The long range goals of our research program supported by NIH GM20488 are to develop and apply new techniques to study biological electron transfer reactions. Despite the importance of these reactions to numerous biological processes, relatively few techniques are available to measure the actual rate of electron transfer between two redox centers in a protein complex. We have recently introduced a new method to study biological electron transfer that utilizes a covalently attached tris(bipyridine)ruthenium group [Ru(ll)]. Several strategies have been developed for the design and synthesis of ruthenium-labeled redox proteins that are optimized for the measurement of inter protein electron transfer. Over twenty singly labeled derivatives of cytochrome c have now been prepared and characterized. One of the most remarkable properties of Ru(ll) is that it can be photoexcited to a metal-to-ligand charge-transfer state, Ru(lI*), which is a strong reducing agent and can rapidly transfer an electron to the heme group Fe(llI) in cytochrome c. Rate constants up to 3 x 10(7) s-1 are observed for derivatives with separations of about 12 Angstroms between the ruthenium and heme groups. We are using this new technique to measure intracomplex electron transfer between cytochrome c and its physiological partners, cytochrome c oxidase, cytochrome c1, cytochrome c peroxidase. The rate constants for these reactions range from 10(4) to over 10(6) s-1 , and are up to three orders of magnitude larger than previous estimates. The specific aims for the next grant period are to: 1) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c peroxidase that brings together rapid kinetics, site-directed mutagenesis, and X-ray crystallography. The rate constant, reorganization energy, interaction domain, and pathway of each electron transfer step in the mechanism will be determined. 2) Carry out a detailed study of the electron transfer reaction between cytochrome c and cytochrome c oxidase. A major goal will be to determine the pathway and kinetics of electron transfer from cytochrome c through Cu-a and heme a to the heme a3--Cu-B binuclear center under coupled turnover conditions. 3) Carry out a detailed study of the electron transfer reaction between cytochrome c and the cytochrome bc1 complex. A major goal will be to determine the pathway and kinetics of electron transfer from the Rieske iron-sulfur center to cytochrome c1 and to cytochrome c.