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A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119 -129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b 5 . Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b 5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b 5 . All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b 5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.The cytochromes P450 (P450s) 1 are a ubiquitous superfamily of mixed function oxidases that catalyze the oxidation of a large number of hydrophobic endogenous and xenobiotic substrates. Known substrates number in the thousands, whereas unique P450 sequences are counted in the hundreds at this time (2-4).The versatility of these oxidases and their potential for industrial purposes has generated a great deal of interest in understanding their structure, function, and redox reactions. The reaction catalyzed by P450 is shown in Reaction 1.where RH is the substrate and ROH is the oxidized product.The enzymatic cycle includes substrate binding, first electron transfer, oxygen binding, second electron transfer, substrate oxidation, and finally product dissociation. The redox partners for the microsomal P450s are cytochrome P450 reductase (P450 reductase) which contains both a FAD and FMN cofactor and cytochrome b 5 (cyt b 5 ). The crystal structure of P450 reductase has recently been published, and the two domains of the enzyme have been individually expressed and characterized (5, 6). In contrast, the crystal structure of cyt b 5 has been known for many years but has just recently been refined (7,8). The first and second electrons are donated to P450 by P450 reductase. Because of its redox potential (Х ϩ 25 mV), cyt b 5 can only donate the second electron to P450 (9). In fact, it has been suggested that cyt b 5 may be able to transfer the second electron to selected P450s even faster than P450 reductase, thereby decreasing the amount of superoxide produced (1...

Functional and dysfunctional enzymatic pathways of cytochrome P450s after formation of the reduced ferrous dioxygen species have been investigated using nonlocal density functional quantum chemical methods, employing a methyl mercapto iron porphine model of the cytochrome P450 heme complex. The goal of this study was to assess the validity of proposed pathways to both compound I and peroxide involving protonation of the distal and proximal oxygen atoms of the reduced ferrous dioxygen species. Optimized geometries, energies, and electrostatic and electronic properties of each putative heme intermediate in these pathways were calculated and these properties examined for consistency with the proposed role of the intermediate in compound I or peroxide formation. Single protonation of the distal oxygen resulted in significant weakening of the O-O bond. Addition of a second proton to the distal oxygen and energy optimization led directly to compound I and water products, without any apparent activation barrier or formation of a diprotonated intermediate. These results provide direct robust support for the proton-assisted mechanism of dioxygen bond cleavage to form compound I. The dysfunctional pathway to the formation of peroxide was explored by examining the properties of the distal and proximal singly protonated species. The proximal tautomer is thermodynamically less favorable than the distal species by 18.4 kcal/mol. Electrostatic features of both singly protonated species suggest preferred proton delivery to the remaining unprotonated oxygen in each case, favoring peroxide formation. Moreover, addition of a second proton to either of these singly protonated species results in formation of a stable hydrogen peroxide heme complex. These results, taken together, suggest that the simultaneous availability of two protons on the distal oxygen is a requirement for P450 enzymatic efficacy, while asynchronous delivery of protons to the dioxygen site favors decoupling.

I. Introduction and Background 407 II. The Heme Site of the Resting Form of CYP450s 410 III. Heme Site of the Substrate Bound Ferric CYP450s 412 IV. The Heme Site of the Ferrous Dioxygen Species and the Putative Transient Reduced Ferrous Dioxygen Species in CYP450s 413 V. Investigation of a Proposed Pathway to Formation of Compound I from the Twice Reduced Dioxygen Species of CYP450s 414 VI. Electronic Structure of a P450 Compound I Heme Species 415 VII. Role of the Protein in Compound I Formation 416 VIII. Conclusions 417 IX. Glossary of Abbreviations 417 X. Acknowledgment 417 XI. References 417 Gilda H. Loew received her B.A. degree in Chemistry and Physics from New York University, Her M.A. degree in Chemistry from Columbia University, and her Ph.D. degree in Chemical Physics from the University of California Berkeley. She has authored 304 technical publications and is co-inventor on three patents.