MUTAGENESIS-ASSISTED FUNCTIONAL STUDIES OF CYTOCHROME C

解决细胞色素c的三个主要功能特性的程序是 概述。(1)细胞色素c结构和功能的调节 按pH计算的特性。(A)细胞色素c还原的pH依赖性 潜势与物种高度相关。电位法、电化学法 以及旨在测试可能的来源的突变体的核磁共振特征 这一物种变异将被实施。(B)尽管形成了一个 细胞色素c具有pK~8.5-9的碱性构象是众所周知的，现在 似乎是细胞色素c的一种生理功能，其机制是 这种构象变化和碱的活性中心结构 人们对细胞色素c知之甚少。突变型细胞色素 碱性构象将通过动力学、EPR、NIR-MCD和核磁共振进行研究 方法，突变的细胞色素的功能性质在 细胞色素c构象中氧化还原相关改变的关键残基 将被进一步定性。(2)细胞色素的相互作用和反应 C与其他血红素蛋白结合。(A)将使用电位法和核磁共振方法 研究细胞色素c与细胞色素b5和细胞色素c的结合 过氧化物酶和部分细胞色素b5与血红蛋白的结合。这个 电位测量技术提供了以前无法获得的信息 关于质子结合中络合作用的变化和精确 络合物形成的平衡常数。(B)特别修改 特定赖氨酸修饰的野生型和突变型细胞色素c衍生物 可通过核磁共振进行拆分的残留物将用于鉴定 参与蛋白质-蛋白质识别的残基。(C)厌氧停用- 流动动力学将与布朗动力学计算相结合，以研究 两对血红素蛋白之间的双分子电子转移 静电和热力学因素对 观察到的比率。(D)在细胞色素上修饰两种蛋白质的突变体 C-细胞色素c-过氧化物酶蛋白-蛋白质界面的研究 不同形态细胞色素c-(锌细胞色素c)的动力学分析 过氧化物酶)复合体。(3)电子一体化的新战略 以细胞色素为中心的供体/受体c.创建第二个的两种方法 提出了细胞色素c的氧化还原活性部位。这些措施涉及(A) 突变型表面半胱氨酸与黄素的共价连接 残留物和(B)Cys-102周围环境的重建 酵母细胞色素c产生一个(类型I(蓝色))铜中心。

A program addressing three major functional properties of cytochrome c is outlined. (1) Regulation of cytochrome c structure and functional properties by pH. (a) The pH-dependence of cytochrome c reduction potentials is highly species dependent. Potentiometric, electrochemical, and NMR characteristics of mutants designed to test possible origins of this species variation will be undertaken. (b) Although formation of an alkaline conformation of cytochrome c with a pK~8.5-9 is well known and now appears to be a physiological function of cytochrome c, the mechanism of this conformational change and the active site structure of alkaline cytochrome c are poorly understood. Mutant cytochromes with altered alkaline conformations will be studied by kinetic, EPR, NIR-MCD, and NMR methods, and the functional properties of mutant cytochromes altered at residues critical to the redox-linked change in cytochrome c conformation will be further characterized. (2) Interaction and reaction of cytochrome c with other heme proteins. (a) Potentiometric and NMR methods will be used to study binding of cytochrome c to cytochrome b5 and cytochrome c peroxidase and, in part, binding of cytochrome b5 to hemoglobin. The potentiometric technique provides previously unavailable information concerning complexation-linked changes in proton binding and precise equilibrium constants for complex formation. (b) Specifically modified wild-type and mutant cytochrome c derivatives modified at specific Lys residues to permit their resolution by NMR will be used to identify residues involved in protein-protein recognition. (c) Anaerobic stopped- flow kinetics will be used with Brownian dynamics calculations to study the bimolecular electron transfer between pairs of heme proteins to assess the relative contributions of electrostatic and thermodynamic considerations to the observed rates. (d) Mutants of both proteins modified at the cytochrome c-cytochrome c peroxidase protein-protein interface will be studied by kinetic analysis of various forms of the cytochrome c-(Zncytochrome c peroxidase) complex. (3) New strategies for incorporation of electron donor/acceptor centers in cytochrome c. Two methods for creating a second redox-active sites in cytochrome c are proposed. These involve (a) covalent attachment of flavins through alkylation of mutant surface Cys residues and (b) reconstruction of the environment surrounding Cys-102 in yeast cytochrome c to create a (type I(blue)) copper center.