metals such as Co, Fe, and Ni, the relaxation time values are strongly spin dependent and the spin-averaged relaxation times are much shorter than in the noble metals.10,11 Although in solutions of high viscosity, relaxation rates are predominantly slower than common RP lifetimes, in some systems they may still compete with coherent spin-state mixing. These systems include small, highly symmetric radicals, transition metals and molecules with large hfc interaction.12 For a relatively strongly coupled RP, in which the two radicals are fixed at a distance of less than approximately 10 Å, the energy difference in zero applied field between the singlet and triplet spin states J can be large. If 2J>>EHFI (the energy of hyperfine interaction), then hyperfine interactions cannot mix the singlet and triplet states, however, there might be a region on the energy surface where the S and T-1 states are degenerate. If the appropriate magnetic field is selected such that the Zeeman interaction energy splits the three triplet spin states by exactly this amount, mixing between the S and T-1 can happen for a negative exchange interaction. Hence, a narrow region of MF values will produce an increase in ISC. This mechanism is particularly

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important for RPs whose relative positions are fixed in space, for example in the photosynthetic reaction centres13,14 and when the hyperfine couplings are very large.15 The MFEs resulting from the hyperfine coupling mechanism have a saturated region. In higher fields the dominant mechanism is the ∆g mechanism, in which the cage product yield has a maximum (minimum), when the RP is generated from a precursor in a S (T) state as a function of increasing field If the g-values of the two radicals are different, the S-T0 state mixing takes place, the frequency of which is dependent upon the strength of the local field. The differences of the g-values for most radicals are very small, as g-values of most radicals differ little from that of the free electron, thus the applied field has to be of a significant strength. In some cases, however, these values can be quite different, and one of such example is vitamin B12.

B12 is a perfect candidate for the investigation of MFEs with features not usually found in typical organic RPs: competition between ultrafast spin-state interconversion and rapid relaxation, and the contribution of the Δg at relatively low MFs (i.e., < 1 T). Such investigations, however, required specialised apparatus capable of combining ultrafast kinetic measurements and a large range of MF-exposure amplitudes. A novel TA experimental setup, designed to study ultrafast MFEs over a large MF range was successfully designed, constructed and tested. This process involved the design of a MF-generating apparatus alongside the development of an optical setup, and enabled the measurements with and without the application of a broad range of external field magnitudes to be performed. The newly designed MF-generating apparatus allowed for the field strength to be changed easily and quickly within a wide range of fields (0-950 mT). Furthermore, the use of permanent magnets rather than electric coils removed the influence of heat, which can not only have a negative effect on biological samples, but also influence the reaction kinetics. The new optical set up allowed the collection of TA data with better signal to noise ratio than that collected using the existing Helios setup. The new set up also enabled the easy measurement of kinetics on the same sample with and without a magnetic field. Further developments were attempted to facilitate the data collection process further, but these proved to be beyond the capabilities of the laser system. The published MFE on the photoreaction of FAD, which has previously been characterised, was chosen as a biological test system for the new instrument generating the MF,16,17 due to the observation of a field dependence on a timescale similar to that

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of the available setup. The full pH dependence over a timeframe of 400 μs was performed allowing us to fully characterize the mechanism and intermediates, which was necessary for the MF investigations. The MF dependence measurements in external fields of up to 950 mT were then performed. The results, including the saturation of the Zeeman effect, were reproduced and a probable MFE due to the Δg mechanism in higher field strengths was observed with no apparent need to include the effects of the relaxation processes. The decrease of the recombination rate of the component assigned to be some combination of a triplet excited state and the RP18 was observed only at the highest MF achievable by the neodymium disc magnets. The observation of the MFEs on ultrafast timescale provided insights about the nature of different kinetic parameters. The pH-dependence measurements were particularly important, since the protonation of the flavin and adenine radicals is said to affect the dynamics of the RP as well as the excited state.17 We have not only successfully reproduced previously reported experiments, but also investigated the effects of MF in a wide range of fields. Experiments described in this thesis were performed only on free FAD and it is currently unknown how the mechanism of the interconversion between the triplet excited state and the RP affects the FAD system when bound to a protein. It has been, however, suggested that the MFE in triplet excited state of FAD has a potential for different applications, since the field effect may be transferred into other systems by using a quencher molecule of the triplet flavin.17 These investigations are said to be underway. The Selene setup was then used to investigate MFEs and ultrafast spin dynamics in free AdoCbl. The data obtained for the photolysis of AdoCbl were in agreement with those previously reported by Sension and co-workers.19,20,21,22 The MFE investigations have been extended and measurements were performed in wide range of fields up to 900 mT. The saturation of the Zeeman effect was observed and the obtained MFE curve was in agreement with the theoretically predicted shape calculated using the previously described method.23 The recombination rate decrease due to the Δg mechanism was clearly observed, but the theoretical simulations of this process proved to be beyond the scope of this thesis. Investigations of MFEs on photolysis of free AdoCbl at low pH have also been performed. The lower axial ligand in base-off AdoCbl is replaced with a molecule of water leading to changes in electronic structure and opening of new, rapid channels for internal conversion, the nature of which has not yet been fully

