Structural Determination of Mutant FMN Containing Domain of Human Cytochrome P450 Reductase Using X-ray Crystallography

Abstract ID 100832 Poster Board 436 Cytochrome P450 enzymes are heme-containing monooxygenases critical for human health due to their role in both the metabolism of drugs, and the biosynthesis of steroid hormones, bile acids, eicosanoids, and vitamins. These enzymes perform this wide range of substrate oxidation using their catalytic heme, which requires electrons from redox partner proteins. For 48 of the 57 human P450 enzymes, this redox partner is cytochrome P450 reductase. This reductase is a highly dynamic protein with two flavin-containing domains that pass two electrons to the P450 heme. The FMN-containing domain directly binds the P450 for electron transfer to the P450 heme. Both clinical and experimental data suggest that mutations in this reductase can differentially affect P450 enzyme function depending on the P450 enzyme. This has been observed as P450-dependent increases or decreases in substrate metabolism as well as changes in regiospecificity of the oxidation (1, 2). These results imply that the reductase FMN domain interacts differently with distinct P450 enzymes. Therefore, the current study aims to utilize X-ray crystallography to determine the structures of some of these relevant FMN domain mutations. The goal of this work is to understand what if any effect these mutations have on the structure of the FMN containing domain of reductase and how those structural changes might affect P450 interactions and thus function. Mutant FMN domain proteins were expressed in E. coli and purified, followed by crystallization in conditions similar those published for the wild type reductase FMN-containing domain (3). Crystals diffracted to 1.5 Å resolution or better and were solved using molecular replacement. These structures should provide structural information to better explain the differential functional effects that have been so widely documented in vitro and in clinical patients. Pandey, A. V., and Flück, C. E. (2013) NADPH P450 Oxidoreductase: Structure, Function, and Pathology of Diseases. Pharmacol Ther138, 229-254 10.1016/j.pharmthera.2013.01.010. Esteves, F., Almeida, C. M. M., Silva, S., Saldanha, I., Urban, P., Rueff, J., Pompon, D., Truan, G., and Kranendonk, M. (2023) Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism. Biomolecules13, 10.3390/biom13071083. Zhao, Q., Smith, G., Modi, S., Paine, M., Wolf, R. C., Tew, D., Lian, L. Y., Primrose, W. U., Roberts, G. C., and Driessen, H. P. (1996) Crystallization and Preliminary X-Ray Diffraction Studies of Human Cytochrome P450 Reductase. J Struct Biol116, 320-325 10.1006/jsbi.1996.0048