Oxidation-State-Dependent Binding Properties of the Active Site in a Mo-Containing Formate Dehydrogenase.

William E Robinson,Judy Hirst,Erwin Reisner,Arnau Bassegoda

doi:10.1021/jacs.7b03958

Abstract

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 under debate, and whether the primary Mo coordination sphere remains saturated or one of the ligands dissociates to allow direct substrate binding during turnover is disputed. Herein, 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 and 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.

Highlights

Metal-dependent formate dehydrogenase enzymes (FDHs) have recently come to prominence as efficient and reversible electrocatalysts for CO2 reduction.[1,2]. Both the Mo-dependent FDH from Escherichia coli (EcFDH-H)[2] and the W-dependent FDH from Syntrophobacter f umaroxidans[1] interconvert CO2 and formate reversibly when immobilized on graphite-based electrodes, and the Mo-containing FDHs from Desulfovibrio desulf uricans[3] and Rhodobacter capsulatus,[4] along with the W-containing formylmethanofuran dehydrogenase from Methanothermobacter wolfeii,[5] have been reported to reduce CO2 to formate
Either 2.5 or 5 μL of EcFDH-H solution were applied to its surface and left to dry for 10 min, before the electrode was inserted into the electrochemical cell
We have shown that inhibition of FDH catalysis is strongly dependent on the oxidation state of the enzyme, suggesting that inhibitors and substrates interact intimately with the Mo center in the active site

Summary

Introduction

Metal-dependent formate dehydrogenase enzymes (FDHs) have recently come to prominence as efficient and reversible electrocatalysts for CO2 reduction.[1,2] Both the Mo-dependent FDH from Escherichia coli (EcFDH-H)[2] and the W-dependent FDH from Syntrophobacter f umaroxidans[1] interconvert CO2 and formate reversibly when immobilized on graphite-based electrodes, and the Mo-containing FDHs from Desulfovibrio desulf uricans[3] and Rhodobacter capsulatus,[4] along with the W-containing formylmethanofuran dehydrogenase from Methanothermobacter wolfeii,[5] have been reported to reduce CO2 to formate. State-of-the-art electrocatalysts such as [Fe4N(CO)12]− 14 and a series of CpCo-diphosphine complexes[15] were shown to reduce CO2 to formate with high activity and Faradaic efficiency in the presence of water and were proposed to catalyze the reaction through metal-hydride intermediates that CO2 can abstract or insert into. Nickel bisdiphosphine (“DuBois”) catalysts oxidize formate in organic solution at up to 15.8 s−1 16,17 and have been proposed to operate by a β-deprotonation mechanism in which the formate proton is removed by a pendent base, not by hydride transfer to Ni.[17] all of these molecular electrocatalysts require overpotentials of hundreds of millivolts to perform unidirectional catalysis, in stark contrast to the reversible catalysis of FDHs.[1,2] the FDH active site provides an attractive biological blueprint to inform the design of efficient synthetic electrocatalysts for formate oxidation and CO2 reduction. The principles by which enzymes such as FDH have evolved into such efficient and reversible catalysts are increasingly well understood,[18] the FDH catalytic mechanism itself is currently controversial, and only limited structural and functional data are available

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