Peroxynitrite-mediated tyrosine nitration catalyzed by superoxide dismutase.

Peroxynitrite (ONOO-), the reaction product of superoxide (O2-) and nitric oxide (NO), may be a major cytotoxic agent produced during inflammation, sepsis, and ischemia/reperfusion. Bovine Cu,Zn superoxide dismutase reacted with peroxynitrite to form a stable yellow protein-bound adduct identified as nitrotyrosine. The uv-visible spectrum of the peroxynitrite-modified superoxide dismutase was highly pH dependent, exhibiting a peak at 438 nm at alkaline pH that shifts to 356 nm at acidic pH. An equivalent uv-visible spectrum was obtained by Cu,Zn superoxide dismutase treated with tetranitromethane. The Raman spectrum of authentic nitrotyrosine was contained in the spectrum of peroxynitrite-modified Cu,Zn superoxide dismutase. The reaction was specific for peroxynitrite because no significant amounts of nitrotyrosine were formed with nitric oxide (NO), nitrogen dioxide (NO2), nitrite (NO2-), or nitrate (NO3-). Removal of the copper from the Cu,Zn superoxide dismutase prevented formation of nitrotyrosine by peroxynitrite. The mechanism appears to involve peroxynitrite initially reacting with the active site copper to form an intermediate with the reactivity of nitronium ion (NO2+), which then nitrates tyrosine on a second molecule of superoxide dismutase. In the absence of exogenous phenolics, the rate of nitration of tyrosine followed second-order kinetics with respect to Cu,Zn superoxide dismutase concentration, proceeding at a rate of 1.0 +/- 0.1 M-1.s-1. Peroxynitrite-mediated nitration of tyrosine was also observed with the Mn and Fe superoxide dismutases as well as other copper-containing proteins.     

Stopped-flow kinetic analysis for monitoring superoxide decay in aqueous systems.

We have utilized a commercially available, computer-driven stopped-flow spectrophotometer to rapidly measure the self-dismutation or catalyzed decay of superoxide in aqueous buffers. In the self-dismutation assay, a dimethyl sulfoxide solution of superoxide is mixed in less than 2 ms with an aqueous buffer. The decay of superoxide is monitored directly by its absorbance at 245 nm and the data is processed by computer. By careful purification of the water and the use of metal-free buffers, a decay of superoxide that fits second-order kinetics is obtained without using metal ion chelators in the buffer. The second-order rate constant for superoxide decreased with increasing pH and decreased by a factor of 3.3 by using D2O in place of H2O in the buffer. The rapid mixing time makes it possible to determine rate constants for active superoxide dismutase catalysts at a pH as low as 7. A first-order decay of superoxide is obtained when the aqueous buffer contains bovine Cu/Zn superoxide dismutase or aquo copper(II), which are known catalysts of superoxide dismutation. The rate of superoxide decay was established to be first-order in catalyst. The catalytic rate constant for bovine Cu/Zn superoxide dismutase was determined to be 2.3 x 10(9) M-1 s-1 in H2O and D2O-based buffers and was independent of pH over the range 7-9. Aquo copper(II) gave a catalytic rate constant of 1.2 x 10(8) M-1 s-1, but was ineffective in the presence of EDTA. The catalytic rate constants obtained by stopped-flow kinetics are in excellent agreement with studies carried out by the direct method of pulse radiolysis.     

Manganese and iron porphyrins catalyze peroxynitrite decomposition and simultaneously increase nitration and oxidant yield: implications for their use as peroxynitrite scavengers in vivo.

