Cytochromes P450 are a superfamily of heme-thiolate monooxygenases, found in almost all living organisms that play an important role in the biosynthesis and biodegradation of endogenous compounds. In humans, P450s are the major enzymes involved in drug metabolism and bioactivation, accounting for 75% of the total metabolism. The classical P450 reaction is the selective introduction of an oxygen atom, derived from molecular dioxygen, into a substrate unactivated carbon center. The mechanism involves the consumption of two reducing equivalents and the activation of dioxygen at the heme center, leading to the formation of a highly oxidative porphyrin radical ferryl species, namely Compound I. Most P450 monooxygenases are characterized by low activity, limited stability, need of an expensive cofactor (NAD(P)H) and general dependence on auxiliary electron carrier proteins called reductases. We have developed an efficient light-driven P450 BM3 hybrid enzyme system that circumvents the use of the reductase and the expensive NADPH cofactor. This approach utilizes the photochemical properties of Ru(II) photosensitizers covalently attached to strategically positioned non-native single cysteine residues of P450 BM3 heme domain mutants able to perform P450 reactions upon light activation. The high photocatalytic activity and initial reaction rates obtained with the optimized hybrid P450 BM3 enzymes validated our light- activated approach and lays the foundation for the current proposal. We are planning on solving the crystal structure of the highly active hybrid enzyme (Aim 1) in order to understand the factors controlling the efficient electron transfer as well as to improve the design of future hybrd enzymes. We will also take advantage of the unique properties of the hybrid enzymes to study P450 protein interactions thought to play an important role in human enzyme activity (Aim 2). The last two aims will be dedicated to expanding the scope of the light- activated approach using P450 BM3 mutants able to generate various human drug metabolites (Aim 3) and using a mammalian P450 enzyme, CYP2B4, known to hydroxylate bulky hydrophobic xenobiotics (Aim 4). This light-activated P450 enzyme approach will be used as a human P450 model to facilitate the identification of toxic metabolites early in the drug development process and to enable the diversification of lead compounds through the generation of a broad range of hydroxylated derivatives.