Liebl U, Sled V, Brasseur G, Ohnishi T, Daldal F
Department of Biology, Plant Science Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Biochemistry. 1997 Sep 30;36(39):11675-84. doi: 10.1021/bi970776l.
The iron-sulfur (Fe-S) protein subunit of the bc1 complex, known as the Rieske protein, contains a high-potential [2Fe-2S] cluster ligated by two nitrogen and two sulfur atoms to its apoprotein. Earlier work indicated that in Rhodobacter capsulatus these atoms are provided by two cysteine (C133 and C153) and two histidine (H135 and H156) residues, located at the carboxyl-terminal end of the protein [Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry 31, 3342-3351]. These ligands are part of the conserved sequences C133THLGC138 (box I) and C153PCHGS158 (box II) and affect the properties of the Fe-S protein and its [2Fe-2S] cluster. In this work, the role of amino acid side chains at positions 134 and 136, adjacent to the cluster ligands in box I, was probed by using site-directed mutagenesis and biophysical analyses. These positions were substituted with R, D, H, and G to probe the effect of charged, polar, large, and small amino acid side chains on the properties of the [2Fe-2S] cluster. Of the mutants obtained T134R, -H, and -G were photosynthetically competent (Ps+) but contained Fe-S proteins with redox midpoint potentials (Em7) 50-100 mV lower than that of a wild type strain. In contrast, T134D was Ps- and contained no detectable [2Fe-2S] cluster, although it reverted frequently to Ps+ by substitution of D with N. On the other hand, all L136 mutants were Ps-, the EPR characteristics of their [2Fe-2S] cluster were perturbed, and they were unable to sense the Qpool redox state or to bind stigmatellin properly. The overall data indicated that replacement of the amino acid side chain at position 134 of the Fe-S protein affects mainly the Em7 and oxygen sensitivity of the [2Fe-2S] cluster without abolishing its function, while substitutions at position 136 perturb drastically its ability to monitor the Qpool redox state and its interaction with the Qo site inhibitor stigmatellin. These two distinct phenotypes of box I T134 and L136 mutants are discussed with regard to the recently published three-dimensional structure of the water soluble part of the bovine heart mitochondrial Rieske Fe-S protein.
bc1复合体的铁硫（Fe-S）蛋白亚基，即 Rieske 蛋白，含有一个高电位的[2Fe-2S]簇，该簇通过两个氮原子和两个硫原子与脱辅基蛋白相连。早期研究表明，在荚膜红细菌中，这些原子由位于蛋白羧基末端的两个半胱氨酸（C133 和 C153）和两个组氨酸（H135 和 H156）残基提供[Davidson, E., Ohnishi, T., Atta-Asafo-Adjei, E., & Daldal, F. (1992) Biochemistry 31, 3342 - 3351]。这些配体是保守序列 C133THLGC138（框 I）和 C153PCHGS158（框 II）的一部分，影响 Fe-S 蛋白及其[2Fe-2S]簇的性质。在这项工作中，通过定点诱变和生物物理分析探究了框 I 中与簇配体相邻的 134 和 136 位氨基酸侧链的作用。这些位置被替换为 R、D、H 和 G，以探究带电、极性、大体积和小体积氨基酸侧链对[2Fe-2S]簇性质的影响。在获得的突变体中，T134R、-H 和 -G 具有光合活性（Ps+），但其 Fe-S 蛋白的氧化还原中点电位（Em7）比野生型菌株低 50 - 100 mV。相比之下，T134D 无光合活性（Ps-），且未检测到[2Fe-2S]簇，尽管它通过将 D 替换为 N 经常回复为 Ps+。另一方面，所有 L136 突变体均为 Ps-，其[2Fe-2S]簇的电子顺磁共振特征受到干扰，并且它们无法感知 Q 池的氧化还原状态或正确结合鱼藤酮。总体数据表明，Fe-S 蛋白 134 位氨基酸侧链的替换主要影响[2Fe-2S]簇的 Em7 和氧敏感性，但不消除其功能，而 136 位的替换则极大地扰乱了其监测 Q 池氧化还原状态的能力及其与 Qo 位点抑制剂鱼藤酮的相互作用。结合最近发表的牛心线粒体 Rieske Fe-S 蛋白水溶性部分的三维结构，讨论了框 I 的 T134 和 L136 突变体的这两种不同表型。
