Photooxidation of cytochrome P450-BM3

Abstract

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...

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].…”

Reactive Intermediates in Cytochrome P450 Catalysis

“…The rapid removal of one electron from the P450 active site effectively runs the catalytic cycle in reverse, generating compound II (an iron(IV)-hydroxide species) from ferric enzyme. As with reductive flash-quench, however, the technique has yet to yield P450-I (28,29).…”

Manipulation of enzyme properties by noncanonical amino acid incorporation

“…Cytochrome P450s are a family of mono-oxygenases catalyzing the hydroxylation of aliphatic and aromatic compounds and the epoxidation of alkenes [53]. Cytochrome P450 from Bacillus megaterium (BM-3) is a variant that catalyzes hydroxylation of long-chain substrates [54,55].Arnold et al…”