The importance of electron tunneling mediation pathways between localization sites in a number of specific proteins will be studied. The influence of protein primary, secondary, tertiary, and quaternary structure, as well as the importance of bound water, protein/protein docking geometries, and protein dynamics on donor-acceptor coupling and electron transport (ET) rates will be probed. In addition to analyzing physiological systems, we will also design mutant proteins to test the predictive ability of the electronic coupling model (PATHWAYS). PATHWAYS accounted well for the donor-acceptor coupling in numerous redox labeled metalloproteins; our goal here is to apply the technique to biological ET systems. The PATHWAY calculations are of considerable relevance to a number of specific biochemical issues in both natural and genetically engineered organisms. Specific ET systems that are proposed to be studied are: (l) the photosynthetic reaction center -- influence of pathways on stabilization of the charge separated state and the role of dynamical modulation of tunneling pathways will be investigated, (2) intermolecular and intersubunit ET reactions -- in Zn/Fe hybrid hemoglobin (a model system for long range ET across a tunable interface), and in the cytochrome c/ cytochrome c peroxidase couple - a well studied physiological bimolecular ET problem, (3) cytochrome c itself -- the role of tunneling pathways on the proposed "trigger mechanism" for its function; the importance of internal water molecules for mediating electronic coupling, and (4) the unusually long lived tyrosyl radical in ribonucleotide reductase, a key enzyme involved in DNA biosynthesis. One of the motives for this work is that the accessibility of genetically engineered and semisynthetic proteins allow us to design mechanistic probes of electron transfer reaction mechanisms. Using PATHWAYS, as was done successfully for a number of chemically modified ET proteins, we expect to be able to map the dominant electron transfer pathways in physiologically important processes, predict how mutations are expected to influence rates, and map the regions in proteins that dominate the electronic coupling between given sites. Our goal is not simply to explain existing experimental numbers, but to assist in the design of new experiments that provide detailed information about the relationship between structure and function. This work will be carried out as a collaborative project between the University of Pittsburgh Department of Chemistry and the University of California, San Diego Department of Physics. This proposal is intended to allow the continuation of collaborative studies by the co- investigators that led to the pathway concept and its resent numerical implementation.