RESONANCE RAMAN STUDIES OF HEME PROTEINS

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We have observed detailed and informative resonance Raman spectra from the heme proteins cytochrome P450 and cytochrome causing near ultraviolet laser excitation. We propose to extend our measurements into the visible using our tunable dye laser and further into the ultraviolet using harmonic generation and optical multichannel analyzer (OMA) detection. We plan to specifically focus on Raman spectral studies of important catalytic intermediate states such as the reduced and oxygenated P450 complexes in order to help deduce mechanisms involved in oxygen binding and cleavage in this important class of metabolic enzymes. The long term objective is to use resonance Raman scattering to gain new information about the active site structure and function of cytochrome P450 and other heme proteins and to achieve a better understanding of metalloporphyrin optical absorption spectra. Photosensitive intermediate states will be studied using OMA detection and our dual arm Raman difference spectrometer will be utilized in a variety of experiments involving cryogenic temperatures, isotopic labels, pH and pressure perturbations, etc. The theoretical implications of 3-body normal mode oscillator models will be further pursued and the "transform" based theories relating absorption and Raman excitation profiled measurements will be applied to heme protein systems such as cytochrome c, hemoglobin and cytochrome P450. Fluorescence excitation profile measurements of heme systems will be made using our newly developed technique for absolute quantum yield determination. Photoacoustic spectroscopy will be used to aid in our understanding of these fluorescence experiments and the accompanying rapid nonradiative relaxation channels present in heme protein systems.