Rousset M, Montet Y, Guigliarelli B, Forget N, Asso M, Bertrand P, Fontecilla-Camps J C, Hatchikian E C
Unité de Bioénergétique et Ingéniérie des Protéines, Institut de Biologie Structurale et Microbiologie, Centre National de la Recherche Scientifique, 31, Chemin Joseph Aiguier, 13402 Marseille CDX 20, France.
Proc Natl Acad Sci U S A. 1998 Sep 29;95(20):11625-30. doi: 10.1073/pnas.95.20.11625.
The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.
通过定点诱变研究了脱硫弧菌属[NiFe]氢化酶中高电位[3Fe-4S]1+,0簇的作用，该簇位于近端和远端低电位[4Fe-4S]2+,1+簇之间的中间位置。果糖脱硫弧菌[NiFe]氢化酶的脯氨酸238占据了[3Fe-4S]簇中缺失的第四个铁位点的潜在配体位置，用半胱氨酸残基取代。从酶活性、电子顺磁共振、铁硫中心的氧化还原性质和晶体结构方面研究了突变酶的性质。基于光谱学和X射线晶体学研究，我们表明果糖脱硫弧菌氢化酶的[3Fe-4S]簇在P238突变体中转化为[4Fe-4S]中心。[3Fe-4S]到[4Fe-4S]簇的转化导致修饰簇的中点电位降低约300 mV，而两个天然[4Fe-4S]簇和NiFe中心的光谱和氧化还原性质没有明显改变。与野生型酶相比，中间铁硫簇中点电位的显著降低对P238C突变体的催化活性只有轻微影响。讨论了这些结果对高电位[3Fe-4S]簇在分子内电子传递途径中作用的影响。
