Quantitative Production of Compound I from a Cytochrome P450 Enzyme at Low Temperatures. Kinetics, Activation Parameters, and Kinetic Isotope Effects for Oxidation of Benzyl Alcohol

Abstract

Cytochrome P450 enzymes are commonly thought to oxidize substrates via an iron(IV)-oxo porphyrin radical cation transient termed Compound I, but kinetic studies of P450 Compounds I are essentially non-existent. We report production of Compound I from cytochrome P450 119 (CYP119) in high conversion from the corresponding Compound II species at low temperatures in buffer mixtures containing 50% glycerol by photolysis with 365 nm light from a pulsed lamp. Compound I was studied as a reagent in oxidations of benzyl alcohol and its benzylic mono-and dideuterio isotopomers. Pseudo-first-order rate constants obtained at −50 °C with concentrations of substrates between 1.0 and 6.0 mM displayed saturation kinetics that gave binding constants for the substrate in the Compound I species (K bind ) and first-order rate constants for the oxidation reactions (k ox ). Representative results are K bind = 214 M −1 and k ox = 0.48 s −1 for oxidation of benzyl alcohol. For the dideuterated substrate C 6 H 5 CD 2 OH, kinetics were studied between −50 °C and −25 °C, and a van't Hoff plot for complexation and an Arrhenius plot for the oxidation reaction were constructed. The H/D kinetic isotope effects (KIEs) at −50 °C were resolved into a large primary KIE (P = 11.9) and a small, inverse secondary KIE (S = 0.96). Comparison of values extrapolated to 22 °C of both the rate constant for oxidation of C 6 H 5 CD 2 OH and the KIE for the non-deuterated and dideuterated substrates to values obtained previously in laser flash photolysis experiments suggested that tunneling could be a significant component of the total rate constant at −50 °C. Cytochrome P450 enzymes (P450s or CYPs) are heme-containing enzymes that are widely distributed in nature and catalyze a wide range of reactions including numerous oxidations. 1,2 In humans, P450s are the major oxidants of drugs, pro-drugs, and xenobiotics, [3][4][5] generating great interest from the pharmaceutical perspective in regard to toxicology and drug dosing. P450s also are of medicinal interest due to the relationships of P450 over-expression with cancer and other disease states. [6][7][8] The active oxidizing forms of P450 enzymes have not been detected under turnover conditions, but they are widely believed to be iron(IV)-oxo species with the porphyrin oxidized to a radical cation. 1,9 Such species, termed Compounds I, are analogous to known intermediates formed in other heme-containing enzymes, such as peroxidases and catalases, upon reaction of the enzymes with hydrogen peroxide. 10 Unlike peroxidases and catalases, P450s typically are activated by a sequence of reactions comprised of reduction of the enzyme to the ferrous state, oxygen binding, a second-reduction step, and protonation steps. Nonetheless, activations of men@uic.edu. a Current address: School of Pharmacy, Luzhou Medical College, Luzhou, Sichuan, China, 646000 Supporting Information Available: Detailed kinetic results, NMR spectra of BA-d 1 and BA-d 2 , mass spectra for BA isotopomers. This material is available fre...

Reactive Intermediates in Cytochrome P450 Catalysis

“…The Mössbauer, UV-visible, and resonance Raman spectra of the PN-generated intermediate match those of a ferric nitrosyl complex. Importantly, high-field Mössbauer measurements reveal that the PN-generated species is S ϭ 0, as expected for an {FeNO} 6 complex, but inconsistent with an authentic S ϭ 1 iron(IV)-oxo (or hydroxide) species (50).…”

“…Dioxygen then binds to the ferrous heme, forming a species that is best described as a ferric superoxide complex (4). The subsequent reduction of this species forms a ferric peroxo species (5), which is protonated at the distal oxygen to generate a ferric hydroperoxo complex (6). The delivery of an additional proton to the distal oxygen cleaves the O-O bond, yielding compound I (7) and a water molecule.…”

“…Over the course of 2009, several articles on the ferric-looking P450-I intermediate were published, some even reporting its quantitative production in P450 BM3 and CYP119A1 (4,6,14,15). These species were reported to be reactive but not overly so: oxidizing benzyl alcohol at a rate of 4 s Ϫ1 at 22°C.…”

“…4B, in which the data from In closing the subject, it is interesting to note the use of a shifted P450-I spectrum by Newcomb and co-workers (53) as their reference and not the ferric-like spectrum that they have argued for previously. One wonders about the implications for their previous work (4,6,(11)(12)(13)(14)(15). Furthermore, given that the PN/LFP technique does not yield P450-I, it would appear to be difficult to follow P450-I reaction kinetics using this method (4,6,(11)(12)(13)(14)(15).…”

“…relatively unreactive) preparations of P450-I (4,6,(11)(12)(13)(14)(15). These reports are surprising, given the highly reactive and historically elusive nature of P450-I (1, 5).…”