The focus of this research is to understand the mechanisms of biological electron transfer and, in a broader sense, the relationship between structure and function in redox proteins and proteins in general.
The specific aims are three-fold: first, utilizing site-directed mutagenesis coupled with structural, kinetic and thermodynamic studies to elucidate the factors mediating physiologically relevant electron transfer by the cytochromes c; second, to understand how the oxidation-reduction potential is controlled in the cytochromes c as well as the factors which control their stability and dynamic rearrangements; and, third, to investigate other families of redox protein which have unique or distinct properties relative to c-type cytochromes. Redox proteins are critical components in a variety of metabolic processes, including: respiration, photosynthesis, nitrogen fixation, denitrification, methanogenesis, steroid biosynthesis, DNA biosynthesis, chemical carcinogenesis and drug detoxification. In this context, the proposed studies are designed to elucidate the general principles that control biological electron transfer using c-type cytochromes (in particular cytochrome c2) in well defined and tractable model systems. It is anticipated that the proposed studies will elucidate both conceptually and technologically fundamental principles that will be applicable to most electron transfer systems and in particular physiologically relevant reactions which involve protein-protein interactions. In addition, we should obtain fundamental information on structure-function relations in proteins. These include a detailed understanding of biological recognition, that is, how physiologically relevant reactions between proteins occur in a complex metabolic mileu, and factors involved in the stabilization of native structures and the role of dynamic processes within proteins in modulating their structure and function. The approach is to couple studies of function (electron transfer kinetics in physiologically relevant reactions as well as model systems) with site-directed mutagenesis, structural studies (NMR and x-ray), stability measurement (redox potentials, spectral pK values, guanidine denaturation, hydrogen-deuterium exchange kinetics and ligand binding) and computer modeling of reactive species.
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