John H. Horner

Highly Reactive Porphyrin-Iron-Oxo Derivatives Produced by Photolyses of Metastable Porphyrin-Iron(IV) Diperchlorates

Photolyses of metastable porphyrin-iron(IV) diperchlorates in laser flash photolysis reactions gave highly reactive transients. The systems studied were 5,10,15,20-tetraphenylporphyrin (TPP), 5,10,15,20-tetramesitylporphyrin (TMP), and 2,3,7,8,12,13,17,18-octaethylporphyrin (OEP). The new species, which decayed within milliseconds in acetonitrile solutions, were shown to react with organic substrates by oxo-transfer reactions involving insertions into carbon-carbon double bonds of alkenes and styrenes or benzylic carbon-hydrogen bonds of arenes. The order of reactivity was OEP > TPP > TMP. Second-order rate constants for reactions with several substrates at 22 degrees C were determined; representative values of rate constants for the TPP derivative were k = 8.6 x 10(5) M(-1) s(-1) for styrene, k = 2.5 x 10(6) M(-1) s(-1) for cyclohexene, and k = 7.7 x 10(4) M(-1) s(-1) for ethylbenzene. These porphyrin-iron-oxo transients reacted 4-5 orders of magnitude faster than the corresponding iron(IV)-oxo porphyrin radical cations with rate constants similar to those of porphyrin-manganese(V)-oxo derivatives. Rate constants for oxidations of benzylic C-H positions of arenes correlated with the C-H bond dissociation energies, and Hammett correlations for reactions with substituted styrenes had rho(+) values ranging from -0.5 to -0.7, reflecting electrophilic character of the oxidants and their high reactivity. On the basis of their unique UV-visible spectra, high reactivities, and oxo-transfer properties, the new transients are tentatively identified as porphyrin-iron(V)-oxo perchlorates, electronic isomers (or valence tautomers) of well-known iron(IV)-oxo porphyrin radical cations.

Spectra and kinetic studies of the compound I derivative of cytochrome P450 119.

The Compound I derivative of cytochrome P450 119 (CYP119) was produced by laser flash photolysis of the corresponding Compound II derivative, which was first prepared by reaction of the resting enzyme with peroxynitrite. The UV-vis spectrum of the Compound I species contained an asymmetric Soret band that could be resolved into overlapping transitions centered at approximately 367 and approximately 416 nm and a Q band with lambda(max) approximately 650 nm. Reactions of the Compound I derivative with organic substrates gave epoxidized (alkene oxidation) and hydroxylated (C-H oxidation) 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)) and the first-order oxidation rate constant (k(ox)), were obtained for all of the substrates. The oxidations of lauric acid and methyl laurate displayed saturation kinetic behavior, which permitted the determination 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 (kH/kD) 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. Studies of competition kinetics employing CYP119 activated by hydrogen peroxide indicated that the same oxidizing transient is formed in the photochemical reaction and in the hydrogen peroxide shunt reaction.

Tunneling in C-H Oxidation Reactions by an Oxoiron(IV) Porphyrin Radical Cation: Direct Measurements of Very Large H/D Kinetic Isotope Effects

Rate constants for oxidations of benzyl alcohol-d0 and -d7 by oxoiron(IV) tetramesitylporphyrin radical cation perchlorate in acetonitrile were measured in single turnover kinetic studies. The kinetic isotope effect (kH/kD) increased from 28 at 23 degrees C to 360 at -30 degrees C due to extensive hydrogen atom tunneling that was analyzed in terms of a parabolic energy barrier to tunneling. Similarly, large KIE values were found for oxidations of ethylbenzene-d0 and -d10 at room temperature. The large KIE values are a function of the porphyrin identity, and porphyrins containing electron-withdrawing groups display normal KIEs. KIEs found under catalytic turnover conditions are somewhat smaller than those obtained in single turnover reactions. The results should serve as benchmarks for computational studies of C-H oxidations by porphyrin and heme-iron-oxo systems.

