HEME PROTEIN-HEME PROTEIN ELECTRON TRANSFER MECHANISMS

电子在血红素蛋白之间的转移是一种 代谢过程的范围从氧化磷酸化到 从光合作用到外源物质和药剂的加工。 理解结构和机制的一种卓有成效的方法 决定血红素蛋白间电子转移速率的因素 已经在申请人的实验室中开发的是在 详细说明蛋白质之间的相互作用和电子转移的动力学 已知的三维结构。细胞色素b-5(b-5)、细胞色素c (C)、血红蛋白(Hb)和细胞色素c过氧化物酶(CCP) 在此类研究中的使用由于详细的结晶学分析 它们的结构是可用的。这项工作的具体目标是 今后三年如下：(1)确定具体选址 试剂EDC在1：1共价偶联络合物中的交联作用 按分段分列的进度报告中所述的b-5和c 测序技术。(2)研究两种亚铁血红素的功能性质 通过测定这些络合物的还原电势和 用温和的还原剂Fe(EDTA)2-还原它们的动力学。(3) 评估酵母c的Arg-18和Phe-87在与其相互作用中的作用 B-5通过使用酵母蛋白的基因工程突变体 已经在这些位置上被取代了。(4)评估问卷的有效性 已发表的b-5和Hb之间通过使用 一种新的人类Hb突变体，其残基被认为是 对这两种蛋白质的相互作用至关重要。(5)制备衍生产品 在Lys-13上特异修饰的马心c(与 Arg-18在酵母c)中表达，并研究它们与b-5相互作用的稳定性 和CCP比较来自约束性研究的信息的性质 对比动力学研究，比较诱变与化学诱变的价值 在这种工作中的修改。(6)使用b-5和Hb的血红素替代 用荧光猝灭法研究蛋白质相互作用 个别亚铁血红素丙酸基团在这种相互作用中的作用。(7) 确定蛋白质相互作用对共结合的影响 Hb和Ccp分别与b-5和c形成的络合物的性质(8) 制备b-5与Hb和c形成的络合物的晶体 C和Ccp之间作为衍射分析或单晶的前奏 光谱研究。

The transfer of electrons between heme proteins is fundamental to a variety of metabolic processes ranging from oxidative phosphorylation to photosynthesis to the processing of xenobiotics and pharmaceutical agents. One fruitful approach to understanding the structural and mechanistic factors that determine the rate of electron transfer between heme proteins that has been developed in the applicant's laboratory is to characterize in detail the interaction and kinetics of electron transfer between proteins of known three-dimensional structure. Cytochrome b-5 (b-5), cytochrome c (c), hemogrlobin (Hb), and cytochrome c peroxidase (CCP) are of particular use in such studies owing to the detailed crystallographic analyses of their structures that are available. The specific aims of this work for the next three years are as follows: (1) Determine the specific sites of crosslinking induced by the reagent EDC in covalently-coupled 1:1 complexes of b-5 and c described in the PROGRESS REPORT by fragmentation and sequencing techniques. (2) Study the functional properties of the two heme centers in these complexes by determining their reduction potentials and the kinetics of their reduction by the mild reducing agent Fe(EDTA)2-. (3) Assess the roles of Arg-18 and Phe-87 of yeast c in its interaction with b-5 through the use of genetically-engineered mutants of the yeast protein that have been substituted at these positions. (4) Assess the validity of a published model for the complex formed between b-5 and Hb through the use of a new mutant of human Hb that is altered at a residue believed to be crucial to the interaction of the two proteins. (5) Prepare derivatives of horse heart c that are specifically modified at Lys-13 (homologous to Arg-18 in yeast c) and study the stability of their interactions with b-5 and CCP to compare the nature of information derived from binding studies vs. kinetics studies and to compare the value of mutagenesis vs. chemical modification in such work. (6) Use heme substitution of b-5 and Hb to study protein-protein interaction by fluorescence quenching and to study the roles of individual heme propionate groups in such interactions. (7) Determine the effect of protein-protein interaction on CO-binding properties of Hb and CCP in their respective complexes with b-5 and c. (8) Prepare crystals of the complexes formed between b-5 and Hb and c and between c and CCP as a prelude to diffraction analysis or single-crystal spectroscopic studies.