Abstract
This chapter describes methodologies for employing sensitizer-linked substrates and ligands as probes of P450 structure and mechanism. Sensitizer-linked substrates target cytochromes P450 in a background of other heme enzymes, and their demonstrated biosensing and substrate screening capabilities may facilitate future drug design efforts. Unlike many structural probes of the P450 hydrophobic pocket, sensitizer-linked substrates bind reversibly and may undergo modest turnover during biological catalysis. Resonance Raman spectroscopy interrogates the heme environment in P450 Fe 2+ -CO Ru-substrate complexes. Ru-substrates complement existing mechanistic probes by providing an efficient ET pathway to the P450 heme through the hydrocarbon linker. Because of rate-limiting ET in the natural enzymatic system reactive Fe-peroxy and ferryl intermediates in P450 catalytic cycles are not readily observable. A general procedure for generating sensitizer-linked substrates is discussed in the chapter through a figure. This protocol may be generalized to include any combination of photosensitizer, linker, and substrate/ligand that targets the desired P450 active site. Ones have synthesized numerous compounds: Ru and Os photosensitizers with substituted bipyridine, terpyridine, phenanthroline, and imidazole ligands; alkyl, polyethylene glycol, perfluorobiphenyl, and polyxylyl linkers; attached via amide, ether, or amino bonds to substrates (adamantane, ethyl benzene, borneol, norbornane, thioanisole styrene) or imidazole ligands.
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