Porphyrin-manganese(V)-oxo and porphyrin-manganese(IV)-oxo species were produced in organic solvents by laser flash photolysis (LFP) of the corresponding porphyrin-manganese(III) perchlorate and chlorate complexes, respectively, permitting direct kinetic studies. The porphyrin systems studied were 5,10,15,20-tetraphenylporphyrin (TPP), 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TPFPP), and 5,10,15,20-tetrakis(4-methylpyridinium)porphyrin (TMPyP). The order of reactivity for (porphyrin)Mn(V)(O) derivatives in self-decay reactions in acetonitrile and in oxidations of substrates was (TPFPP) > (TMPyP) > (TPP). Representative rate constants for reaction of (TPFPP)Mn(V)(O) in acetonitrile are k = 6.1 x 10(5) M(-1) s(-1) for cis-stilbene and k = 1.4 x 10(5) M(-1) s(-1) for diphenylmethane, and the kinetic isotope effect in oxidation of ethylbenzene and ethylbenzene-d(10) is k(H)/k(D) = 2.3. Competitive oxidation reactions conducted under catalytic conditions display approximately the same relative rate constants as were found in the LFP studies of (porphyrin)Mn(V)(O) derivatives. The apparent rate constants for reactions of (porphyrin)Mn(IV)(O) species show inverted reactivity order with (TPFPP) < (TMPyP) < (TPP) in reactions with cis-stilbene, triphenylamine, and triphenylphosphine. The inverted reactivity results because (porphyrin)Mn(IV)(O) disproportionates to (porphyrin)Mn(III)X and (porphyrin)Mn(V)(O), which is the primary oxidant, and the equilibrium constants for disproportionation of (porphyrin)Mn(IV)(O) are in the order (TPFPP) < (TMPyP) < (TPP). The fast comproportionation reaction of (TPFPP)Mn(V)(O) with (TPFPP)Mn(III)Cl to give (TPFPP)Mn(IV)(O) (k = 5 x 10(8) M(-1) s(-1)) and disproportionation reaction of (TPP)Mn(IV)(O) to give (TPP)Mn(V)(O) and (TPP)Mn(III)X (k approximately 2.5 x 10(9) M(-1) s(-1)) were observed. The relative populations of (porphyrin)Mn(V)(O) and (porphyrin)Mn(IV)(O) were determined from the ratios of observed rate constants for self-decay reactions in acetonitrile and oxidation reactions of cis-stilbene by the two oxo derivatives, and apparent disproportionation equilibrium constants for the three systems in acetonitrile were estimated. A model for oxidations under catalytic conditions is presented.

Cytochrome P450 enzymes (P450s) comprise a large class of enzymes that effect numerous oxidations in nature. The active oxidants in P450s are thought to be iron(IV)-oxo porphyrin radical cations termed Compounds I, and these intermediates have been sought since the discovery of P450s 40 years ago. We report formation of the Compound I derivative of a P450 enzyme by laser flash photolysis oxidation of the corresponding Compound II species, an iron(IV)-oxo neutral porphyrin intermediate. The Compound II derivative in turn was produced by oxidation of the P450 with peroxynitrite, which effected a net one-electron, oxo-transfer reaction to the iron(III) atom of the resting enzyme. For the P450 studied in this work, CYP119 from the thermophile Sulfolobus solfactaricus, the P450 Compound II derivative was stable for seconds at ambient temperature, and the Compound I transient decayed with a lifetime of ca. 200 ms.