Twelve substituted metalloporphyrins (MPs), some of which have been previously characterized with respect to superoxide dismutase and peroxynitrite decomposing activities, were evaluated for their ability to scavenge peroxynitrite in vitro at 37 degrees C. Because the overall effectiveness of MPs as catalytic peroxynitrite scavengers is a function of (1) how fast they react with peroxynitrite, (2) how fast they cycle back to the starting compound, and (3) how well they contain or quench the reactive intermediates generated, all of these properties were evaluated and compared directly under the same conditions. Of the various MPs tested, only the iron and manganese porphyrins showed significant reactivity with peroxynitrite. The Mn(IV) intermediates resulting from oxidation by peroxynitrite were relatively stable and rereduction to the Mn(III) forms was rate-limiting to catalytic decomposition of peroxynitrite. However, in the presence of oxidizeable substrates like phenolics, rereduction of Mn(IV) forms occurred very rapidly and both the Mn- and Fe-porphyrins catalyzed nitration and oxidation by peroxynitrite. Mn- and Fe-porphyrins enhanced the yield of nitrated phenolics by peroxynitrite as much as 5-fold at pH 7.4 and up to 12-fold at pH 9. 1, while total oxidative yield was more than doubled. Nitration enhancement by MPs was effectively inhibited by ascorbate, glutathione, or serum, although much higher concentrations of ascorbate were required to inhibit nitration catalyzed by either Mn or Fe tetramethylpyridyl porphyrin. Catalysis of peroxynitrite nitration by MPs appears to proceed via a radical-mediated reaction mechanism whereby the phenolic substrate rapidly reduces Mn(IV) = O or Fe[IV] = O to the +3 state to yield phenoxyl radical which then combines with the other primary product, nitrogen dioxide. Based on the rate constants and the proposed reaction mechanism, it is reasonable to suggest that Mn and Fe porphyrins could detoxify peroxynitrite in vivo by efficiently trapping the relatively unreactive peroxynitrite anion and, in effect, channeling it into a single reaction pathway which could then be more effectively scavenged by cellular reductants like ascorbate.     

Peroxynitrite oxidation of sulfhydryls. The cytotoxic potential of superoxide and nitric oxide.

Peroxynitrite anion (ONOO-) is a potent oxidant that mediates oxidation of both nonprotein and protein sulfhydryls. Endothelial cells, macrophages, and neutrophils can generate superoxide as well as nitric oxide, leading to the production of peroxynitrite anion in vivo. Apparent second order rate constants were 5,900 M-1.s-1 and 2,600-2,800 M-1.s-1 for the reaction of peroxynitrite anion with free cysteine and the single thiol of albumin, respectively, at pH 7.4 and 37 degrees C. These rate constants are 3 orders of magnitude greater than the corresponding rate constants for the reaction of hydrogen peroxide with sulfhydryls at pH 7.4. Unlike hydrogen peroxide, which oxidizes thiolate anion, peroxynitrite anion reacts preferentially with the undissociated form of the thiol group. Peroxynitrite oxidizes cysteine to cystine and the bovine serum albumin thiol group to an arsenite nonreducible product, suggesting oxidation beyond sulfenic acid. Peroxynitrous acid was a less effective thiol-oxidizing agent than its anion, with oxidation presumably mediated by the decomposition products, hydroxyl radical and nitrogen dioxide. The reactive peroxynitrite anion may exert cytotoxic effects in part by oxidizing tissue sulfhydryls.     

Peroxynitrite formation from macrophage-derived nitric oxide.

Peroxynitrite formation by rat alveolar macrophages activated with phorbol 12-myristate 13-acetate was assayed by the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate. The inhibitor of nitric oxide synthesis N-methyl-L-arginine prevented the Cu,Zn superoxide dismutase-catalyzed nitration of 4-hydroxyphenylacetate by stimulated macrophages, while Cu-depleted Zn superoxide dismutase did not catalyze the formation of 3-nitro-4-hydroxyphenylacetate either in vitro or in the presence of activated macrophages. The rate of phenolic nitration by activated macrophages was 9 +/- 2 pmol x 10(6) cells-1 x min-1 (mean +/- STD). Only 8% of synthetic peroxynitrite was trapped by superoxide dismutase, which suggested that the rate of peroxynitrite formation may have been as high as 0.11 nmol x 10(6) cells-1 x min-1. This upper estimate was consistent with N-methyl-L-arginine increasing the amount of superoxide detected with cytochrome c by 0.12 nmol x 10(6) cells-1 x min-1. The rate of nitrite and nitrate accumulation was 0.10 +/- 0.001 nmol x 10(6) cells-1 x min-1, suggesting that the majority of nitric oxide produced by activated macrophages may have been converted to peroxynitrite. The formation of a relatively long lived, strong oxidant from the reaction of nitric oxide and superoxide in activated macrophages may contribute to inflammatory cell-mediated tissue injury.     

Evidence for catalytic dismutation of superoxide by cobalt(II) derivatives of bovine superoxide dismutase in aqueous solution as studied by pulse radiolysis.

