Naturally occurring mutations found in one of the two Ω-loop substructures in human cytochrome c are associated with low blood platelet count (thrombocytopenia). Both Ω-loops participate in the formation of conformers associated with cytochrome c peroxidase activity and apoptotic function. At alkaline pH values, the Met80 ligand to the ferric heme iron dissociates, and a lysine residue in the 71–85 Ω-loop coordinates to the iron. The alkaline isomerization has been the focus of extensive kinetic studies, and it is established that a deprotonation triggers the release of the Met80 ligand (pK_trigger). A second deprotonation stabilizes a pentacoordinate heme form (pK_a2). In this study, site-directed variants at the 41 and 48 positions in the 40–57 Ω-loop and at the 81 and 83 positions in the 71–85 Ω-loop reveal that conformational transitions in the 71–85 Ω-loop, leading to the alkaline or peroxidatic conformers, are controlled by the 40–57 Ω-loop. We find that the variants causing thrombocytopenia, G41S and Y48H, lower the pK_trigger and increase pK_a2. Our results are presented in a mechanistic framework, depicted by a cube, that accounts for the pH dependencies of the equilibrium and kinetic parameters governing the alkaline transition of the native protein and Ω-loop variants. The data are most consistent with the trigger for Met80 replacement by a lysine being a deprotonation within a hydrogen bonded unit that links the two Ω-loops rather than an individual group. Such a proposal aligns with the entatic contribution made by the same unit in controlling the Met80–Fe(III) bond strength.

中文翻译：

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自然发生的与疾病相关的突变，在人类细胞色素c的40-57Ω环中引发碱性异构化

在人类细胞色素c的两个Ω环亚结构之一中发现的自然发生的突变与血小板计数低（血小板减少）有关。两个Ω环均参与与细胞色素c过氧化物酶活性和凋亡功能相关的构象异构体的形成。在碱性pH值下，血红素铁的Met80配体解离，并且71-85Ω环中的赖氨酸残基与铁形成坐标。碱性异构化一直是广泛动力学研究的重点，并且已确定去质子化可触发Met80配体的释放（p K_触发）。第二次去质子稳定了五配位血红素形式（p K _a2）。在这项研究中，定点变异在40-57Ω环的41和48位以及71-85Ω环的81和83位揭示了71-85Ω环的构象转变，导致呈碱性或过氧化物构象的分子，由40–57Ω环控制。我们发现引起血小板减少症，G41S和Y48H的变异体降低了p K_触发并增加了p K _a2。我们的结果显示在一个机械结构的框架中，用立方体表示，该框架说明了平衡的pH依赖性以及控制天然蛋白质和Ω-loop变体的碱性转变的动力学参数。数据与赖氨酸是Met80替代触发因素最一致，赖氨酸是连接两个Ω环而不是单个基团的氢键合单元内的去质子作用。这样的提议与同一单元在控制Met80-Fe（III）结合强度方面所做的积极贡献相吻合。