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characterized. Previous studies reported the lack of significant recombination in this conformation of AdoCbl, and data shown in this thesis seem to be supportive of this claim. Although there is no evidence for the presence of the geminate recombination in this or previous data, the amplitude of the signals is very small, due to absorption spectrum being shifted towards the shorter wavelengths. The existence of this recombination cannot be therefore fully excluded and may be demonstrated in future studies. The studies of the influence of externally applied MFs on biochemical reaction have received a fair amount of attention, since the magnetic interactions are much smaller than the thermal fluctuations, which means that very weak field may alter chemical reactions. Until recently investigations of MFEs on reactions proceeding via 24 RPs were measured in fields no stronger than 2 T and as a result, studies focusing on effects of higher fields remained incomplete.25 Recently, however, these investigations have been extended to higher fields in order to clarify the effects of spin-orbit coupling, the Zeeman interactions and the processes of spin relaxation on the geminate recombination. Theoretically, the ∆g mechanism should only be able to affect the geminate recombination in very high external MFs, but the specific field value will depend on the differences in the g-values of the two radicals. This mechanism has been demonstrated to affect the RP dynamics even in systems, where the difference is as small as 0.0055 when an external field of 30 T was applied. It seems therefore probable that, due to a Δg value as big as 0.3 for the RP generated upon the photolysis of free AdoCbl, much lower fields will be sufficient to facilitate this mechanism as demonstrated in Chapter 6. Exposure to EMFs is not a new phenomenon, but during the 20th century, environmental exposure to man-made fields has been steadily increasing as growing electricity demand, ever-advancing technologies and changes in social behaviour have created more and more artificial sources. Over the course of the past few decades, numerous EMF sources have become the focus of health concerns, including power lines, microwave ovens, computer and TV screens, security devices, radars and most recently mobile phones and their base stations. It has been postulated that long term exposure to fields is responsible for the increased probability of childhood leukaemia, although to date the evidence remains unclear. Staff working with MRI scanners are exposed every day to much higher MF, often for extended periods of time, and it is

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estimated that every year 60 million scans are being performed worldwide.26 Currently used MFs are also much higher than in the past- it has increased from 40 mT in 1980s to 27 almost 12 T nowadays. Since 1992, functional MRI (fMRI) has become one of the most often used techniques for mapping neuronal functions. The experimental data have 28,29,30,31 revealed that the sensitivity of this technique significantly increases, when stronger MFs are applied and systems operating at fields up to 8 T have become more common. In a study from 200132 the concerns associated with the exposure to such high fields were discussed, however, the only effects reported by the participants were dizziness and headache, when the field was changed rapidly. A similar effect has previously been reported, but it was considered to be insignificant to human’s health.33 In experiments described in this thesis, the MFE due to the Δg mechanism has been observed in two separate systems- FAD and vitamin B12 suggesting that high fields may have influence on biological systems and raising the concerns of the risks associated with the exposure to strong fields. The effect of the Δg mechanism in B12 was demonstrated to take place at lower field compared to most RPs and, since it reduces the MFEs due to the Zeeman interaction, it may possibly act as some sort of protective mechanism. To our information, this is the first reported observation of the MFE due to the Δg mechanism in a biological system. The RPM remains a unique mechanism, which is not overwhelmed by thermodynamic factors and it is therefore currently the only plausible biophysical mechanism to facilitate MF-sensitivity in biology and to potentially underpin the birds’ ability to use the Earth’s MF for navigation.34,35,36 In order for MFEs to be observed a number of conditions need to be fulfilled, but it is probable that evolution has found a way of accommodating it. As previously suggested,37 the influence of external fields on living organisms cannot be fully ruled out and considering the number of biological systems involving the RPs, it should not be neglected in the application of both low and high fields.