By using the technique of pulse radiolysis to generate O2-., it is demonstrated that Co(II) derivatives of bovine superoxide dismutase in which the copper alone and both the copper and zinc of the enzyme have been substituted by Co(II), resulting in (Co,Zn)- and (Co,Co)-proteins, are capable of catalytically dismutating O2-. with 'turnover' rate constants of 4.8 X 10(6) dm3.s-1.mol-1 and 3.1 X 10(6) dm3.s-1.mol-1 respectively. The activities of the proteins are independent of the pH (7.4-9.4) and are about three orders of magnitude less than that of the native (Cu,Zn)-protein. The rate constants for the initial interaction of O2-. with the Co-proteins were determined to be (1.5-1.6) X 10(9) dm3.s-1.mol-1; however, in the presence of phosphate, partial inhibition is apparent [k approximately (1.9-2.3) X 10(8) dm3.s-1.mol-1]. To account for the experimental observations, two reaction schemes are presented, involving initially either complex-formation or redox reactions between O2-. and Co(II). This is the first demonstration that substitution of a metal into the vacant copper site of (Cu,Zn)-protein results in proteins that retain superoxide dismutase activity.     

Some redox properties of myohemerythrin from retractor muscle of Themiste zostericola

Distinct semimetmyohemerythrin species are produced by one-electron oxidation of deoxymyohemerythrin and one-electron reduction of metmyohemerythrin. The former, (semimetmyo)o, changes (greater than or equal to 90%) to the latter, (semimetmyo)R, with k = 1.0 x 10(-2) s-1, delta H = 15.1 kcal mol-1 and delta S = -17 eu. Oxidation of (semimetmyo)o by Fe(CN)6(3)- rapidly produces an unstable metmyohemerythrin form which converts to the final metmyohemerythrin with k = 4.6 x 10(-3) s-1, delta H = 16.8 kcal mol-1, and delta S = -13 eu. The two met forms react at the same rate with N3-, but the unstable form reacts very rapidly with S2O4(2-) in contrast to stable metmyohemerythrin. (Semimetmyo)R or a mixture of metmyohemerythrin and deoxymyohemerythrin equilibrate very slowly to a mixture containing all three species. The rate constants for disproportionation and comproportionation are 0.89 M-1 s-1 and 9.4 M-1 s-1, respectively. EPR spectra near liquid He temperatures and optical absorption spectra have been used to characterize and measure the rates at 25 degrees C, pH 8.2, and I = 0.15 M. The comparative behavior of octameric and monomeric protein is discussed.     

Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNA(Phe)(yeast). Implications for conformational change and activity

The thermodynamics and kinetics of magnesium binding to tRNA(Phe)(yeast) have been studied directly by 25Mg NMR. In 0.17 M Na+(aq), tRNA(Phe) exists in its native conformation and the number of strong binding sites (Ka greater than or equal to 10(4)) was estimated to be 3-4 by titration experiments, in agreement with X-ray structural data for crystalline tRNA(Phe) (Jack et al., 1977). The set of weakly bound ions were in slow exchange and 25Mg NMR resonances were in the near-extreme-narrowing limit. The line shapes of the exchange-broadened magnesium resonance were indistinguishable from Lorentzian form. The number of weak magnesium binding sites was determined to be 50 +/- 8 in the native conformation and a total line-shape analysis of the exchange-broadened 25 Mg2+ NMR resonance gave an association constant Ka of (2.2 +/- 0.2) X 10(2) M-1, a quadrupolar coupling constant (chi B) of 0.84 MHz, an activation free energy (delta G*) of 12.8 +/- 0.2 kcal mol-1, and an off-rate (koff) of (2.5 +/- 0.4) X 10(3) s-1. In the absence of background Na+(aq), up to 12 +/- 2 magnesium ions bind cooperatively, and 73 +/- 10 additional weak binding sites were determined. The binding parameters in the nonnative conformation were Ka = (2.5 +/- 0.2) X 10(2) M-1, chi B = 0.64 MHz, delta G* = 13.1 +/- 0.2 kcal mol-1, and koff = (1.6 +/- 0.4) X 10(3) s-1. In comparison to Mg2+ binding to proteins (chi B typically ca. 1.1-1.6 MHz) the lower chi B values suggest a higher degree of symmetry for the ligand environment of Mg2+ bound to tRNA. A small number of specific weakly bound Mg2+ appear to be important for the change from a nonnative to a native conformation. Implications for interactions with the ribosome are discussed.     

