The molecular mechanisms by which two energy-transducing enzyme systems, mitochondrial cytochrome oxidase and the photosynthetic reaction center, operate will be studied. Cytochrome oxidase maintains electron-transfer activity in the mitochondrion by catalyzing the four-electron reduction of dioxygen to water. It is also the locus of Site III respiratory control and contributes directly to the transmembrane proton gradient by pumping protons stoichiometrically with electron transported to O2. The overall reaction may, therefore, be written as 4 cytc2+ +O2 +8H+ in -> 4 cytc3+ +4H+ out +2H2O. Dioxygen chemistry occurs in a binuclear center that comprises the heme alpha3 iron and its associated copper, CuB. We have proposed recently that the CuB site is directly involved in the proton pump function. By using time-resolved Raman spectroscopy, we intend to continue our work in measuring vibrational spectra for partially metabolized intermediates in dioxygen and H2O2 reduction and to assess mechanisms by which the protein imposes proton control on the rates of these reactions. Site-directed mutants of two bacterial oxidases are now available, and we will continue our work in characterizing the roles of specific amino acid residues in metal binding and in electron and proton transfer. Both magnetic-resonance and vibrational spectroscopic techniques will be used in this work. In photosynthetic reaction centers, photon absorption produces a series of electron-transfer reactions that lead to a charge-separated state in the sub-ns time regime. We intend to employ two-color, pump/probe ps Raman techniques to characterize electronic and vibrational changes that accompany the charge separation. Model compound studies of neutral porphyrin and chlorin species, of their pi-pi* excited states and of both the excited state and charge-transfer states of model diporphyrins will be carried out to facilitate data interpretation for the biological systems. Our initial work will be done with photosynthetic bacterial reaction centers and will proceed to Photosystem II reaction centers from oxygen- evolving higher plant species.
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