The long-range goal of the spectroscopic studies described in this core project are to use the structural information gained from X-ray crystallographic studies of Prostaglandin H Synthase (PGHS) and Cytochrome c Oxidase (CcOX) as platforms for exploring the details of their catalytic mechanisms, and to develop new spectroscopic methods for the study of metal centers in biological systems. Vibrational spectroscopies, Resonance Raman and FTIR, which provide detailed information regarding bond strengths, will be extended to energy regimes where metal-ligand bonding can be better characterized When combined with small-volume, stop-flow kinetic techniques, these optical methods provide a unique pathway for characterizing catalytic intermediates. The advanced EPR methods that will be utilized in this core project, allow one to measure to wave function that house an unpaired electron spin and gain an understanding of the electronic structure of a catalytic site. The EPR development work that will be undertaken in this core project includes the development of a pulse-microwave/pulse-magnetic field method for improving our capability to resolve hyperfine couplings for high-spin paramagnetic metal centers, the application of several new coherence transfer pulsed EPR methods for the measurement of protein radical conformation and ligand hyperfine couplings, and the parallel development of analysis strategies to support these measurements. Because the development work described in this proposal is driven by specific questions being addressed for CcOX and PGHS, the methodologies defined during the course of this project will be generally useful to the biomedical community.
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