MUTAGENESIS ASSISTED FUNCTIONAL STUDIES OF CYTOCHROME C

细胞色素c功能的三个基本方面具有一般性 对许多其他蛋白质的影响将被研究：（1）调节 细胞色素c构象动力学和中点还原电位 在碱性pH下，血红素铁的Met 80配体被 Lys 79或Lys 73以产生两种碱性构象状态。 电化学，停流，和核磁共振分析这两个 构象异构体将进行通过分析的 Lys 79 Ala和Lys 73 Ala变体，以评估其结构和功能 特性.相关研究将考虑（a）构象动力学 Phe 82的突变先前已被证明可以降低PKa， 对于构象平衡，（B）构象和功能 用替代的“碱性”取代残基79和73的结果 （c）构象连接的可滴定配体的身份 在失去Met 80配体之前去质子化的基团。（二） 细胞色素c与其他血红素蛋白质的相互作用与反应。（一） 将研究细胞色素c与细胞色素b5的相互作用 通过应用新型核磁共振技术来识别 参与复合物形成的表面赖氨酸残基，通过详细的 细胞色素电位滴定的静电学模型 c-细胞色素b5复合物和快速动力学技术。(b)的 细胞色素c-细胞色素c过氧化物酶复合物的特征在于： 电位滴定和快速动力学技术，包括 使用严格选择的过氧化物酶突变体， 细胞色素的两个结合位点。这些结果也将 通过静电建模计算进行模拟。(c)复杂 由人红细胞色素b5和人血红蛋白形成， 通过电位滴定分析亚基。平行研究 将评估复杂的形成对光谱和 血红蛋白的配体结合特性。(3)电子的作用 供体结构对分子内电子转移动力学的影响。 通过结合定点的 诱变和化学修饰将被用来研究效果 电子给体结构对分子内电子转移动力学的影响 通过与用类似的细胞色素获得的已发表的结果进行比较， 其中钌络合物用作电子供体的衍生物。

Three fundamental aspects of cytochrome c function that have general implications for many other proteins will be studied: (1) Regulation of cytochrome c conformational dynamics and midpoint reduction potential by pH. At alkaline pH, the Met80 ligand to the heme iron is replaced by either Lys79 or Lys73 to produce two alkaline conformational states. Electrochemical, stopped-flow, and NMR analysis of these two conformational isomers will be undertaken through analysis of the Lys79Ala and Lys73Ala variants to assess their structural and functional properties. Related studies will consider (a) the conformational dynamics of mutations at Phe82 that have previously been shown to lower the PKa for the conformational equilibrium, (b) the conformational and functional consequences of replacing residues 79 and 73 with alternative "alkaline ligands," and (c) the identity of the conformationally-linked titratable group that deprotonates prior to loss of the Met80 ligand. (2) Interaction and reaction of cytochrome c with other heme proteins. (a) The interaction of cytochrome c with cytochrome b5 will be studied through application of a novel NMR technique for identification of surface Lys residues involved in complex formation, through detailed electrostatics modelling of potentiometric titrations of the cytochrome c-cytochrome b5 complex and by rapid kinetics techniques. (b) The cytochrome c-cytochrome c peroxidase complex will be characterized by potentiometric titrations and rapid kinetics techniques that will include use of critically selected mutants of the peroxidase predicted to disrupt each of the two binding sites for the cytochrome. These results will also be simulated by electrostatics modelling calculations. (c) The complex formed by human erythrocyte cytochrome b5 and human hemoglobin and its subunits will be analyzed by potentiometric titrations. Parallel studies will assess the influence of complex formation on the spectroscopic and ligand binding characteristics of hemoglobin. (3) The role of electron donor structure on kinetics of intramolecular electron transfer kinetics. Synthetic flavocytochromes produced by a combination of site-directed mutagenesis and chemical modification will be used to study the effect of electron donor structure on intramolecular electron transfer kinetics by comparison with published results obtained with analogous cytochrome derivatives in which ruthenium complexes serve as the electron donor.