Project Summary The proposed work aims to clarify longstanding mechanistic uncertainties about drug metabolizing cytochrome P450s (CYPs). These CYPs are extraordinarily substrate promiscuous and they are major determinants of xenobiotic detoxication, drug metabolism and drug interactions. Because of their role in drug metabolism, they are also drug targets, wherein their pharmacological manipulation can afford control of therapy with other drugs. One focus here is the knowledge gap concerning the relationship between ligand-dependent heme spin state and catalytic properties. In the CYP canonical catalytic reaction cycle developed for substrate specific isoforms, the substrate displaces heme bound water and causes a shift from low spin to high spin heme with concomitant shift in the heme properties that facilitate reduction and progress through the catalytic cycle. In contrast, with drug metabolizing CYPs, many drugs cause no shift to high spin heme, but they are efficiently metabolized or cause hydrogen peroxide formation, both of which require progression through the catalytic cycle. Recent results demonstrate that many of these substrates hydrogen bond to the axial water, rather than displace it. The catalytic properties of these water-bridged complexes have not been determined. Therefore, the proposed work will determine for CYP3A4 the heme redox properties and the catalytic competence of water-bridged complexes, using computational approaches and advanced biochemical methods including spectropotentiometry and stopped-flow reaction kinetics. Computational approaches will also be employed to understand the effect of water-bridged complexes on heme reduction processes. A second focus of the proposed work aims to clarify the role of conformational dynamics in the complex allosteric behavior of CYPs and their remarkable substrate promiscuity. Both traits are linked to the protein dynamics, which remain poorly characterized, and unclarified by the available crystal structures. The allosteric properties confound prediction of drug clearance and drug interactions, so there is great interest in translational models that better predict drug interactions based on refined allosteric models. Here, the experimental methods of hydrogen-deuterium exchange mass spectrometry (H/DX) and pre-steady state ligand binding methods with CYP3A4 in lipid bilayer nanodiscs are combined with computational approaches such as accelerated Molecular Dynamics simulations (aMD) and steered molecular dynamics. The proposed studies fill a significant gap in understanding the, previously experimentally inaccessible, CYP dynamics in a lipid membrane and the role of conformational dynamics in achieving substrate promiscuity and allostery.