Spin dynamics in biological radical reactions

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Abstract

Biological reactions often involve formation of short-lived intermediates, from which some of them are in form of a radical pair (RP). These intermediates are often difficult to detect, but by observing their spin dynamics, the influence of the environment on reaction mechanism can be elucidated. The aim of this study was to develop the system that will enable following formation and dynamics of these short-lived species. The continuous-flow setup was developed for 9 GHz and 35 GHz EPR frequencies, which enabled observation of short-lived radicals at nanosecond resolution, through an optimised triggering sequence which provided measurement at the shortest possible delay after flash (DAF). The developed system was exploited to study the yet unknown reaction mechanism of antimalarial drugs and, in greater detail, photochemistry of cofactors of B12 enzymes, reactions which proceed through formation of a RP. The first investigated system involved a question about the phototoxicity of antimalarial drugs and potential electron transfer reactions. From our approach, with using Fourier transform EPR (FT-EPR) to test different drug analogues, it is suggested that the amine functionality is the source of electron transfer in these reactions. The second studied biological system involved investigating spin dynamics of B12 -derivatives, using the bespoke continuous-flow system. Methylcobalamin (MeCbl) and 5â€Â™-deoxyadenosylcobalamin (AdoCbl) serve as cofactors of enzymes involved in metabolism of vitamin B12 , through their reactivity in organometallic reactions. T he Co-C bond has a crucial role in these enzymatic reactions. Flash photolysis FT-EPR was used to determine differences between electronic structure of two cofactors and origin of the precursor molecules, since both cofactors produce analogous RPs following photoexcitation. From simulation of the experimental data it was observed that whereas the spin polarisation following the photolysis of AdoCbl suggests signal from predominantly S-born, geminate pairs, MeCbl geminate RPs are born in both S and T spin-states and polarisation from T-born f-pairs also contributes to the signal. Further significant differences in reaction mechanism were identified varying the lower and upper axial ligand, the positions important to B12-dependent enzyme reactions. Finally, the spin chemistry of the reoxidation of insect and plant cryptochromes (Crys) proteins was studied through investigation of magnetic field effects (MFEs). The proteins have been previously suggested as being involved in magnetoreception. Recent behavioural studies and theoretical calculations have indicated involvement of a RP, potentially formed in the reoxidation step. However, using the MFE stopped-flow device in our study showed no evidence for MFEs on this reaction step and the work will guide future studies to fully understand the reoxidation step.

Keyword(s)

chemically induced dynamic electron/nuclear polarisation; cobalamins, cryptochromes ; magnetic field effect; radical pair mechanism; radical pairs, time resolved EPR

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