7.2 Future work

The experimental setup Selene has been tested and demonstrated as a suitable technique for the investigations of ultrafast MFEs, however, there are still a number of ways the technique could be improved or extended. The further development of Selene

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setup, as described in section 5.3 was eventually unsuccessful, but in principle, if a laser system which was significantly more stable was used, then this technique may be able to measure small MFEs on ultrafast timescales with great efficiency. Another direction for future work would be to increase the possible highest MF achievable, which would allow for the observations of the Δg mechanism in other biological and chemical systems. Additional neodymium disc magnets could be added to the existing hardwear, although handling magnets with that MF strength becomes a significant health and safety issue. Another option would be to use a Halbach array, which is designed to create a strong MF in its centre, while minimising any external MFs. This is achieved by having a spatially rotating pattern of magnetisation. Experiments specifically related to the FAD and AdoCbl test systems used here could be extended to measure the complete field dependence over a range of pH, and also for these biological cofactors in protein systems. It is expected, that when bound within a protein the RP will be strongly confined and MFE will be very different to those observed for the free cofactors in solution. The influence of the environment on the observations of MFE can be illustrated by the B12-dependent enzymes. Although the active site is expected to extend the lifetime of the RP and therefore, the probability of the reencounter, the MF-sensitivity in such systems is completely removed. A strong kinetic coupling between Co-C bond homolysis and the following abstraction of the H atom was suggested,38,39,40 and it seems that the intermediate radicals41,42 are rapidly quenched by the substrate. Consequently, the geminate recombination is very limited and it seems very improbable that the AdoCbl-dependent enzymes are affected by the applications of external fields, unless the conditions in vivo are significantly different the experimental ones.43 Another example of such systems with potential for the observations of MFEs are flavoproteins. Although a number of flavin-dependent enzymes are known or suggested to proceed via RP intermediates,44,45 no field sensitivity of the flavin-dependent enzyme reaction kinetics has yet been demonstrated. On the other hand, recently reported MFEs on FAD photochemistry in isolated cryptochromes suggests flavins are capable of supportive conditions for the manifestation of the biological sensitivity to external fields.46 The authors reported the LFE on the photoinduced RP in a cryptochrome (Cry- 1 from Arabidopsis thaliana) in external fields as low as 1 mT and up to 30 mT.

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Although in the past enzyme systems have been studied in order to determine the existence of MF-sensitivity, such as cytochrome P450 reductase (CPR),47 monoamine oxidase (MAO),48 horseradish peroxidase (HRP)38 and EAL,49 recent attempts to reproduce these claims have not been successful.50 The number of systems, which have been suggested or demonstrated to proceed via RP intermediates in combination with reported field effects51,52,53 indicates that they may be viable carriers of MF-sensitivity. It is evident that the understanding of the MFEs on biological systems can only benefit from further research on the subject. The investigations of MFEs in biological and chemical systems should therefore continue and it would benefit from further instrument development, which would allow for the field induced changes to be observed more directly. Adenosylcobalamin is a particularly suitable system for such investigations due to the peculiar magnetic properties of the generated RP containing the Co(II) radical and with features not usually found in typical organic RPs: competition between ultrafast spin-state interconversion and rapid relaxation, and the contribution of the Δg at relatively low MFs (i.e. < 1 T). Vitamin B12 plays a crucial role for humans’ health, as it is necessary for the process of blood formation as well as normal functioning of the brain. It is therefore important to further investigate the spin dynamics and the magnetic sensitivity. Further enzyme systems, such as EAL, are still to be re-investigated for MF-sensitivity in the laboratories in Manchester University.

The mechanism for photolysis in B12 coenzymes and its derivatives remains a phenomenon, the investigations of which required further experiments as well as computational studies.54

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