The objective is the use of theoretical chemistry techniques to provide a link between structure, spectra, and function of cytochrome P450s. Using the recently determined X-ray crystal structure of substrate-bound P450cam and empirical energy, semiempirical, and ab initio methods of quantum chemistry, we will 1) continue studies of the four stable states in the normal oxidative cycles: a) characterize the heme unit of the low-spin resting state, focusing on features that modulate high-spin/low-spin equilibria, and determine electromagnetic properties and optical spectra; b) characterize low-spin, Type II substrate-bound complexes to determine effects of these substrates on optical spectra and how they inhibit the normal oxidative cycle; c) elucidate the origin of the highly anisotropic electronic properties of the high-spin Type I substrate-bound complex. Explore effects of spin-orbit coupling and facile changes in the nature of the cysteine residue on electromagnetic properties and calculate electronic spectra; d) describe effect of reduction to the ferrous P450 complex on the iron-cysteine geometry, electromagnetic properties and spectra; e) determine changes in the cysteine ligand that restore a normal Soret band for oxy and carboxy ferrous P450 complexes, and hence inactivate the enzyme. These changes will include variation of Fe-S bond distance, distortions of cysteine orientation, and protonation of the sulfur. 2) In a new effort, characterize the substrate-binding site and determine the steric and electronic factors that modulate substrate affinity and reactivity. 3) Determine the nature of the biologically active oxygen transfer state and the pathway to its formation from the oxy ferrous complex. 4) Using a more complex model for the biologically active state than in previous studies, a) characterize the mechanism of alkane hydroxylation; b) study the pathways for epoxidation of and N-alkylation by alkenes; c) elucidate the mechanism of oxidation of aromatic substrates to toxic and benign products; d) determine the mechanism and effects of heteroatoms on the process of O- and N-demethylation initiated by (Alpha)C-hydroxylation, leading to active carcinogens. 5) Explore subsequent steps in the anaerobic reduction of halogenated hydrocarbons, particularly the interaction of potentially damaging halomethyl and haloethyl radicals with the ferrous heme unit, and the further reduction of such species, eventually forming a ferrous-carbene complex.
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