Application of EPR Techniques to Biological Systems
Shyue Chu Ke, Department of Physics, National Dong Hwa University
A brief introduction to continuous wave Electron Paramagnetic Resonance (CW EPR), Electron Nuclear Double Resonance (ENDOR), Electron Spin Echo Envelop Modulation (ESEEM), and Fourier Transform-EPR (FT-EPR) techniques will be given.
Application of CW EPR to the study of copper centers in particulate Methane Monooxygenase (pMMO) will be given as an example. pMMO is a copper protein that converts methane into methanol in the presence of dioxygen. The mechanism of oxygen activation and alkane oxidation, the exact nature of the copper centers of pMMO, their geometric arrangements, and how they function during catalysis have remained elusive due to the complexities of the system. With the help of CW EPR and chemical perturbation to the system, we have been able to differentiate the Catalytic copper clusters from the Electron transfer copper centers of pMMO.
Application of ESEEM technique to study catalysis of Vitamin B12 dependent Ethanolamine Deaminase (EDA) will be given as an example. EDA catalyzes radical mediated oxidative deamination of ethanolamine. The first step of the catalysis is thermohomolysis of the adenosylcobamain cobalt-carbon bond creating a low spin Cobalt(II) and 5’-deoxyadenosyl radical pair. In this study, we have characterized the cobalt – 14N superhyperfine and 14N nuclear quadruple couplings in cryotrapped free and enzyme bound cobalamin. Our ESEEM results clearly prove that the α-axial ligand to cobalamin upon binding to EDA is dimethybenzimidazole. A 14% increases in the isotropic hyperfine coupling of the remote dimethybenzimidazole 14N nucleus in enzyme bound versus free base on cobalamin shows an enhanced delocalization of unpaired spin density from cobalt onto the axial ligand, which would contribute to the acceleration of cobalt-carbon bond cleavage.
Finally, techniques of synchronized nanoseconds laser-microwave pulse operation to produce a series of time-resolved snap-shots of the evolving chemistry in biological systems will be discussed.