The Compound I derivative of cytochrome P450 119 (CYP119) was produced by laser flash photolysis of the corresponding Compound II derivative, which, in turn, was prepared by reaction of the resting enzyme with peroxynitrite. The UV-visible spectrum of the Compound I species contains an asymmetric Soret band that can be resolved into overlapping transitions centered at ca. 367 and 416 nm and a Q-band with λ max ≈ 650 nm. Reactions of the Compound I derivative with organic substrates gave epoxideized (alkene oxidations) and hydroxylated (C-H oxidations) products as demonstrated by product studies and oxygen-18 labeling studies. The kinetics of oxidations by CYP119 Compound I were measured directly; the reactions included hydroxylations of benzyl alcohol, ethylbenzene, Tris buffer, lauric acid, and methyl laurate, and epoxidations of styrene and 10-undecenoic acid. Apparent second-order rate constants, equal to the product of the equilibrium binding constant (K bind ) times the first-order oxidation rate constant (k ox ), were obtained for all substrates. The oxidations of lauric acid and methyl laurate displayed saturation kinetic behavior, which permitted solution of both K bind and k ox for these substrates. The unactivated C-H positions of lauric acid reacted with a rate constant of k ox = 0.8 s −1 at room temperature. The CYP119 Compound I derivative is more reactive than model Compound I species, iron(IV)-oxo porphyrin radical cations, and similar in reactivity to the Compound I derivative of the heme-thiolate enzyme chloroperoxidase. Kinetic isotope effects (k H /k D ) for oxidations of benzyl alcohol and ethylbenzene were small, reflecting the increased reactivity of the Compound I derivative in comparison to models. Nonetheless, CYP119 Compound I apparently is much less reactive than the oxidizing species formed in the P450 cam reaction cycle. Competition kinetic studies employing CYP119 activated by hydrogen peroxide indicate that the same oxidizing transient is formed in the photochemical reaction and in the hydrogen peroxide shunt reaction.The ubiquitous cytochrome P450 (CYP or P450) enzymes are heme-containing enzymes with thiolate from protein cysteine as the fifth ligand to iron. 1 P450s serve in several catalytic roles in nature, but the major function is to catalyze oxidation reactions, typically via two-electron, oxo-transfer processes. In Man, the P450s perform both highly specific reactions, such as oxidation of androgens to estrogens, and broad-spectrum oxidations of drugs, pro-drugs, and xenobiotics in the liver. 2 Much of the interest in P450s derives from the pharmaceutical impact of these enzymes and their relationships to disease states, including cancers and liver disease, that result from over-expression of P450s. [3][4][5] men@uic.edu. Supporting Information Available Results of PN mixing studies and addional examples of deconvolutions of spectra from stopped-flow mixing of CYP119 with mCPBA. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2...

A kinetic scale for dialkylaminyl radicals was established by measuring unimolecular rate constants for a series of dialkylaminyl radical clocks that spans eight orders of magnitude and using clock reactions to measure the second order rate constants for reactions of several hydrogen atom donors. N-Hydroxypyridine-2-thione derivatives of carbamic acids (so-called PTOC carbamates) were used as radical precursors in direct, laser-flash kinetic measurements and in indirect, radical chain kinetic studies. The calibrated radical clocks are N-methyl-6,6-diphenyl-5-hexenaminyl, N-methyl-trans-5-phenyl-4-pentenaminyl, N-methyl-5,5-diphenyl-4-pentenaminyl, N-methyl-trans-2-phenylcyclobutanaminyl, and N-methyl-trans-2-phenylcyclopropanaminyl. Calibrated hydrogen atom donors are Bu 3 SnH, t-BuSH, PhSH, and PhSeH. Whereas the tin hydride reactions with dialkylaminyl radicals are slower than reactions with alkyl radicals, the polarity-matched reactions of the electron-rich dialkylaminyl radicals with the electronpoor hydrogen donors t-BuSH, PhSH, and PhSeH have rate constants nearly equal to those for reactions of alkyl radicals with the same donor.

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...

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Rate constants for 5-exo cyclizations of the 6,6-diphenyl-5-hexenyl radical (1a), the 1-methyl-6,6-diphenyl-5-hexenyl radical (1b), and the 1,1-dimethyl-6,6-diphenyl-5-hexenyl radical (1c) were measured by laser flash photolysis methods, and Arrhenius parameters for these cyclizations were determined. Relative rate constants for cyclizations of radicals 1 and reactions with benzeneselenol were determined by indirect kinetic methods, and the relative Arrhenius parameters for the competing reactions were combined with the parameters for the cyclization reactions to give absolute Arrhenius parameters for the PhSeH reactions. At 20 °C, PhSeH reacts with the 1°, 2°, and 3° radicals 1 with nearly the same rate constants, (1.2 ± 0.1) × 109 M-1 s-1. Absolute Arrhenius parameters for reactions of PhSH and t-BuSH with the primary alkyl radical 1a were calculated using literature values for the competition between cyclization of 1a and reactions with the appropriate thiol and the absolute values for cyclization of 1a determined in this work. The results suggest that rate constants for reactions of primary alkyl radicals with t-BuSH are about 20% smaller than those previously reported. In the case of PhSH, the results are in good agreement with one previously reported set of rate constants but about 35% smaller than another set of rate constants that was subsequently incorporated into fast alkyl radical kinetics. The rate constants for alkyl radical reactions calibrated by competition against reaction with PhSeH and PhSH apparently are 30−40% smaller than those previously reported, and the derived rate constants for the fast radical reactions should be adjusted. An especially noteworthy example is ring opening of the cyclopropylcarbinyl radical, the Arrhenius function for which was determined in part from PhSH trapping results. Using the adjusted rate constants for PhSH and recalculating the Arrhenius parameters for the cyclopropylcarbinyl radical ring opening gives log(k/s-1) = (13.045 ± 0.10) − (6.99 ± 0.09)/θ (kcal/mol, errors at 2σ); the rate constant at 20 °C of 6.7 × 107 s-1 is about 13% smaller than that previously calculated.