The objective is to understand the mechanism of biological electron transfer in molecular terms. Moreover, by the nature of the proposed studies a substantial understanding of biological specificity, the factors mediating protein-protein and protein-membrane interactions and structure-function relations in proteins will be obtained. Electron transfer and coupled energy conservation are fundamental to the life process thus an understanding of these phenomena is required if we are to fully understand the living cell. It is well established that driving force, electrostatics, distance/sterics, orientation, intervening media and protein dynamics all play a role in controlling the kinetics of electron transfer between molecules. In order to fully quantitate the role of each of these factors, we are proposing to exploit five specific redox proteins which have generally similar but distinct electron transfer properties. These systems were chosen as they each have different characteristics amenable to analysis in terms of the factors controlling biological electron transfer. The principal approaches are to use transient kinetics as an assay of function, interpretation of kinetics using available structural information and computer graphics, and using the techniques of molecular biology (site directed mutagenesis) to fully exploit these systems.
Our specific aims i nclude the following: a) characterization of examples of Class I c-type cytochromes (Rps. capsulata cytochrome c2), iron-sulfur proteins (white campion ferredoxin) and copper proteins (white campion plastocyanin) in terms of their interaction with physiological and non-physiological reactants to provide information on the factors mediating electron transfers in these systems, b) structural analysis of a group of high potential iron-sulfur proteins to obtain information on the control of oxidation-reduction potentials, protein folding, and phylogeny, c) characterization of cytochrome b/c1, from Rps. capsulata, an example of a complex (multichromophoric) membrane protein which will provide information on intracomplex electron transfer and interaction with cytochrome c2, and c) through comparison of the different protein studies derive a general understanding of biological electron transfer and the means to exploit other less well characterized systems.
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