HEME PROTEIN-HEME PROTEIN ELECTRON TRANSFER MECHANISMS

血红素蛋白质之间的电子转移是各种血红素蛋白质的基础。 从氧化磷酸化到 光合作用到异生物质和药剂的加工。 一个富有成效的方法来理解结构和机制 决定血红素蛋白之间电子转移速率的因素 在申请人的实验室中开发的是表征 详细说明蛋白质之间的电子转移的相互作用和动力学 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）是 由于详细的晶体学分析， 它们的结构是可用的。 这项工作的具体目标是 未来三年的具体安排如下：（1）确定具体选址 在共价偶联的1：1络合物中由试剂EDC诱导的交联 进度报告中所述的B-5和c 测序技术。 (2)研究了两种血红素的功能性质 通过确定它们的还原电位， 用温和还原剂Fe（EDTA）2-还原它们的动力学。 （三） 评估酵母c的Arg-18和Phe-87在其与 B-5通过使用酵母蛋白的基因工程突变体 在这些位置上被取代了。 (4)评估的有效性 已发表的B-5和Hb之间形成复合物的模型， 一种新的人类血红蛋白突变体， 这对两种蛋白质的相互作用至关重要。 (5)制备的衍生物 在Lys-13处特异性修饰马心c（与 c）并研究它们与B-5相互作用的稳定性 和CCP比较从结合研究中获得的信息的性质 vs.动力学研究，并比较诱变与化学诱变的值 在这样的工作中进行修改。 (6)使用B-5和Hb的血红素取代， 通过荧光猝灭研究蛋白质-蛋白质相互作用， 单个血红素丙酸酯基团在这种相互作用中的作用。 （七） 确定蛋白质-蛋白质相互作用对CO结合的影响 Hb和CCP在其各自与B-5和c的复合物中的性质。 （八） 制备B-5与Hb和c之间形成的复合物的晶体， c和CCP之间的关系，作为衍射分析或单晶 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.