The soluble NAD+-reducing [NiFe]-Hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH
The soluble NAD+-reducing [NiFe]-Hydrogenase from Ralstonia eutropha H16 consists of six subunits and can be specifically activated by NADPH
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DOI：
10.1128/jb.187.9.3122-3132.2005
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发表时间：
2005-05-01
影响因子：
3.2
通讯作者：
Friedrich, B
中科院分区：
文献类型：
--
作者：
Burgdorf, T;van der Linden, E;Friedrich, B
兼性富营养化变形杆菌Ralstonia eutropha H16的可溶性[NiFe]-氢化酶（SH）目前被描述为一种异四聚体酶。纯化后的蛋白由两个功能不同的异二聚体组成。HoxHY二聚体代表氢化酶模块，HoxFU二聚体构成nadh -脱氢酶。在双模形式中，SH以H-2为代价介导NAD（+）的还原。我们纯化了一种新的高分子量SH，它含有一个额外的亚基。这个额外的亚基被确定为hoxI的产物，hoxI是SH基因簇（hoxFUYHWI）的成员。Edman降解结合SH高分子量复合物的蛋白质测序，建立了HoxFLTYH的亚基化学计量学(2)。交联实验表明，两个HoxI亚基是最近的邻居。六聚体SH的稳定性取决于缓冲液的pH和离子强度。四聚体形式的SH可以被少量NADH瞬间激活，但不能被NADPH激活。然而，六聚体形式也可以通过添加少量NADPH来激活。这表明HoxI为NADPH提供了一个结合域。NADPH的一个特殊反应位点增加了SH和线粒体NADH之间的相似性：泛醌氧化还原酶（复合体1）。
The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD(+) at the expense of H-2. We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFLTYH(2). Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex 1).