Role of phenylalanine-82 in yeast iso-1-cytochrome c and remote conformational changes induced by a serine residue at this position.
Role of phenylalanine-82 in yeast iso-1-cytochrome c and remote conformational changes induced by a serine residue at this position.
复制标题
苯丙氨酸 82 在酵母 iso-1-细胞色素 c 中的作用以及该位置丝氨酸残基诱导的远程构象变化。
DOI：
10.1021/bi00420a043
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Brayer,GD
中科院分区：
文献类型：
--
作者：
Louie,GV;Pielak,GJ;Smith,M;Brayer,GD
加拿大不列颠哥伦比亚省温哥华市英属哥伦比亚大学生物化学系v6t1w5 1987年10月23日接收；摘要：酵母iso-1细胞色素c Ser-82突变蛋白的还原形式的三维结构分析已经完成，分辨率为2.8-Á。用丝氨酸残基替换phe82会导致突变位点附近和远处的构象变化。在Ser-82附近发生位置移位的基团包括Arg-13、Gly-83和84，以及血红素基团的CBB甲基。远移以吡咯环A的丙酸盐为中心，主要涉及Asn-52、Trp-59和一个内部隐藏的水分子wat166。在82号位置放置丝氨酸侧链也导致形成一个大的溶剂通道，这大大增加了血红素基团的溶剂接近性。这似乎说明了这种蛋白质的还原潜力要低得多。Ser-82对与细胞色素c过氧化物酶络合的稳态活性和电子转移速率的不利影响也可以通过突变位点附近区域的修饰特性来解释。观察到的远程构象变化似乎代表了在电子转移事件中酵母iso1 -细胞色素c转化为完全氧化状态时发生的初始构象变化。这些结果与[Moore， GR (1983) FEBS Lett. 161, 171-175]的建议一致，即氧化态之间构象变化的触发因素在于血红素铁原子与吡罗环A丙酸基团之间相互作用的性质。总的来说，我们的结果支持了野生型蛋白中phe82至少有三个作用的建议。这些包括：限制溶媒对血红素的接近性，从而通过电调节这种蛋白质的还原电位；通过提供沿传递路线的最佳介质来促进电子传递的速率；与氧化还原伙伴形成接触面相互作用，以协助形成生产性电子转移配合物。虽然细胞色素c介导的电子传递机制已被深入研究，但它仍然是一个尚未解决的重要基础生化问题[参见Mathews（1985）和Poulos and Finzel（1984）的综述]。在酵母（酿酒酵母）中，有两种细胞色素c同工酶。最丰富的形式是酵母iso-1-cy-tochrome c，该蛋白的三维原子结构最近在我们的实验室中被阐明（Louie et al., 1988）。酵母iso-1细胞色素c的三级结构与其他真核细胞色素c高度同源（Takano & Dickerson, 1981a; Ochi et al., 1983）。然而，酵母蛋白确实有一些独特的结构特征。这些残基包括位于多肽链n端5至+ 1的替代构象，在中心血红素基团的His-18配体侧形成表面ß环的残基19-26，以及位于形成血红素口袋一部分的螺旋段c端的残基49-56。关于酵母iso1 -细胞色素c的表达和电子转移特性的功能和遗传学研究有广泛的背景。这源于酵母系统的多功能性，它在研究电子转移反应方面比其他系统有几个优势。例如，与其他真核细胞色素c相比，许多这项工作得到了医学研究委员会的资助…
Department of Biochemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T l W5 Received October 23, 1987; Revised Manuscript Received April 27, 1988 abstract: A three-dimensional structural analysis of the reduced form of the Ser-82 mutant protein of yeast iso-1-cytochrome c has been completed to 2.8-Á resolution. Replacement of Phe-82 with a serine residue results in conformational changes both near and remote from the mutation site. Those groups undergoing positional shifts near Ser-82 include Arg-13, Gly-83 and-84, and the CBB methyl of the heme group. Remote shifts are centered about the propionate of pyrrole ring A and principally involve Asn-52, Trp-59, and an internally buried water molecule, WAT-166. Placement of a serine side chain at position 82 also leads to the formation of a large solvent channel which substantially increases the solvent accessibility of the heme group. This would appear to account for the much lower reduction potential observed for this protein. The detrimental effect of Ser-82 on both the steady-state activity and the rate of electron transfer in complexation with cytochrome c peroxidase can also be interpreted interms of the modified character of the region about the mutation site. The remote conformational changes observed appear to represent the equivalent of the initial conformational changes occurring as yeast iso-1-cytochrome c is converted to the fully oxidized state during an electron-transfer event. These results agree well with the proposal [Moore, GR (1983) FEBS Lett. 161, 171-175] that the trigger for conformational changes between oxidation states resides in the nature of the interactions between the heme iron atom and the pyrrole ring A propionate group. Overall, our results support suggestions that Phe-82 in the wild-type protein has at leastthree roles. These include the following: limiting solvent accessibility to the heme, thereby regulating the reduction potential of this protein dielectrically; facilitating the rate of electron transfer by providing the optimal medium along the transfer route; forming contact face interactions with redox partnersto assist in the formation of the productive electron-transfer complex..^^. lthough the mechanism of electron transfer mediated by cytochrome c has been intensively studied, it remains an un-resolved biochemical problem of fundamental importance [see reviews by Mathews (1985) and Poulos and Finzel (1984)]. In yeast (Saccharomyces cerevisiae), two isozymes of cytochrome c occur. The most abundant form is yeast iso-1-cy-tochrome c, and the three-dimensional atomic structure of this protein has been recently elucidated in our laboratory (Louie et al., 1988). The tertiary structure of yeast iso-1-cytochrome c is highly homologous with those of other eukaryotic cyto-chromes c (Takano & Dickerson, 1981a; Ochi et al., 1983). However, the yeast protein does have a number of unique structural features. These include alternative conformations for residues-5 to+ 1 at the N-terminal end of the polypeptide chain, residues 19-26 which form a surface ß loop on the His-18 ligand side of the central heme group, and residues 49-56 which are at the C-terminal end of a helical segment forming a part of theheme pocket. 1 An extensive background of functional and genetic studies related to the expression and electron-transfer properties of yeast iso-1-cytochrome c is available. This arises from the versatility of the yeast system which offers several advantages over others in the study of electron-transfer reactions. For example, in contrast to other eukaryotic cytochromes c, many fThis work was supported by grants from the Medical Research Council of …