Near Ultraviolet Raman Studies of Cytochrome P450

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DESCRIPTION (provided by applicant): We aim to extend our understanding of the structure and function of cytochrome P450 and other heme protein systems by using pump-probe kinetics and time resolved resonance Raman spectroscopy over a wide dynamic range in time (ps-ms). Development of a unique temporally stabilized, and electronically tunable, two-color picosecond laser system will enable both the transient Raman and the kinetic studies. Femtosecond coherence spectroscopy (FCS), which is a direct and unique probe of the coherent protein/cofactor vibrational motion induced by electronic transitions associated with biochemical reactions, will also be utilized in studies of the biochemical reaction coordinates as well as the global protein response. One specific hypothesis to be tested addresses the reason behind the anomalously low femtosecond quantum yield for O2 photolysis and the potential for a transient side-on oxygen binding geometry that arises because of the participation of ligand bending modes in the reaction coordinate for dissociation. Vibrationally hot six coordinate oxymyoglobin species have already been detected using white light continuum probes, and we propose to extend such studies to the P450 system and its catalytic intermediates as well as to expand the probe capability to include picosecond Raman scattering techniques. Experiments at low temperature will also be carried out using newly developed optical techniques. These experiments will include the first picosecond and femtosecond studies of geminate ligand recombination as a function of temperature. Exploratory work with the Raman microscope will continue with spatially specific probes of mitochondria and with studies of P450 embedded in membrane nanodiscs. Overall this project has a wide range of health related implications involving cytochrome P450 in particular and heme proteins in general. Many chemotherapeutic agents as well as polycyclic carcinogens are metabolized by P450 systems. Moreover, all metabolic disorders involving a P450 protein must relate to this research, since a fundamental understanding of the active site structure and function of P450 at the molecular level will result in a better insight for those concerned with treatment of these disorders at any level.