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. 2019 Sep 15:672:108077.
doi: 10.1016/j.abb.2019.108077. Epub 2019 Aug 16.

Coupling efficiency in light-driven hybrid P450BM3 and CYP119 enzymes

Mallory Kato  1 , Marya Melkie  1 , Jeffrey Li  1 , Bridget Foley  1 , Hoang Truc Nguyen  1 , Liridona Leti  1 , Lionel Cheruzel  2
Affiliations

Coupling efficiency in light-driven hybrid P450BM3 and CYP119 enzymes

Mallory Kato et al. Arch Biochem Biophys. .

Abstract

The light-driven hybrid P450 enzyme approach utilizing the photochemical properties of a covalently attached Ru(II)-diimine photosensitizer was extended to the archaeal Sulfolobus acidocaldarius CYP119 enzyme leading to high photocatalytic activity in the hydroxylation of the chromogenic substrate, 11-nitrophenoxyundecanoic acid. The determined kcat was greater than those reported with various natural redox partners. In addition, the sacrificial electron donor, diethyldithiocarbamate, used in the photocatalytic reaction is shown to play a dual role. It acts as an efficient quencher of the Ru(II) excited state leading to a highly reducing species necessary to inject electrons into the heme. It is also known for its antioxidant properties and is shown herein to be a useful probe to determine coupling efficiency in the light-driven hybrid enzymes.

Keywords: Bacterial P450 enzymes; Light-driven process; Reactive oxygen species; Uncoupling pathway.

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Figures

Figure 1:
Figure 1:
A) Key intermediates and products generated in the light-driven hybrid enzymes with the light-harvesting Ru(II)-diimine complexes highlighting the various pathways leading to the formation of the oxidized quencher, DTC2; B) Photocatalytic cycle for the Ru(II)-polypyridyl complex and C) relevant structures of the photosensitizer, the sacrificial electron donor (DTC) and its oxidized form.
Figure 2:
Figure 2:
A) Temperature dependence of the photocatalytic activity of the hybrid enzymes, CYP119-1 (blue) and sL407C-1 (red) and B) kinetic parameters for the hydroxylation of 11-pNCA at 50 °C for the hybrid CYP119-1 enzyme. (Conditions: 1 uM CYP119-1, 100 mM DTC, 375 μM substrate with blue LED irradiation for 1h (A) and 1 min (B)).
Figure 3:
Figure 3:
Plot summarizing the total turnover numbers (left axis, closed symbols) versus the coupling efficiency (right axis, open symbols) for the various light driven hybrid enzymes/substrates (circle: hybrid P450 BM3 variants with 16-PNCA; square: hybrid P450 BM3 mutants with PNP and triangle: hybrid CYP119 mutants with 11-pNCA).
See this image and copyright information in PMC

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