Osmium tetroxide, used in the treatment of arthritic joints, is a fast mimic of superoxide dismutase

Aqueous solutions of osmium tetroxide (OsO4) have been injected into arthritic knees for the past 45 years to chemically destroy diseased tissue, in a procedure termed "chemical synovectomy." Arthritis is an inflammatory disease. The primary inflammatory chemical species are the superoxide anion radical (O2.-) and nitric oxide (.NO), which combine to form the peroxynitrite anion (ONOO-). Here we show that OsO4 does not react with ONOO- but very efficiently catalyzes the dismutation of O2.- to O2 and H2O2. Using the pulse-radiolysis technique, the catalytic rate constant has been determined to be (1.43+/-0.04) x 10(9) M-1 s-1, independent of the pH in the 5.1-8.7 range. This value is about half that for the natural Cu,Zn-superoxide dismutase (Cu,Zn-SOD). Per unit mass, OsO4 is about 60 times more active than Cu,Zn-SOD. The catalytically active couple is OsVIII/OsVII, OsVIII oxidizing O2.- to O2 with a bimolecular rate constant of k=(2.6+/-0.1)x10(9) M-1 s-1 and OsVII reducing it to H2O2 with a bimolecular rate constant of (1.0+/-0.1)x10(9) M-1 s-1. Although lower valent osmium species are intrinsically poor catalysts, they are activated through oxidation by O2.- to the catalytic OsVIII/OsVII redox couple. The OsVIII/OsVII catalyst is stable to biochemicals other than proteins and peptides comprising histidine, cysteine, and dithiols.     

Catalysis of the Haber-Weiss reaction by iron-diethylenetriaminepentaacetate.

To help settle controversy as to whether the chelating agent diethylenetriaminepentaacetate (DTPA) supports or prevents hydroxyl radical production by superoxide/hydrogen peroxide systems, we have reinvestigated the question by spectroscopic, kinetic, and thermodynamic analyses. Potassium superoxide in DMSO was found to reduce Fe(III)DTPA. The rate constant for autoxidation of Fe(II)DTPA was found (by electron paramagnetic resonance spectroscopy) to be 3.10 M-1 s-1, which leads to a predicted rate constant for reduction of Fe(III)DTPA by superoxide of 5.9 x 10(3) M-1 s-1 in aqueous solution. This reduction is a necessary requirement for catalytic production of hydroxyl radicals via the Fenton reaction and is confirmed by spin-trapping experiments using DMPO. In the presence of Fe(III)DTPA, the xanthine/xanthine oxidase system generates hydroxyl radicals. The reaction is inhibited by both superoxide dismutase and catalase (indicating that both superoxide and hydrogen peroxide are required for generation of HO.). The generation of hydroxyl radicals (rather than oxidation side-products of DMPO and DMPO adducts) is attested to by the trapping of alpha-hydroxethyl radicals in the presence of 9% ethanol. Generation of HO. upon reaction of H2O2 with Fe(II)DTPA (the Fenton reaction) can be inhibited by catalase, but not superoxide dismutase. The data strongly indicate that iron-DTPA can catalyze the Haber-Weiss reaction.     

Kinetic studies on spin trapping of superoxide and hydroxyl radicals generated in NADPH-cytochrome P-450 reductase-paraquat systems. Effect of iron chelates

Electron spin resonance (ESR) studies on spin trapping of superoxide and hydroxyl radicals by 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) were performed in NADPH-cytochrome P-450 reductase-paraquat systems at pH 7.4. Spin adduct concentrations were determined by comparing ESR spectra of the adducts with the ESR spectrum of a stable radical solution. Kinetic analysis in the presence of 100 microM desferrioxamine B (deferoxamine) showed that: 1) the oxidation of 1 mol of NADPH produces 2 mol of superoxide ions, all of which can be trapped by DMPO when extrapolated to infinite concentration; 2) the rate constant for the reaction of superoxide with DMPO was 1.2 M-1 s-1; 3) the superoxide spin adduct of DMPO (DMPO-OOH) decays with a half-life of 66 s and the maximum level of DMPO-OOH formed can be calculated by a simple steady state equation; and 4) 2.8% or less of the DMPO-OOH decay occurs through a reaction producing hydroxyl radicals. In the presence of 100 microM EDTA, 5 microM Fe(III) ions nearly completely inhibited the formation of the hydroxyl radical adduct of DMPO (DMPO-OH) as well as the formation of DMPO-OOH and, when 100 microM hydrogen peroxide was present, produced DMPO-OH exclusively. Fe(III)-EDTA is reduced by superoxide and the competition of superoxide and hydrogen peroxide in the reaction with Fe(II)-EDTA seems to be reflected in the amounts of DMPO-OOH and DMPO-OH detected. These effects of EDTA can be explained from known kinetic data including a rate constant of 6 x 10(4) M-1 s-1 for reduction of DMPO-OOH by Fe(II)-EDTA. The effect of diethylenetriamine pentaacetic acid (DETAPAC) on the formation of DMPO-OOH and DMPO-OH was between deferoxamine and EDTA, and about the same as that of endogenous chelator (phosphate).     

