Structural study of [NiFe]hydrogenase

[NiFe]氢化酶的结构研究

基本信息

批准号：
12680654
负责人：
HIGUCHI Yoshiki
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

Hydrogenases catalyze the reversible oxidation of molecular hydrogen and play a key role in hydrogen metabolism in various bacteria. Hydrogenases are classified into [non-metal], [Fe], [NiFe], and [NiFeSe] hydrogenases according to the metal composition of their active sites. The [NiFe]hydrogenase from D. vulgaris Miyazaki F is composed of a heterodimer with a total molecular mass of 91 kDa. The active site of the [NiFe] hydrogenases in the oxidized form is composed of Ni and Fe atoms with four cysteinyl sulfur and four non-protein ligands (S/O, CO, CN or SO). Carbon monoxide (CO) was known as a reversible inhibitor of hydrogenases. The carbon monoxide complex of [NiFe]hydrogenase from D. v. Miyazaki has been prepared and characterized by X-ray crystallography, and absorption and Raman spectroscopy. The exogenously added CO was found 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. One of the CO-bound structures showed an additional electron density peak between the CO and Sγ(Cys546), suggesting that an intermediate of the catalytic reaction was trapped at the active site. 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 key role during the initial process to bind H_2.

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。根据其活性位点的金属组成，将氢酶分为[非金属]，[Fe]，[Nife]和[Nifese]氢酶。来自D. vulgaris宫崎F的[Nife]氢酶由总分子量为91 kDa的异二聚体组成。氧化物形式的[Nife]氢化酶的活性位点由Ni和Fe原子组成，该原子具有四个半胱氨酸硫和四个非蛋白质配体（S/O，CO，CN左右）。一氧化碳（CO）被称为氢化酶的可逆抑制剂。来自D.v。miyazaki的[Nife]氢酶的一氧化碳复合物已通过X射线晶体学以及抽象和拉曼光谱术进行了制备和表征。在Ni-Fe活性位点的Ni原子的电子密度分布中观察到了外源添加的CO明显变化。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。一个共同结构的结构之一显示CO和Sγ之间的额外电子密度峰（CYS546），表明催化反应的中间体被困在活性位点。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结合H_2的初始过程中起着关键作用。