Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies

Abstract

Background In order to perform selective C-H functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)2PhenA (bpy = 2,2′-bipyridine and PhenA = 5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective C-H bond hydroxylation of several substrates. Methods A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. Results The crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. Conclusions In the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. General Significance The results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.

Correlating the para-Substituent Effects on Ru(II)-Polypyridine Photophysical Properties and on the Corresponding Hybrid P450 BM3 Enzymes Photocatalytic Activity

“…Subtle changes in intensity and blue-shifted emission are noticed, which can be attributed to changes in the local environment of the photosensitizers covalently attached to the protein. Based on the recent crystal structure of P2 , 5 the photosensitizer is located in a bowl-shaped cavity with the ancillary bipyridine ligands pointing toward the bulk solvent away from any protein residues. 5 We thus infer that introduction of the X substituent does not perturb the position and orientation of the photosensitizer across the series of functionalized hybrid enzymes.…”

“…4b The DTC anion is able to efficiency quenched the Ru(II) excited state via electron transfer (Figure 1, Step 2) and generate a highly reductive species. 5, 15 …”

“…4 Using sodium diethyldithiocarbamate (DTC) as the reductive quencher under established flash-quench conditions (Figure 1), a highly reductive species is photogenerated capable of rapidly delivering electrons to the heme active site to initiate the enzymatic process. 5 …”

Light‐Driven Hydrogen Evolution by Nickel‐Substituted Rubredoxin

“…In the wild‐type (WT) protein, the native, free cysteine residue at position 31, which immediately precedes the metal‐binding loop of residues cysteine‐32 through cysteine‐35, was used for labeling. This covalent attachment approach represents a robust method for incorporation of Ru‐based phototriggers into a protein scaffold, enabling light‐driven fuel production and detailed mechanistic investigations (Figure a) …”

Photoenzymes and Related Topics: An Update

“…Various ruthenium‐based photosensitizers have been coupled to this enzyme to convert it to a photoenzyme (e.g. ), one of the most efficient being Ru(2,2′‐bipyridine) 2 (5‐acetamido‐1,10‐phenanthroline) . Other P450 enzymes have also been engineered for photochemical syntheses .…”