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]氢化酶由两个亚基组成，大亚基中有一个Ni-Fe活性位点，Ni由四个半胱氨酸硫配位（其中两个在Fe和Ni之间形成桥梁）。双原子配体，以及氧化形式的第三个桥 [NiFe] 氢化酶的纯化形式（无活性氧化）、Ni-C（活性还原）和 CO 抑制形式。最近，D. v. Miyazaki F已经报道了氧化形式是Ni-A和Ni-B的混合物，并且酶通过Ni-B被激活从Ni-A到Ni-C。 1. 在本项目中，我们发现了使用 50 mM Na_2S 并暴露于 O_2 中从 Ni-B 制备纯 Ni-A 的方案，并阐明了 Ni-A 和 Ni-B 的晶体结构。乙有单原子非蛋白质桥接配体 (X_<B1>)，而 Ni-A 具有双原子种类 (X_<A1>-X_<A2>) 此外，半胱氨酸的硫被发现具有修饰的原子种类。 (X_<546>)在Ni-A和Ni-B.2中为了阐明H_2在活性位点的激活机制，我们成功制备了大单晶。 (1.0 mm^3)的酶在D_2O溶液中的中子衍射实验表明-晶体衍射约10 A。较大晶体的结晶条件正在得到改善。3． HypE 编码的蛋白质参与与 Fe 配位的 CN 的生物合成。分别在 2.0 和 2.6 A 分辨率下进行结构比较，结果表明 ATP 的结合不会导致 C 末端残基 Cys341 的整体结构发生变化，其硫醇基团应该在腈基团合成之前被氨基甲酰化。，完全埋藏在蛋白质内，并且位于结合的 ATP 的·-磷酸基团附近。所获得的结构表明催化反应发生在这种构型中，但a。 Cys341.4 的氨基甲酰化是一种转录因子，负责在某些细菌中表达 CO 耐受性氢化酶。我们已经确定了 C 中咪唑 (Im) 结合的 CooA 的晶体结构。氢甲酰胺 (Ch-CooA)，熔点为 2.2 A。Ch-CooA 的结构表明，Im 与血红素 Fe 结合，并取代 N 末端，就像即使发生配体交换，Im 结合的 Ch-CooA 仍处于非活性形式。这些结果表明，Im 结合导致的 N 末端的释放不足以激活 CooA。该结构提供了对 CooA 的新见解。实现激活所需的结构变化较少。

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）

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

DOI：
发表时间：
2006
期刊：
Biochemistry 45
影响因子：
0
作者：
N.Yahata;K.Ozawa;A.Nakahara;H.Ogata;Y.Higuchi;H.Akutsu
通讯作者：
H.Akutsu

6-アミノカプロン酸環状2量体加水分解酵素(EI)・基質複合体のX線結晶構造解析

6-氨基己酸环状二聚体水解酶 (EI)/底物复合物的 X 射线晶体结构分析

DOI：
发表时间：
2007
期刊：
影响因子：
0
作者：
安平健吾;柴田直樹;門上剛;樋口芳樹;加藤太一郎;武尾正弘;根来誠司
通讯作者：
根来誠司

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

硫酸還元菌の酸化還元タンパク質の遺伝子とそのタンパク質の構造

硫酸盐还原菌氧化还原蛋白基因及结构