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.

High-valent iron-oxo species are thought to be intermediates in the catalytic cycles of oxygenases and peroxidases. An attractive route to these iron-oxo intermediates involves laser flash-quench oxidation of ferric hemes, as demonstrated by our work on the ferryl (compound II) and ferryl porphyrin radical cation (compound I) intermediates of horseradish peroxidase. Extension of this work to include cytochrome P450-BM3 (CYP102A1) has required covalent attachment of a Ru II photosensitizer to a nonnative cysteine near the heme (Ru II K97C -Fe III P450 ), in order to promote electron transfer from the Fe III porphyrin to photogenerated Ru III . The Ru II K97C -Fe III P450 conjugate was structurally characterized by X-ray crystallography (2.4 Å resolution; Ru-Fe distance, 24 Å). Flash-quench oxidation of the ferric-aquo heme produces an Fe IV -hydroxide species (compound II) within 2 ms. Difference spectra for three singly oxidized P450-BM3 intermediates were obtained from kinetics modeling of the transient absorption data in combination with generalized singular value decomposition analysis and multiexponential fitting.ruthenium bipyridine | enzyme catalysis T he cytochromes P450 constitute a superfamily of thiolateligated heme enzymes so named because the Soret absorption band in their CO-bound derivatives peaks near 450 nm. These monooxygenases catalyze a dazzling array of regio-and stereospecific oxidation reactions, including the hydroxylation of aliphatic and aromatic hydrocarbons and the epoxidation of alkenes (1, 2). P450s take two reducing equivalents from NAD(P)H and deliver one atom from dioxygen to the organic substrate; the other oxygen atom is released as water. The consensus mechanism for P450 catalysis (Fig. 1) implicates a ferryl porphyrin radical cation [compound I (CI): Fig. 1, intermediate 6] as the active oxygenating agent (3), but this elusive species has not been observed in P450 under single-turnover or steady-state catalytic conditions. In the postulated mechanism, CI is proposed to abstract a hydrogen atom from the substrate to form transient Fe IV -hydroxide complex (compound II, CII), followed by radical recombination to produce oxygenated product (4, 5). Mechanistic studies of P450 catalysis in cryogenic matrices have suggested that the barrier to formation of CI (5 → 6) is higher than that for its reaction with substrate (6 → 7 → 1) (6). Consequently, recent efforts have focused on alternate routes to P450 CI that bypass the hydroperoxide intermediate (5). One approach employs generation of CII using peroxynitrite, followed by laser flash photolysis to yield CI (7). This technique has been used in studies of the spectra and reactivity of the putative CI species, but the interpretation of the results remains open to question.In earlier work, we employed ½RuðbpyÞ 3 2þ (bpy ¼ 2;2 0 -bipyridine) in a bimolecular flash-quench photochemical oxidation protocol to generate CII and CI in horseradish peroxidase (HRP) and the heme octapeptide from cyctochrome c (MP8) (8, 9). This approach was uns...

Proton wires: One‐dimensional water chains play an important role in proton permeation through transmembrane proteins. The X‐ray crystal structures as well as solid‐state 2H and 17O NMR and calorimetry studies of two imidazole hydrates that stabilize infinite one‐dimensional water chains are reported (see picture; N blue, O red, H white). The 2H NMR spectroscopic studies suggest that water molecules in both compounds undergo different reorientational dynamics.

We have developed hybrid P450 BM3 enzymes consisting of a Ru(II)-diimine photosensitizer covalently attached to non-native single cysteine residues of P450 BM3 heme domain mutants. These enzymes are capable, upon light activation, of selectively hydroxylating lauric acid with 40 times higher total turnover numbers compared to the peroxide shunt.

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.