In the context of our overall objective of understanding the mechanisms of biological electron transfer, both structural and kinetic studies on c-type cytochromes and other redox proteins will be carried out. We have successfully developed the appropriate genetic system for the site-directed mutagenesis of Rhodobacter capsulatus cytochrome c2 and have characterized a number of mutants. Studies to this point have focused on the effect of mutations on electron transfer kinetics, redox potential, protein stability, protein dynamics and protein structure. In addition, substantial progress has been made on characterization of the structural and redox of properties of the high potential iron sulfur proteins and the cytochromes c'. Based on our findings to date, we are proposing a comprehensive and integrated plan to investigate the mechanism of electron transfer primarily utilizing mutants of cytochrome c2 and by studying their interaction with both wild-type and mutant photosynthetic reaction centers, cytochrome c peroxidase and cytochrome c1. Although the principal focus will be on cytochrome c2, we will carry out parallel but less extensive efforts with other families of redox proteins such as HIPIP, cytochrome c', plant ferredoxin, and multi-heme cytochromes. We are combining the use of site-directed mutagenesis with a range of approaches in order to define in molecular terms electron transfer between cytochrome c2 and electron donors and acceptors. Thus, studies on the kinetics of electron transfer will be combined with structural studies (NMR and x-ray crystallography), studies on protein stability, redox potentials and protein dynamics (NMR). It is anticipated that the proposed studies will permit us to address the role of electrostatics, sterics, distance between redox centers, relative orientation of interacting redox centers, intervening media through which electrons are transferred, and protein dynamics in biological electron transfer. The proposed studies will also address issues related to the control of biological redox potentials, the importance of specific amino acids to structural domains, to protein stability, and the factors controlling biological specificity and recognition that mediate protein-protein interactions.
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