Winter Gordon, Buhrke Thorsten, Jones Anne K, Friedrich Bärbel
Institut für Biologie, Humboldt-Universität zu Berlin, Chausseestr. 117, 10115 Berlin, Germany.
Arch Microbiol. 2004 Oct;182(2-3):138-46. doi: 10.1007/s00203-004-0680-6. Epub 2004 Aug 31.
The H(2)-splitting active site of [NiFe] hydrogenases is tightly bound to the protein matrix via four conserved cysteine residues. In this study, the nickel-binding cysteine residues of HoxC, the large subunit of the H(2)-sensing regulatory hydrogenase (RH) from Ralstonia eutropha, were replaced by serine. All four mutant proteins, C60S, C63S, C479S, and C482S, were inactive both in H(2) sensing and H(2) oxidation and did not adopt the native oligomeric structure of the RH. Nickel was bound only to the C482S derivative. The assembly of the [NiFe] active site is a complex process that requires the function of at least six accessory proteins. Among these proteins, HypC has been shown to act as a chaperone for the large subunit during the maturation process. Immunoblot analysis revealed the presence of a strong RH-dependent HypC-specific complex in extracts containing the C60S, C63S, and C482S derivatives, pointing to a block in maturation for these mutant proteins. The lack of this complex in the extract containing C479S indicates that this specific cysteine residue might be crucial for the interaction between HoxC and HypC.
[NiFe]氢化酶的H(2)分裂活性位点通过四个保守的半胱氨酸残基与蛋白质基质紧密结合。在本研究中，来自真养产碱菌的H(2)传感调节氢化酶（RH）大亚基HoxC的镍结合半胱氨酸残基被丝氨酸取代。所有四种突变蛋白C60S、C63S、C479S和C482S在H(2)传感和H(2)氧化方面均无活性，且未采用RH的天然寡聚结构。镍仅与C482S衍生物结合。[NiFe]活性位点的组装是一个复杂的过程，需要至少六种辅助蛋白的功能。在这些蛋白质中，HypC已被证明在成熟过程中作为大亚基的伴侣蛋白。免疫印迹分析显示，在含有C60S、C63S和C482S衍生物的提取物中存在一种强烈的RH依赖性HypC特异性复合物，表明这些突变蛋白的成熟受阻。在含有C479S的提取物中缺乏这种复合物，表明这个特定的半胱氨酸残基可能对HoxC和HypC之间的相互作用至关重要。
