Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117,10115 Berlin, Germany.
J Biol Chem. 2012 Nov 9;287(46):38845-53. doi: 10.1074/jbc.M112.376947. Epub 2012 Sep 27.
Hydrogen-cycling [NiFe] hydrogenases harbor a dinuclear catalytic center composed of nickel and iron ions, which are coordinated by four cysteine residues. Three unusual diatomic ligands in the form of two cyanides (CN(-)) and one carbon monoxide (CO) are bound to the iron and apparently account for the complexity of the cofactor assembly process, which involves the function of at least six auxiliary proteins, designated HypA, -B, -C, -D, -E, and -F. It has been demonstrated previously that the HypC, -D, -E, and -F proteins participate in cyanide synthesis and transfer. Here, we show by infrared spectroscopic analysis that the purified HypCD complexes from Ralstonia eutropha and Escherichia coli carry in addition to both cyanides the CO ligand. We present experimental evidence that in vivo the attachment of the CN(-) ligands is a prerequisite for subsequent CO binding. With the aid of genetic engineering and subsequent mutant analysis, the functional role of conserved cysteine residues in HypD from R. eutropha was investigated. Our results demonstrate that the HypCD complex serves as a scaffold for the assembly of the Fe(CN)(2)(CO) entity of [NiFe] hydrogenase.
氢循环 [NiFe]氢化酶含有一个双核催化中心，由镍和铁离子组成，由四个半胱氨酸残基配位。三种不寻常的双原子配体以两个氰化物 (CN(-)) 和一个一氧化碳 (CO) 的形式与铁结合，显然解释了辅因子组装过程的复杂性，其中涉及至少六种辅助蛋白的功能，分别命名为 HypA、-B、-C、-D、-E 和 -F。先前已经证明 HypC、-D、-E 和 -F 蛋白参与氰化物的合成和转移。在这里，我们通过红外光谱分析表明，从 Ralstonia eutropha 和 Escherichia coli 纯化的 HypCD 复合物除了含有两种氰化物外，还含有 CO 配体。我们提供的实验证据表明，在体内，CN(-)配体的附着是随后 CO 结合的前提。借助遗传工程和随后的突变分析，研究了 R. eutropha HypD 中保守半胱氨酸残基的功能作用。我们的结果表明，HypCD 复合物可作为 [NiFe]氢化酶 Fe(CN)(2)(CO)实体组装的支架。
