The two complementary goals of these continuing computational studies of the ubiquitous family of metabolizing heme proteins the cytochrome P450s are: i) Continued elucidation of the most enigmatic portion of the enzymatic cycle common to all P450 isozymes involving putative transient species; the pathway to formation of the catalytically active, ferryl (Fe=O), Compound I species from the twice reduced dioxygen species (figure 2) ii) Further characterization of the molecular origin of substrate and product specificity in the portion of the enzymatic cycle that is unique for each P450 isozyme, by comparative structure function studies of bacterial and mammalian fatty acid hydroxylases. To accomplish the first goal, three types of computational studies are planned. i) Calculation and analysis of the optimized geometries and electronic structure of the transient heme species using the techniques of ab initio quantum chemistry to directly assess their involvement in the proposed pathway, ii) Calculation of the optical spectra of the putative transient species using semi-empirical quantum chemical method to aid experimentalists attempting to identify them by spectroscopic methods and iii) Further probing the dual role of the protein in Compound I formation namely proton donation to the distal oxygen and a link from it to an ultimate source of protons identified in recent molecular dynamic simulation of one isozyme P450eryF, by using the same methods to a) extend the studies to two other P450 isozymes, P450cam and P450BM-3 with known substrate bound structures and b) determine if disruption of this pathway could be the origin of the effect of specific mutations already known to lead to dysfunction. Additional mutations of each of these isozymes predicted from our studies to lead to dysfunction will be suggested to our experimental collaborators for further assessment. The second goal involves comparative structure function studies of bacterial P450BM-3 and mammalian P450 4A1 and P4504A11 fatty acid hydroxylases focusing on how differences in the binding site architecture of these isozymes can alter the substrate and product specificity of their common substrates, fatty acids. While these bacterial and mammalian isozymes have some differences in preferred substrate chain length, they have strikingly different product preferences; 4A1 and 4A11 for omega and BM-3 for omega-1, omega-2 and omega-3 regioselective hydroxylations. The preferred omega-hydroxylated metabolites of the fatty acid substrates of P4504A11 are directly implicated in important biological functions, the regulation of blood flow and vascular tone in vital organs such as kidney. These studies require use of 3D structures of all three isozymes and are now made possible by very recent experimental and computational advances, specifically: i) the X-Ray structure determination of a substrate bound P450-BM3 by our collaborator Tom Poulos, and ii) the ability to construct reliable models of 4A1 and 4A11 by methods recently developed and assessed in our laboratory for other P450 isozymes. In addition our collaborator Dr. Paul Ortiz de Montellano has agreed to make mutants of the 4A1 and 4A11 isozyme suggested by our proposed 3D structure and binding site that will further test the reliability of the models to make robust predictions.
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