X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative

The cytochrome P450 enzyme CYP119, its compound II derivative, and its nitrosyl complex were studied by iron K-edge x-ray absorption spectroscopy. The compound II derivative was prepared by reaction of the resting enzyme with peroxynitrite and had a lifetime of ≈10 s at 23°C. The CYP119 nitrosyl complex was prepared by reaction of the enzyme with nitrogen monoxide gas or with a nitrosyl donor and was stable at 23°C for hours. Samples of CYP119 and its derivatives were studied by x-ray absorption spectroscopy at temperatures below 140 (K) at the Advanced Photon Source of Argonne National Laboratory. The x-ray absorption near-edge structure spectra displayed shifts in edge and pre-edge energies consistent with increasing effective positive charge on iron in the series native CYP119 < CYP119 nitrosyl complex < CYP119 compound II derivative. Extended x-ray absorption fine structure spectra were simulated with good fits for k = 12 Å−1 for native CYP119 and k = 13 Å−1 for both the nitrosyl complex and the compound II derivative. The important structural features for the compound II derivative were an iron-oxygen bond length of 1.82 Å and an iron-sulfur bond length of 2.24 Å, both of which indicate an iron-oxygen single bond in a ferryl-hydroxide, FeIVOH, moiety.

Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species, but not redox activity, as critical elements in cytotoxicity

Azurin is a copper‐containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour‐suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054–7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo‐azurin devoid of copper and site‐directed mutants that are redox negative because of either replacement of a cysteine residue (Cys‐112) involved in co‐ordination with copper or mutational replacement of two methionine residues (Met‐44 and Met‐64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys‐112 Asp mutant azurin can form complexes with the tumour‐suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox‐negative Met‐44LysMet‐64Glu mu‐tant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.

Ocular Surface Extracellular DNA and Nuclease Activity Imbalance: A New Paradigm for Inflammation in Dry Eye Disease

PURPOSE We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation. METHODS Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva. We used quantitative PCR to evaluate eDNA signaling pathways and inflammatory cytokine expression. We also determined the amount of ocular surface eDNA and evaluated tear fluid nuclease activity. RESULTS eDNA, NETs, and neutrophils were present on the ocular surface in DED patients and abundant in mucoid films. NETs consisted of eDNA, histones, cathelicidin, and neutrophil elastase. Tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of eDNA on the ocular surface was increased significantly. Expression of genes downstream of eDNA signaling, such as TLR9, MyD88, and type I interferon, as well as the inflammatory cytokines interleukin-6 and tumor necrosis factor-α, was significantly increased in DED patients. CONCLUSIONS Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.

Polar substituent and solvent effects on the kinetics of radical reactions with thiols

Abstract The rates of thiol trapping of radicals depend upon polar substituents (Table 1) and solvent effects (Table 2). The rate accelerating effect of water on the reactions of biologically relevant radicals is of special importance.

Carbamoyl radicals from Se-phenylselenocarbamates: Intramolecular additions to alkenes

Abstract A series of 5 exo -trig cyclizations of carbamoyl radicals generated from readily available Se-phenylselenocarbamates is reported. Kinetic studies indicate that the rate constant of this cyclization exceeds 1×10 8 s −1 in reveral cases.

Kinetics and Activation Parameters for Oxidations of Styrene by Compounds I from the Cytochrome P450BM-3 (CYP102A1) Heme Domain and from CYP119

Cytochrome P450 (CYP or P450) enzymes are ubiquitous in nature where they catalyze a vast array of oxidation reactions. The active oxidants in P450s have long been assumed to be iron(IV)-oxo porphyrin radical cations termed Compounds I, but P450 Compounds I have proven to be difficult to prepare. The recent development of an entry to these transients by photo-oxidation of the corresponding iron(IV)-oxo neutral porphyrin species (Compounds II) permits spectroscopic and kinetic studies. We report here application of the photo-oxidation method for production of Compound I from the heme domain of CYP102A1 (cytochrome P450(BM-3)), and product and kinetic studies of reactions of styrene with this Compound I transient and also Compound I from CYP119. The studies were performed at low temperatures in 1:1 (v:v) mixtures of glycerol and phosphate buffer. Single-turnover reactions at 0 degrees C gave styrene oxide in good yields. In kinetic studies conducted between -10 and -50 degrees C, both Compounds I displayed saturation kinetics permitting determinations of binding constants and first-order oxidation rate constants. Temperature-dependent functions for the binding constants and rate constants were determined for both Compounds I. In the temperature range studied, the Compound I transient from the CYP102A1 heme domain bound styrene more strongly than Compound I from CYP119, but the rate constants for oxidations of styrene by the latter were somewhat larger than those for the former. The temperature-dependent functions for the first-order oxidation reactions are as follows: log k = 13.2-15.2/2.303RT and log k = 13.3-14.6/2.303RT (kilocalories per mole) for Compounds I from the CYP102A1 heme domain and CYP119, respectively.

Rate constants for aminyl radical reactions

Abstract Rate constants for cyclization of the N -methyl-5,5-diphenyl-4-pentenaminyl radical ( 4 ) and for reaction of this radical with t -BuSH were determined by direct methods.