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.

Hnd，来自 果糖脱硫弧菌是一种四聚体酶，可以进行基于黄素的电子分叉。它将H ₂的氧化与NAD ⁺的能主还原和铁氧还蛋白的负主还原偶联在一起。我们以前表明，Hnd保留了活性，即使与其他电子分叉氢化酶不同，也需氧纯化。在这项研究中，我们描述了O ₂下酶的纯化无气氛及其生化和电化学表征。尽管由于其多聚体组成而造成的复杂性，Hnd仍可催化并直接与电极交换电子。我们通过Hnd的蛋白膜电化学方法，通过有氧或厌氧纯化Hnd，然后比较在两种条件下纯化的酶的电化学性质，表征了该电子分叉氢化酶的催化和抑制性质通过蛋白质膜电化学。氢化酶通常在没有双氧的条件下在氧化条件下失活，然后可以在还原条件下某种程度上重新活化。我们证明，Hnd的这种高潜在失活/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，表明两种酶的共存和相互转化。我们还表明，Hnd的催化性能（Km对于H ₂，在CO和O ₂活性位点处的扩散和反应与标准氢化酶（那些不能催化电子分叉的酶）相当。这些结果表明，电子分叉所需的其他亚基的存在，既不会改变活性部位的催化行为，也不会改变气体扩散动力学，但会引起异常高速率的失活/再活化。