The cytochromes P450 are a family of enzymes that catalyze the monooxygenation of a variety of endogenous and exogenous substrates. They contain a heme moiety, which is involved in binding, activating, and splitting molecular oxygen for insertion into the substrate.  The heme is reduced by electrons derived from NADPH and delivered to the cytochrome P450 by the flavin-containing cytochrome P450 reductase. Cytochrome b5 facilitates reactions by some cytochromes P450 via a variety of mechanisms, many of which are still being elucidated. The mammalian isoforms are all integral membrane proteins. These components are shown in the picture to the right.

     The aims of this proposal are to examine the similarities and differences among four members of the CYP4A gene subfamily from rabbit with respect to substrate hydroxylation specificity and the determination of structural properties that determine these specificities. To this end, CYP4A4, 4A5, 4A6, and 4A7 have each been expressed in E. coli to obtain sufficient quantities for characterization by various biophysical techniques and kinetic studies. Possible contributions of individual residues to the substrate specificities and binding interactions exhibited by these 85% sequence-similar P450s are being examined using site-directed mutagenesis. Macromolecular interactions among the redox partners of these cytochrome P450-mediated electron transport systems are being examined by analytical ultracentrifugation and surface plasmon resonance. These enzymes have been implicated in hemodynamic regulation.