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...

Classical trajectory calculations have been carried out to simulate the unimolecular decomposition of formaldehyde in the ground electronic state (Se). Global potential-energy surfaces were constructed using the empirical valence-bond (EVB) approach. Two sets of ab initio input were used to characterize two different EVB potential-energy surfaces, and trajectory calculations using one of these gives excellent agreement with experimental data for the product-state distributions of H2 and CO. The trajectory study of vector correlations with prompt dissociation of the parent molecule provides understanding of the dissociation dynamics in the molecular frame. From comparison with some of the experimental results and information from a few ab initio calculations, some improvements for the current potential surfaces are suggested.

A reaction path Hamiltonian is constructed that is based on a straight-line, least motion path that interpolates linearly between equilibrium reactant and product geometries of the molecular system. Conservation of linear and angular momentum are correctly accounted for. The resulting Hamiltonian has a Cartesian-type kinetic energy, the Coriolis coupling terms originally present in the kinetic energy having been transformed to potential energy coupling (hence the term "diabatic" reaction path Hamiltonian). Curvature coupling terms that appear in the original reaction path Hamiltonian, which is based on the minimum energy reaction path, are absent here because the present reaction path is straight. This new, diabatic reaction path Hamiltonian should be especially useful for describing H-atom transfer reactions in polyatomic systems, a case for which the minimum energy reaction path provides a poor description.6298

A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.