Eukaryotic cytochromes P450 (P450s) are ubiquitous, integral membrane enzymes that catalyze the mono-oxygenation of a large number of both exogenous and endogenous compounds. These reactions include pathways for the purging of environmental toxins and carcinogens, the synthesis of sterols, steroids and prostanoids and, in some cases, the metabolic activation of chemical carcinogens. Both mitochondrial and microsomal P450s are synthesized with leader sequences that target them to their respective membranes. Truncation mutants lacking the N-terminal membrane sequence are still associated with membranes when expressed in Escherichia coli or yeast, suggesting that microsomal P450s contain additional membrane binding domains that may be similar to those of mitochondrial P450s. We have made a N-terminal deletion mutant of P450 2C5, into which we have introduced further mutations in a domain which is suspected to be involved in internal membrane binding. These additional mutations prevent membrane association and promote solubility in the absence of detergents, whilst the molecule retain catalytic activities (hydroxylation of progesterone) that are highly similar to those of the unmodified enzyme. Several of these engineered P450s have proved suitable for crystallization, and we have grown large crystals (0.5 x 0.5 x 0.3 mm3) that diffract X-rays to a resolution of 2.9A. By utilizing anomalous scattering from the heme iron in muldwavelength anomalous dispersion (MAD) experiments, we have located the iron and cross- phased single-site gold derivative. Initial intermediate- resolution (3.3A) electron density maps reveal that despite exhibiting a similar overall fold to the bacterial P450s, the structure of this mammalian, microsomal P450 is also significantly different. A large number of laboratories study microsomal P450s and will benefit greatly from a structural model. We propose to continue these crystallographic studies of mammalian P450s to high resolution in order to further characterize the membrane attachment surface, to detail the conformational changes that occur when substrate binds and when the complex is reduced, determine what changes occur in the structure of the protein that when substrate binds and the complex is reduced, and to extend the crystallization methodology to related microsomal P450s of pharmaceutical interest. The structures to be determined in these studies will address questions about the interaction of P450 with the membrane and with P450 reductase, as well as substrate binding and recognition. In addition, these studies will yield significant new information regarding modes of membrane binding, and may help to advance the methodology enabling the engineering of membrane proteins for structural studies.
