An Efficient Light-Driven P450 BM3 Biocatalyst

Abstract

P450s are heme thiolate enzymes that catalyze the regio- and stereoselective functionalization of unactivated C-H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product / mol enzyme / min.

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…Whereas self-sufficient P450s show the highest turnover numbers and coupling efficiency up to now, fusion to redox partners or modern electron donor systems allow also other CYPs to run with increased efficiency [94,125,131,132]. Drawbacks of the CYP family are poor (regio-)selectivities, low productivities, uncoupling, and the dependence on NADPH as reducing agent [14,17,18].…”

Photoassisted Oxidation of Sulfides Catalyzed by Artificial Metalloenzymes Using Water as an Oxygen Source

“…A better strategy to limit those reactions, and then to increase the stereoselectivity of the reaction, could consist of covalently attaching the Ru complex to the protein, in a place close the catalyst, in order to minimize oxidation of the protein backbone, as it has already been realized in the case of cytochromes P450 [12,13].…”

“…For instance, approaches like the use of alternative oxygen atom donors (PhIO, ROOH, H2O2, KHSO5, etc.) [10], chemical or electrochemical reductions [11], and, more recently, reductase proteins that were substituted with ruthenium-based photosensitizers capable of gathering electrons upon light irradiation and transferring them to cytochrome (P450) enzymes [12,13].…”

“…The former pathway relies on the photo-reduction by diethyldithiocarbamate (DTC) of a Ru II -chromophore that is covalently attached to the apo-P450, which then catalyzes the reductive activation of oxygen leading, in the presence of H + , to a high-valent P450Fe V -oxo species that performs the oxidation of fatty acids [12,13]. Conversely, the oxidative pathway relies on the ability to quench the excited state of a [Ru II (bpy)3] 2+ chromophore with an irreversible electron acceptor such as [Co III (NH3)5Cl] 2+ , in order to form the highly oxidative [Ru III (bpy)3] 3+ , which then catalyzes the twoelectron oxidation of an iron-bound water molecule with formation of the high-valent iron-oxo species (Scheme 1, Path a) [14].…”

Tandem Reactions Combining Biocatalysts and Chemical Catalysts for Asymmetric Synthesis

“…Cheruzel and co-workers developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer to afford light-driven hydroxylation of lauric acid with improved TON and initial reaction rate compared with normal P450 systems (Scheme 10) [70]. This process also circumvented the use of reductases and NAD(P)H cofactors by employing Ru(II) mediated electron transfer processes to reduce Fe(III) to Fe(II) in the presence of dithiocarbamate (DTC).…”