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

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.

Creating a Low‐Potential Redox Polymer for Efficient Electroenzymatic CO2 Reduction

“…Zuschriften previously reported that Mo-FDH requires reductive activation by formate,v iologen, or an artificial reducer to recover its formate oxidation or carbon dioxide reduction activity, although this was not necessary for this Cc-PAA bioelectrode as Mo-FDH is reductively regenerated in situ upon applying areductive potential (i.e., À0.6 Vv s. SHE). [9b, 14,24] Control experiments were performed using Mo-FDH that had been denatured by aerobic heat treatment (100 8 8Cf or 1h), which confirmed that the reductive catalytic wave corresponding to electroenzymatic CO 2 reduction was only observed when active Mo-FDH was used (see the Supporting Information, Figure S1). Thep eak current densities of the denatured Mo-FDH/Cc-PAA control bioelectrode increased slightly,w hich, as described in the introduction, is likely due to changes in the inter-redox moiety spacing in unfolded (denatured) Mo-FDH.…”

Molybdenum-Dependent Formate Dehydrogenase for Formate Bioelectrocatalysis in a Formate/O2Enzymatic Fuel Cell

“…Work by Bassegoda et al 13 and Robinson et al 15 reported the reversible interconversion of CO 2 and formate without using any mediator with the FDH adsorbed on graphite-epoxy rotating disk working electrode. While the direct electrochemical approach adopted by the authors is well-suited to study finer thermodynamic and kinetic properties of metalloenzymes (i.e., FDH), mediated electrochemical approaches facilitated by redox polymers (BPV-LPEI) are better suited for application in devices where increased enzyme loading and current densities can be realized at an electrode surface.…”

“…and Robinson et al 15 showed that Mo-FDH from E.coli adsorbed on the surface of a graphite-epoxy rotating disk working electrode can catalyze the reversible interconversion of CO 2 and formate with direct electron transfer at ∼ −0.4 V vs. SHE (pH 6.8).…”

“…15,19 Thus, we evaluated the activation of FDH using 2-mercaptoethanol (2-ME) as a reducing agent. Furthermore, we also determined the performance of a HCOO − /O 2 EFC employing the MET-type Mo-FDH bioanode and the DET-type laccase biocathode by separating the anodic and cathodic chambers of the EFC with a Nafion 212 proton exchange membrane (while also maintaining the anoxic requirements of the anodic compartment).…”

“…9 Robinson et al have proposed a reductive activation to create a stabilized form of the SeCys dissociated state and it was mentioned that this state is also possible upon incubation of the protein with reducing agents (such as sodium dithionite (DT)). 15,19 Thus, we evaluated the activation of FDH using 2-mercaptoethanol (2-ME) as a reducing agent. Furthermore, we also determined the performance of a HCOO − /O 2 EFC employing the MET-type Mo-FDH bioanode and the DET-type laccase biocathode by separating the anodic and cathodic chambers of the EFC with a Nafion 212 proton exchange membrane (while also maintaining the anoxic requirements of the anodic compartment).…”