Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches.

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility-MS (IM-MS), collision induced unfolding (CIU) and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry and domain interactions in the ~240 kDa BM3 dimeric complex. We obtained high sequence coverage (88-99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.