Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From Desulfovibrio fructosovorans

Abstract

Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H2 to both the exergonic reduction of NAD+ and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases. In this study, we describe the purification of the enzyme under O2-free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions via protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions. We demonstrate that the kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H2, diffusion and reaction at the active site of CO and O2) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation.

Uncovering Microbiome Adaptations in a Full-Scale Biogas Plant: Insights from MAG-Centric Metagenomics and Metaproteomics

“…When summing up spectral counts of subunits encoded by the hndABCD operon (K18330, K17992, K18331, K18332), the Hnd hydrogenase complex is the enzyme with the second-highest spectral counts. While only hydrogen-oxidizing activity is reported for the enzyme of Desulfovibrio fructosovorans [95][96][97][98], similar enzymes in other organisms might catalyze hydrogen evolution [99][100][101]. The results indicate that MAG 47 acts as a proteolytic amino acid degrader in the community of D1 (Figure 8), which is in line with previous findings regarding members of the Bacteroidales order [102].…”

An essential role of the reversible electron-bifurcating hydrogenase Hnd for ethanol oxidation in Solidesulfovibrio fructosivorans

“…Some of them have been shown to perform the reverse reaction and confurcate electrons to produce H 2 ( Schut and Adams, 2009 ; Schuchmann and Müller, 2012 ; Wang et al, 2013 ; Zheng et al, 2014 ; Kpebe et al, 2018 ). Contrary to what was previously thought, Hnd reduces NAD + rather than NADP + and a ferredoxin from H 2 and it retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases ( Kpebe et al, 2018 ; Jacq-Bailly et al, 2020 ). It contains, in addition to the hydrogenase catalytic subunit (HndD) which is closely related to the CpI hydrogenase from Clostridium pasteurianum , a [2Fe-2S] subunit (HndA), a subunit homologous to the NuoF flavin subunit of complex I (HndC), and a fourth subunit (HndB) which probably does not contain any redox center ( Malki et al, 1995 ; Kpebe et al, 2018 ).…”

Reversible catalysis

“…Chem. doi: 10.1038/s41570-021-00268- 3 (2021) hydrogenases: the active site of NiFe hydrogenases can be over-oxidized into various dead-end species 33 and certain FeFe hydrogenases [34][35][36] also inactivate under moderately oxidizing conditions, which prevents H 2 oxidation and effectively biases the enzyme in the reductive direction. A distinct explanation of the origin of the catalytic bias is related to kinetics 3 .…”