Journal
JOURNAL OF INORGANIC BIOCHEMISTRY
Volume 115, Issue -, Pages 50-56Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.jinorgbio.2012.05.012
Keywords
Hybrid P450BM3 enzymes; Light-initiated hydroxylation; Ru(II)-diimime photosensitizers; Flash quench technique; Ferric heme reduction
Funding
- National Institute of Health [GM095415]
- Research Corporation for Science Advancement
- National Science Foundation [0923573]
- Direct For Biological Sciences
- Div Of Biological Infrastructure [0923573] Funding Source: National Science Foundation
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We have developed a series of hybrid P450 BM3 enzymes to perform the light-activated hydroxylation of lauric acid. These enzymes contain a Ru(II)-diimine photosensitizer covalently attached to single cysteine residues of mutant P450 BM3 heme domains. The library of hybrid enzymes includes four non-native single cysteine mutants (K97C, Q397C, Q109C and L407C). In addition, mutations around the heme active site, F87A and I401P. were inserted in the Q397C mutant. Two heteroleptic Ru(II) complexes, Ru(bpy)(2)phenA (1) and Ru(phen)2phenA (2) (bpy = bipyridine, phen = 1,10-phenanthroline, and phenA = 5-acetamido-1,10-phenanthroline), are used as photosensitizers. Upon visible light irradiation, the hybrid enzymes display various total turnover numbers in the hydroxylation of lauric acid, up to 140 for the L407C-1 mutant, a 16-fold increase compared to the F87A/Q397C-1 mutant. CO binding studies confirm the ability of the photogenerated Ru(I) compound to reduce the fraction of ferric high spin species present in the mutants upon substrate binding. Published by Elsevier Inc.
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