Oxidation-State-Dependent Binding Properties of the Active Site in a Mo-Containing Formate Dehydrogenase.
Journal of the American Chemical Society
American Chemical Society
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Robinson, W., Bassegoda, A., Reisner, E., & Hirst, J. (2017). Oxidation-State-Dependent Binding Properties of the Active Site in a Mo-Containing Formate Dehydrogenase.. Journal of the American Chemical Society, 139 9927-9936. https://doi.org/10.1021/jacs.7b03958
Molybdenum-containing formate dehydrogenase H from Escherichia coli (EcFDH-H) is a powerful model system for studies of the reversible reduction of CO2 to formate. However, the mechanism of FDH catalysis is currently debated, and whether the primary Mo coordination sphere remains saturated or one of the ligands dissociates to allow direct substrate binding during turnover is disputed. Here, we describe how oxidation state-dependent changes at the active site alter its inhibitor binding properties. Using protein film electrochemistry we show that formate oxidation by EcFDH-H is inhibited strongly and competitively by N3–, OCN–, SCN–, NO2– and NO3–, whereas CO2 reduction is inhibited only weakly and not competitively. During catalysis the Mo center cycles between the formal Mo(VI)=S and Mo(IV)-SH states and by modeling chronoamperometry data recorded at different potentials, substrate and inhibitor concentrations we demonstrate that both formate oxidation and CO2 reduction are inhibited by selective inhibitor binding to the Mo(VI)=S state. The strong dependence of inhibitor-binding affinity on both Mo oxidation state and inhibitor electron-donor strength indicates that inhibitors (and substrates) bind directly to the Mo center. We propose that inhibitors bind to the Mo following dissociation of a selenocysteine ligand to create a vacant coordination site for catalysis, and close by considering the implications of our data for the mechanisms of formate oxidation and CO2 reduction.
This research was supported by BBSRC (BB/I026367/1 and BB/J000124/1), EPSRC NanoDTC Cambridge (EP/L015978/1), an ERC Consolidator Grant ‘MatEnSAP‘ (682833), and by The Medical Research Council (U105663141).
ECH2020 EUROPEAN RESEARCH COUNCIL (ERC) (682833)
External DOI: https://doi.org/10.1021/jacs.7b03958
This record's URL: https://www.repository.cam.ac.uk/handle/1810/265281