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

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