This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.We plan to utilize single crystal XAS to study two representative heme-containing enzymes cytochrome P450 from Pseudomonas putida, which catalyzes the regio- and stereo-specific hydroxylation of camphor, and cytochrome c peroxidase from Saccharomyces cerevisiae, which catalyzes the two-electron oxidation of hydrogen peroxide. Like most Fe-heme enzymes, these are thought to go through several Fe-oxo intermediates, including an oxy-ferryl state (compound I), as part of their catalytic cycle. Although, these enzymes have been extensively characterized by both crystallography and spectroscopy, questions still remain about the nature of their reactive intermediates. By examining stable, metastable, and radiolytically reduced states of these enzymes using Fe K-edge single crystal XAS, we plan to characterize the structure of their catalytic intermediates through EXAFS analysis concomitant with electronic state determination through analysis of the edge region. Single crystal XAS can be used in anisotropic systems to selectively enhance specific molecular vectors through crystallographic alignment with the beam polarization vector, thereby providing enhanced geometric and electronic details about a molecule. In the cytochrome P450 study, polarized XAS will be used to probe the axial Fe-substrate oxygen and Fe-proximal ligand interactions separately from the Fe-heme interactions. The program outlined herein should provide for a detailed understanding of heme enzyme catalysis through 1) the polarized single crystal XAS study of wild-type and effector bound conformations of cytochrome P450 in the resting state and, potentially, in six other forms produced by camphor incubation, chemical oxidation/reduction, oxygenation, and radiolytic reduction; and 2) the single crystal XAS study of cytochrome c peroxidase in the ferric resting-state, ferryl compound I state, and in the radiolytically generated products of the compound I state.
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