Cytochromes P450 constitute a unique superfamily of heme-thiolate enzymes that catalyze the insertion of an oxygen atom, derived from molecular dioxygen, into a C-H bond of a variety of organic substrates, often with high degrees of regio- and stereoselectivity. These proteins have been of great interest due to their unique synthetic potential. However, most P450 monooxygenases are characterized by low activity, limited stability and general dependence on auxiliary electron carrier proteins called reductases.Our research has been focused on an alternative approach to perform P450 reactions based on light activation. We have developed the first generation of hybrid enzymes containing a Ru(II) photosensitizer covalently attached to non-native single cysteine residues of P450 BM3 heme domain mutants. We recently reported that these hybrid enzymes are capable of selectively hydroxylating lauric acid with 40-fold increase in TON compared to the wild type heme domain using the peroxide shunt.Building on these results, we have been working to improve the activity and stability of the hybrid enzymes by optimizing the reaction conditions and by expanding our library of mutants from the two initial hybrid enzymes, generating the second generation of hybrid enzymes. This fifteen mutant library of hybrid enzymes consist of new non-native single cysteine residues of P450-BM3 heme domains as well as new photosensitizers. We will present the results obtained with this second generation of hybrid enzymes regarding the improved reactivity and stability along with insights on their mechanism.

Second generation of hybrid P450 enzymes as light-driven biocatalyst for the selective hydroxylation of C-H bond

243rd National Meeting (2012)

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