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

[NiFe]氢化酶Ni-Fe活性位点氢气激活机制的结构研究

• 批准号: 14380317
• 负责人: HIGUCHI Yoshiki
• 金额: $ 7.68万
• 依托单位: University of Hyogo (2004)Himeji Institute of Technology (2002-2003)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380317/
• 关键词: [NiFe] hydrogenase; ultra-high resolution x-ray analysis; Sulfate-reducing bacteria; CO-bound hydrogenase; quasi-dynamic crystal analysis; Clean energy; Fuel cell; Metalloenzyme

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

氢酶催化分子氢的可逆氧化物，并在各种细菌的氢代谢中起关键作用。二氢（二氢基因是氢化酶的底物和产物）将来是燃料的良好候选者，因为化石燃料是一种有限的资源，并被认为会导致环境破坏。氢化酶产生二氢的反应机制可能对开发用于氢燃料的新化学工程工艺有用，而二氢食品消耗的反应机制可能适用于新型的燃料电池。尽管各种证据表明外部CO与[Nife]氢化酶的Ni原子相互作用，但鉴于活性位点的异质 - 硫酸性质以及两种金属之间的亲密通信，它们并不是完全结论性的。在这项研究中，我们介绍了通过X射线晶体学通过X射线晶体学的D.V.Miyazaki [Nife]氢化酶的Ni原子的第一个直接证据。一氧化碳C…从Desulfibrio vulgaris fulgaris miyazaki f的[Nife]氢化氢化合物的更多粘合剂通过X-Ray Cremporce和Abusement和Resonmocrys和Resoneptrance表征。在1.2-1.4Å分辨率下确定[Nife]氢化酶的九种晶体结构。外源添加的CO被分配为在Ni-Fe活性位点上与Ni原子结合。对于所有测试的晶体，在共同结合的结构和共灭裂结构之间的Ni和Sγ原子的电子密度分布中观察到了明显的变化。在Ni和Sγ（CYS546）附近发现的新型结构特征表明，Ni-Fe活性位点的这两个原子在最初的H2结合过程中起作用。[Nife]氢化氢化氢化氢化氢氢化氢盐具有两个不同的氧化态，NI-A-A（未准备就绪）和Ni-B（Ready）。我们通过使用Na_2s和O_2成功将Ni-B转换为NI-A，并确定两种状态的高分辨率晶体结构。 Ni-B在Ni-Fe活性位点具有单子非蛋白质桥接配体，而Ni-A具有双偏二菌种。 NI-A的桥接物种的末端原子在CO复合物（抑制状态）中占据了与精美CO的C的相似位置。提出了静止（NI-A）和抑制（CO复合物）状态的酶结构的共同特征。这些发现提供了有关新型仿生二氢生产和燃料电池设备的新系统设计的有用信息。较少的

项目成果

Cloning and expression of the enolase gene from Desulfovibrio vulgaris (Miyazaki F)

普通脱硫弧菌 (Miyazaki F) 烯醇化酶基因的克隆和表达

• 发表时间: 2004
• 期刊: Biochim.Biophys.Acta 1676
• 作者: M.Kitamura;Y.Takayama;S.Kojima;K.Kohono;H.Ogata;Y.Higuchi;H.Inoue
• 通讯作者: H.Inoue

N.Mizuno, G.Voordouw, K.Miki, A.Sarai, Y.Higuchi: "Three-dimensional crystal structure of dissimilatory sulfite reductase D (DsrD) protein - possible interaction with B- and Z-DNA by its winged-helix motif"Structure. 11. 1133-1140 (2003)

N.Mizuno、G.Voordouw、K.Miki、A.Sarai、Y.Higuchi：“异化亚硫酸还原酶 D (DsrD) 蛋白的三维晶体结构 - 可能通过其翼状螺旋与 B-和 Z-DNA 相互作用

構造生物 Vol.9 No.2(5)

结构生物学第9卷第2期(5)

• 发表时间: 2003
• 作者: 緒方英明;樋口芳樹
• 通讯作者: 樋口芳樹

T.Chatake, N.Mizuno, G.Voordouw, Y.Higuchi, S.Arai, I.Tanaka, N.Niimura: "Crystallization and preliminary neutron analysis of the dissimilatory sulfite reductase D (DsrD) protein from the sulfate-reducing bacterium Desulfovibrio vulgaris"Acta Crystallogr.

T.Chatake、N.Mizuno、G.Voordouw、Y.Higuchi、S.Arai、I.Tanaka、N.Niimura：“来自硫酸盐还原菌的异化亚硫酸盐还原酶 D (DsrD) 蛋白的结晶和初步中子分析

Hydration Structures in Proteins and Neutron Diffraction Experiment on Dissimilatory Sulfite Reductase D(DsrD)

蛋白质水合结构及异化亚硫酸还原酶D(DsrD)的中子衍射实验

• 发表时间: 2004
• 期刊: J.Synchrotron Rad. 11
• 作者: T.Chatake;A.Ostermann;K.Kurihara;F.G.Parak;N.Mizuno;G.Voordouw;Y.Higuchi;I.Tanaka;N.Niimura
• 通讯作者: N.Niimura