The unique photochemical properties of Ru(II)-diimine complexes have helped initiate a series of seminal electron transfer studies in metalloenzymes. It has thus been possible to experimentally determine rate constants for long-range electron transfers. These studies have laid the foundation for the investigation of reactive intermediates in heme proteins and for the design of light-activated biocatalysts. Various metalloenzymes, such as hydrogenase, carbon monoxide dehydrogenase, nitrogenase, laccase and cytochrome P450 BM3 have been functionalized with Ru(II)-diimine complexes. Upon visible light-excitation, these photosensitized metalloproteins are capable of sustaining photocatalytic activity to reduce small molecules such as protons, acetylene, hydrogen cyanide and carbon monoxide or activate molecular dioxygen to produce hydroxylated products. The Ru(II)-diimine photosensitizers are hence able to deliver multiple electrons to metalloenzymes buried active sites circumventing the need for the natural redox partners. In this review, we will highlight the key achievements of the light-driven biocatalysts, which stem from the extensive electron transfer investigations.

The incorporation of a p-nitrophenoxy moiety in substrates has enabled the development of colorimetric assays to rapidly screen for O-demethylation activity of P450 enzymes. For the light-driven hybrid P450 BM3 enzymes, where a Ru(II) photosensitizer powers the enzyme upon visible light irradiation, we have investigated a family of p-nitrophenoxy derivatives as useful chromogenic substrates compatible with the light-driven approach. The validation of this assay and its adaptability to a 96-well plate format will enable the screening of the next generation of hybrid P450 BM3 enzymes towards C-H bond functionalization of non-natural substrates.

We report herein the selective hydroxylation of 10-undecenoic acid with a light-activated hybrid P450 BM3 enzyme. Under previously developed photocatalytic reaction conditions, only a monohydroxylated product is detected by gas chromatography. Hydroxylation occurs exclusively at the allylic position as confirmed from a synthesized authentic standard. Investigation into the stereochemistry of the reaction indicates that the R enantiomer is obtained in 85% ee. The (R)-9-hydroxy-10-undecenoic acid obtained enzymatically is a valuable synthon en route to various natural products further expanding the light-activated P450 BM3 biocatalysis and highlighting the advantages over traditional methods.

Ru(II)-diimine complexes covalently attached near the heme active site of P450 BM3 enzymes have been used to rapidly inject electrons and drive selective C-H functionalization upon visible light irradiation. Herein, we have generated a series of hybrid P450 BM3 enzymes containing a photosensitizer of general formula [Ru(4,4′-X2bpy)2(PhenA)]2+ where X = Cl, H, tBu, Me OPhe, OMe, or NMe2, bpy = 2, 2′-bipyridine, and PhenA = 5-acetamido-1,10-phenanthroline. We then probed the effect of electron-withdrawing and -donating groups at the para position of the 4,4′-X2bpy ligands on the corresponding hybrid enzymes photocatalytic activity. A three-fold improvement in initial reaction rate was noted when varying the substituent from Cl to tBu however, the reaction rates decrease thereafter with the more electron donating groups. In order to rationalize those effects, we investigated the variation of the substitutent on the photophysical properties of the corresponding [Ru(4,4′-X2bpy)2(bpy)]2+ model complexes. Several linear correlations were established between the E(III/II) potential, the MLCT emission and absorption energies as well as the logarithm of the luminescence quenching rate vs. the summative Brown-Okamoto parameter (Σσp+). Moreover, a downward curved Hammett plot is observed with the hybrid enzyme initial reaction rate revealing mechanistic details about the overall light-driven enzymatic process.

The merging of photoredox trifluoromethylation with hybrid P450 BM3 variants has enabled the selective light-driven functionalization of several arenes. This approach capitalizes on the unique photochemical properties of the Ru­(II)-diimine photosensitizer to initiate single electron transfer events. Under photoredox conditions, a CF3 radical promoted by the d6 metal complex can add to arenes. In the hybrid P450 BM3 enzymes, the covalently attached Ru­(II)-diimine photosensitizer provides the necessary electrons to perform, upon visible light activation, P450 oxyfunctionalizations on the trifluoromethylated substrates.