Lionel Cheruzel, San Jose State University
Cytochrome P450s are unique heme thiolate enzymes that catalyze the regio and stereoselective functionalization of unactivated C-H bonds in a wide range of substrates, using molecular dioxygen, two protons, and two electrons provided by a reductase domain. As an alternative approach to deliver the necessary electrons and perform P450 reactions upon visible light excitation, we have developed hybrid P450 enzymes contain a Ru(II)-diimine photosensitizer covalently attached to strategically positioned non-native cysteine residues of P450 heme domains. High photocatalytic activity (i.e. high total turnover numbers and initial reaction rates) could be achieved in the hydroxylation of natural long-chain fatty acid substrates. The crystal structure of an efficient hybrid enzyme reveals that the photosensitizer is ideally positioned to deliver electrons to the active site utilizing the natural electron transfer pathway. A combination of rational and directed evolution approaches has been used to develop the next generation of hybrid enzymes showing enhanced photocatalytic activity towards a wide range of non-natural substrates.