Electrochemical characterization of a complex FeFe hydrogenase, the electron-bifurcating Hnd from Desulfovibrio fructosovorans

Aurore Jacq-Bailly (1) , Martino Benvenuti (1) , Natalie Payne (1) , Arlette Kpebe (1) , Christina Felbeck (1) , Vincent Fourmond (1) , Christophe Léger (1) , Myriam Brugna (1) , Carole Baffert (1)

1 BIP - Bioénergétique et Ingénierie des Protéines

Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H 2 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 O 2-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 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 H 2 , diffusion and reaction at the active site of CO and O 2) 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.

Mots clés: direct electrochemistry; FeFe hydrogenase; electron bifurcation; Desulfovibrio fructosovorans; inactivation

Domaines: Sciences du Vivant [q-bio]

Aurore Jacq-Bailly, Martino Benvenuti, Natalie Payne, Arlette Kpebe, Christina Felbeck, et al.. Electrochemical characterization of a complex FeFe hydrogenase, the electron-bifurcating Hnd from Desulfovibrio fructosovorans. Frontiers in Chemistry, 2021, ⟨10.3389/fchem.2020.573305⟩. ⟨hal-03103487⟩