Jacq-Bailly Aurore, Benvenuti Martino, Payne Natalie, Kpebe Arlette, Felbek Christina, Fourmond Vincent, Léger Christophe, Brugna Myriam, Baffert Carole
CNRS, Aix Marseille University, BIP, Marseille, France.
Front Chem. 2021 Jan 8;8:573305. doi: 10.3389/fchem.2020.573305. eCollection 2020.
Hnd, an FeFe hydrogenase from , is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H 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-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 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 H, diffusion and reaction at the active site of CO and O) 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.
来自[具体来源]的Hnd是一种铁铁氢化酶，是一种能够进行基于黄素的电子分叉的四聚体酶。它将H的氧化与NAD的放能还原和铁氧化还原蛋白的吸能还原偶联起来。我们之前表明，与其他电子分叉氢化酶不同，即使在有氧条件下纯化，Hnd仍保留活性。在本研究中，我们描述了在无氧气氛下该酶的纯化及其生化和电化学特性。尽管由于其多聚体组成而具有复杂性，但Hnd能够催化并直接与电极交换电子。我们通过在有氧或无氧条件下纯化Hnd，然后比较在两种条件下纯化的酶的电化学特性——蛋白质膜电化学，来表征这种电子分叉氢化酶的催化和抑制特性。氢化酶通常在没有氧气的氧化条件下失活，然后在还原条件下可以在一定程度上重新激活。我们证明，Hnd这种高电位失活/重新激活的动力学表现出原始特性：它取决于酶的纯化条件并随时间变化，这表明该酶的两种形式共存并相互转化。我们还表明，Hnd的催化特性（对H的Km、CO和O在活性位点的扩散和反应）与标准氢化酶（那些不能催化电子分叉的氢化酶）相当。这些结果表明，电子分叉所需的额外亚基的存在既不改变活性位点的催化行为，也不改变气体扩散动力学，但会导致异常的高电位失活/重新激活速率。
