The hemoproteins called cytochrome P450 function in the oxygenation of steroids, fatty acids, and fat soluble vitamins, to name but a few examples. Most important is the role of P45Os in the metabolism of many xenobiotics where they may generate reactive metabolites that have toxic effects, in particular drugs, pollutants, food additives etc. The number of P45Os cloned and sequenced now exceeds 200; the majority of these are present in mammalian tissues. In recent years the heterologous expression of specific P45Os has facilitated their enzymatic characterization permitting the study of structure/function relationships. Recently, this laboratory has successfully engineered two artificial fusion proteins containing the heme domain of specific P45Os linked to the flavoprotein domain of NADPH-P450 reductase and expressed these in high yield in E. coli. Methods have been developed for the purification to homogeneity of these enzymatically active fusion proteins. It is proposed to study the mechanism of domain interactions by engineering fusion proteins containing different reductase moieties (rat, human, plant, bacterial (BM3) and yeast) with different P45Os (human lA2, 2D6, 3A4 and 4A9). fusion proteins will be expressed containing linkers of different length, amino acid charge, and hydrophobicity. The goal is to identify the role of specific amino acids, presumably on the surface of the flavoprotein reductase and/or the specific P45Os, that limit the rate of electron transfer from the flavin domain to the heme domain. Specific amino acids of the P450 or reductase will be modified by mutagenesis to confirm their critical roles in domain interaction with the objective of developing methods for enhancing electron transfer. Experiments will be carried out designed to engineer fusion proteins containing three or more functional proteins required for P450 activity, such as the preparation of a three component fusion protein containing P45017A, NADPH-P450 reductase and cytochrome b5. The effect of structure modifications, will be measured by determining patterns of substrate metabolism, the tight coupling of NADPH oxidation with substrate metabolism, the spectrophotometric measurement of steady state reduction of the flavin and heme components of a fusion protein, and the stopped-flow analysis of P450 reduction. Availability of large amounts of purified P450 proteins also offers the opportunity to initiate attempts to crystallize a mammalian P450.
