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.
