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Activation mechanism of the Active Site of [NiFe] hydrogenas

[NiFe]氢活性位点的激活机制

基本信息

批准号：
16074214
负责人：
HIGUCHI Yoshiki
金额：
$ 6.91万
依托单位：
University of Hyogo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

项目摘要

[NiFe] hydrogenase is composed of two subunits and has a Ni-Fe active site in the large subunit. Ni is coordinated by four cysteine sulfurs (two of them form a bridge between Fe and Ni). Fe has additional non-protein diatomic ligands, and a third bridge in the oxidized form. The as-purified (inactive-oxidized), Ni-C (active-reduced) and CO-inhibited forms of the [NiFe] hydrogenase from D. v. Miyazaki F were already reported. Recently, it was found that the oxidized form is a mixture of Ni-A and Ni-B, and the enzyme is activated from Ni-A to Ni-C through Ni-B.1. In this project, we have discovered the protocol to prepare pure Ni-A from Ni-B by using 50 mM Na_2S and exposure to O_2, and elucidated the crystal structures of Ni-A and Ni-B. We found that Ni-B has a monatomic non-protein bridging ligand (X_<B1>), whereas the Ni-A has a diatomic species (X_<A1>-X_<A2>). In addition, the sulfurs of cysteines are found to have a modified atomic species (X_<546>) in both Ni-A and Ni-B.2. In orde … More r to clarify the activation mechanism of H_2 at the active site, we have succeeded in preparing the large single crystal (1.0 mm^3) of the enzyme in D_2O solution. Neutron diffraction experiments showed that- the crystal diffract about 10 A. The crystallization condition for the larger crystals is being improved.3. The Ni-Fe active site is matured by a series of the proteins coded in the hyp operon. HypE is involved in the biosynthesis of CN which is coordinated to Fe. We determined the crystal structures of HypE in the absence and presence of ATP at 2.0 and 2.6 A resolution, respectively. Comparison of the structures reveals that the binding of ATP does not entail an overall structural change. The residue Cys341 at the C-terminus, whose thiol group is supposed to be carbamoylated prior to the nitrile group synthesis, is completely buried within the protein, and is located in the vicinity of the ・-phosphate group of the bound ATP. The obtained structure suggests that the catalytic reaction occurs in this configuration but that a conformational change is required for the carbamoylation of Cys341.4. CooA is a transcription factor, and is responsible for the expression of CO-tolerant hydrogenases in some bacteria. We have determined the crystal structure of an imidazole (Im)-bound CooA from C. hydrogenoformans (Ch-CooA) at 2.2 A. The structure of Ch-CooA reveals that Im binds to the heme Fe, and replaces the N-terminus, as does CO. Even though the ligand exchange, Im-bound Ch-CooA remains in the inactive form. These results indicate that the release of the N-terminus resulting from Im-binding is not sufficient to activate CooA. The structure provides new insights into the structural changes required to achieve activation. Less

[NiFe]氢酶由两个亚基组成，在大亚基中有一个Ni-Fe活性中心。镍是由四个半胱氨酸硫配位的(其中两个形成了铁和镍之间的桥梁)。铁具有额外的非蛋白质双原子配体，并以氧化形式存在第三个桥。Dv.Miyazaki F的[NiFe]氢酶的纯化形式(失活-氧化)、Ni-C(活性-还原)和CO-抑制形式已有报道。最近发现，酶的氧化形式是Ni-A和Ni-B的混合物，酶通过Ni-B从Ni-A激活到Ni-C。在这个项目中，我们发现了用50 mM Na2S和氧气暴露从Ni-B制备纯Ni-A的方案，并阐明了Ni-A和Ni-B的晶体结构。我们发现，Ni-B具有单原子非蛋白质桥联配体(X_&lt；B1&gt；)，而Ni-A具有双原子物种(X_&lt；A1&gt；-X_&lt；A2&gt；)。此外，半胱氨酸的硫在Ni-A和Ni-B中都具有修饰的原子物种(X_&lt；546&gt；)。在Orde…为了阐明H_2在活性中心的活化机理，我们成功地在D_2O溶液中制备了大单晶(1.0 mm^3)。中子衍射实验表明，晶体的衍射率约为10A，较大晶体的结晶条件得到改善。Ni-Fe活性部位由Hyp操纵子编码的一系列蛋白质成熟。HYPE参与了与铁配位的CN的生物合成。我们分别在2.0和2.6A分辨率下测定了在没有和存在ATP的情况下HYPE的晶体结构。结构的比较表明，ATP的结合并不会导致整体结构的变化。C-末端的残基Cys341，其硫醇基团在氰基合成之前被氨甲酰化，完全埋藏在蛋白质中，并位于结合的ATP的·-磷酸基团附近。得到的结构表明催化反应在这种构型下发生，但Cys341.4的氨甲酰化需要构象变化。CooA是一种转录因子，与某些细菌中CO耐受氢酶的表达有关。我们在2.2A测定了氢化链霉菌与咪唑(Im)结合的CooA(CH-CooA)的晶体结构。CH-CooA的结构表明，Im与血红素Fe结合，并取代N-末端，CO也是如此。即使配体交换，Im结合的CH-CooA仍处于非活性状态。这些结果表明，Im结合导致的N-末端的释放不足以激活CooA。该结构为实现激活所需的结构变化提供了新的见解。较少

项目成果

期刊论文数量（116）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A single-crystal ENDOR and density functional theory study of the oxidized states of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

普通脱硫弧菌 Miyazaki F 的 [NiFe] 氢化酶氧化态的单晶 ENDOR 和密度泛函理论研究

DOI：
发表时间：
2006
期刊：
J. Biol. Inorg. Chem. 11
影响因子：
0
作者：
M.van Gastel;M.Stein;M.Brecht;O.Schroder;F.Lendzian;R.Bittl;H.Ogata;Y.Higuchi;W.Lubitz
通讯作者：
W.Lubitz

Redox-Dependent Interaction of Cytochrome c_3 with [NiFe] Hydrogenase from D. vulgaris Miyazaki F

细胞色素 c_3 与 D. vulgaris Miyazaki F 的 [NiFe] 氢化酶的氧化还原依赖性相互作用