Structural study of the activation mechanism of dihydrogen at the Ni-Fe active site of [NiFe] hydrogenase

氢化酶催化分子氢的可逆氧化，在多种细菌的氢代谢中起关键作用。由于化石燃料是一种有限的资源，并且被认为会造成环境破坏，因此氢化酶的底物和产物二氢是未来燃料的良好候选者。氢化酶生产二氢的反应机理对氢燃料化学工程新工艺的开发具有潜在的价值，而消耗二氢的反应机理对新型燃料电池具有潜在的应用价值。虽然各种各样的证据表明，外源性CO与[NiFe]氢化酶的Ni原子相互作用，但鉴于活性位点的异双核性质和两种金属之间的密切联系，它们并不完全是决定性的。在这项研究中，我们通过x射线晶体学首次提供了CO与D.V.Miyazaki [NiFe]氢化酶Ni原子配位的直接证据。用x射线晶体学、吸收光谱和共振拉曼光谱对Miyazaki Desulfovibrio vulgaris Miyazaki F中[NiFe]氢化酶的co c…More配合物进行了表征。在1.2-1.4 Å分辨率下测定了co -结合和co -解放形式的[NiFe]氢化酶的9种晶体结构。外源添加的CO被指定在Ni- fe活性位点与Ni原子结合。在所有测试的晶体中，Ni和Sγ(Cys546)原子在co结合和co释放结构之间的电子密度分布发生了明显的变化。在Ni和Sγ(Cys546)原子附近发现的新结构特征表明，Ni- fe活性位点的这两个原子在初始h2结合过程中起作用。[NiFe]氢化酶有两种不同的氧化状态，Ni-A（未氧化）和Ni-B（氧化）。我们成功地用Na_2S和O_2将Ni-B转化为Ni-A，并测定了这两种状态的高分辨率晶体结构。Ni-B在Ni-Fe活性位点具有单原子非蛋白桥接配体，而Ni-A具有双原子。Ni-A桥接种的末端原子在CO络合物中与外源CO的C原子占据相似的位置（抑制状态）。提出了酶在静息（Ni-A）和抑制（CO配合物）状态下结构的共同特征。这些发现为设计新的仿生二氢生产系统和燃料电池装置提供了有用的信息。少

Hydrogenases catalyze the reversible oxidation of molecular hydrogen and play a key role in hydrogen metabolism in various bacteria. Dihydrogen, the substrate and product of hydrogenases, is a good candidate for fuel in the future, since fossil fuels are a limited resource and considered to cause environmental disruption. The reaction mechanism of dihydrogen production by hydrogenases is potentially useful for development of new chemical engineering processes for hydrogen fuels, whereas that of dihydrogen consumption is potentially applicable for new types of fuel cells. While various lines of evidence indicated that the extrinsic CO interacts with the Ni atom of [NiFe]hydrogenase, they were not entirely conclusive, given the hetero-binulcear nature of the active site and the intimate communication between the two metals. In this study, we present the first direct evidence of CO coordination to the Ni atom of D.V.Miyazaki [NiFe]hydrogenase by X-ray crystallography.The carbon monoxide c … More omplex of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe] hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 Å resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. Distinct changes were observed in the electron density distribution of the Ni and Sγ(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sγ(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H2-binding process.[NiFe] hydrogenase has two different oxidized states, Ni-A (unready) and Ni-B (ready). We have succeeded in converting Ni-B to Ni-A with the use of Na_2S and O_2, and determination of the high resolution crystal structures of the both states. Ni-B possesses a monatomic non-protein bridging ligand at the Ni-Fe active site, whereas Ni-A has a diatomic species. The terminal atom of the bridging species of Ni-A occupies a similar position as C of the exogenous CO in the CO complex (inhibited state). The common features of the enzyme structures at the resting (Ni-A) and inhibited (CO complex) states are proposed. These findings provide useful information on the design of new systems of biomimetic dihydrogen production and fuel cell devices. Less