Multifrequency (9, 34 and 94 GHz) EPR investigations of paramagnetic intermediates in heterodisulfide reductase and related enzymes

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In this project we will employ state-of-the-art methods of electron paramagnetic resonance spectroscopy (EPR) at 9-, 34-, and 94 GHz frequencies to investigate the reaction mechanism of heterodisulfide reductase, a key enzyme in the energy metabolism of methane-producing archaea. The enzyme catalyses the reversible reduction of the heterodisulfide (CoM-S-S-CoB) to the thiol coenzymes, coenzyme M (CoM-SH) and coenzyme B (CoB-SH) in the final step of methanogenesis and employs an unusual 4Fe4S cluster to carry out substrate chemistry. Multifrequency ENDOR spectroscopy combined with selective isotopic labeling will permit determination of a detailed structure of the essential 4Fe4S-cluster and its interaction with the substrate. Freeze-quench EPR experiments on the millisecond time scale will be employed to identify further intermediates. In a major collaborative effort with other members of this consortium, including external collaborations, on Mössbauer spectroscopy, X-ray crystallography and quantum chemical calculations, the work will potentially emerge into a consistent mechanistic picture for this enzyme at the molecular level. The work will provide a basis to further investigate the hypothetical role of this enzyme in the energy conservation mechanism of other non-methanogenic organisms.