This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. As part of an NIH funded project, we are developing the use of synthetic ?molecular wires?, substrate analogs tethered to photo-sensitizers or affinity tags, for the study of several heme enzymes including cytochrome P450s, peroxidases, and inducible nitric oxide synthase (iNOS). These wires are being designed to bind specifically to the active site of the target enzyme. Once bound, these structures may be used to photochemically trigger the injection or withdrawal of electrons from the redox active heme cofactor to allow the mechanism and turnover to be triggered in a novel way. In addition, these tethered substrate analogs may be used to select for differential binding behavior from a library of mutant proteins, allowing a new approach to the molecular evolution of novel substrate specificity. Crucial to the success of this program, and one of its key features, is the structural characterization of these artificial wires bound to their targets. In preliminary studies, we have been successful in obtaining structures of a series of synthetic wire variants bound to the active site of P450cam. This includes variations in the substrate, linker and sensitizer portion of the wire, and these structures have proven invaluable in driving our efforts to evolve P450s for novel substrate oxidation. We have also obtained a preliminary structure of a peptide based molecular wire bound to an electron transfer channel mutant of CcP, which demonstrates that we will be able to replace the electron transfer pathway in the enzyme with synthetic structures. Finally, we have recently obtained data on iNOS crystals grown in the presence of synthetic pterin based wires that show evidence of binding at the pterin site of the enzyme. We propose to extend these initial efforts by determining structures of a larger array of wires bound to WT and mutant forms of these three enzymes.
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