ESR spin-trapping studies on the reaction of Fe2+ ions with H2O2-reactive species in oxygen toxicity in biology

Using ESR spin-trapping techniques with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), we confirmed the 1:1 stoichiometry for the formation of hydroxyl radicals with Fe2+ in the Fenton reaction under experimental conditions wherein [H2O2] is 90 microM and [Fe2+] is very low, 1 microM or less. The stoichiometry decreased markedly as the Fe2+ concentration was increased. The efficiency of hydroxyl radical generation varied with the nature of the iron chelators used and increased in the order of phosphate alone approximately ADP less than EDTA less than diethylenetriaminepentaacetic acid (DETAPAC). The second order rate constant for the Fenton reaction was measured to be 2.0 x 10(4) M-1 s-1 for phosphate alone, 8.2 x 10(3) M-1 s-1 for ADP, 1.4 x 10(4) M-1 s-1 for EDTA, and 4.1 x 10(2) M-1 s-1 for DETAPAC. Measuring the radicals formed as spins trapped in the presence of ethanol, we estimated the amount of total oxidizing intermediates formed in the Fenton reaction, which we concluded consists of hydroxyl radicals and an iron species. The oxidizing species of iron which might be assigned as ferryl, FeO2+, or Fe(IV) = O was generated effectively in the presence of ADP even at low Fe2+ concentrations. In general, as the Fe2+ concentration was increased, the ferryl species predominated over the hydroxyl radical except for the case of Fe(II)-DETAPAC, which generated only hydroxyl radicals as the oxidizing species. Three possible pathways are proposed for the Fenton reaction, the dominant ones depending very much on the nature of the iron chelator being used.     

Electron self-exchange in Pseudomonas cytochromes

Electron self-exchange has been measured by an NMR technique for cytochromes c551 from Pseudomonas aeruginosa and Pseudomonas stutzeri. The rate for P. aeruginosa cyt c551 is 1.2 x 10(7) M-1 s-1 at 40 degrees C in 50 mM phosphate at pH 7. For P. stutzeri, under the same conditions, the rate is 4 x 10(7) M-1 s-1. For both cytochromes, the rate was independent of ionic strength up to 0.5 M in added NaC1, the enthalpy of activation was 20 +/- 4 kcal mol-1, and the entropy of activation was 38 +/- 10 cal mol-1 deg-1.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Secretion of Bacillus subtilis levansucrase. Fe(III) could act as a cofactor in an efficient coupling of the folding and translocation processes.

The refolding of levansucrase denatured by urea was studied as a possible model for the second step of the secretion pathway of this protein. The folding-unfolding transition was monitored by measuring intrinsic fluorescence and resistance to proteolysis. Both methods provided the same estimation for the unfolding free energy of levansucrase, delta GD, which was 30.1 +/- 1.7 kJ.mol-1 (7.2 +/- 0.4 kcal.mol-1) at pH 7 in 0.1 M-potassium phosphate buffer. The rate of refolding was greatly enhanced by Fe3+, whereas the Fe3+ chelator EDTA prevented correct refolding. Fe3+ allowed the protein to reach its folded form in medium in which the dielectric constant had been lowered by ethanol. The efficiency in vivo of the export of levansucrase bearing an amino acid modification which blocks the second step of the translocation pathway was greatly increased by high concentrations of Fe3+ in the culture medium. Assuming that the protein folding governs the second step of the secretion process of levansucrase, we discuss from an irreversible thermodynamic point of view the possible role of Fe3+ in the efficient coupling of the two events.     

Superoxide radical production during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium perchlorate.

1-Methyl-4-phenyl-2,3-dihydropyridinium perchlorate (MPDP+), an intermediate in the metabolism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was found to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected by their ability to reduce ferricytochrome c. Superoxide dismutase inhibited this reduction in a dose-dependent manner. The rate of reduction of ferricytochrome c was dependent not only on the concentration of MPDP+ but also on the pH of the system. Thus, the rate of autoxidation of MPDP+ and the sensitivity of this autoxidation to superoxide dismutase-inhibitable ferricytochrome c reduction were both augmented, as the pH was raised from 7.0 to 10.5. The rate constant (Kc) for the reaction of superoxide radical with ferricytochrome c to form ferricytochrome c was found to be 3.48 x 10(5) M-1 s-1. The rate constant (KMPDP+) for the reaction of MPDP+ with ferricytochrome3+ c was found to be only 4.86 M-1 s-1. These results, in conjunction with complexities in the kinetics, lead to the proposal that autoxidation of MPDP+ proceeds by at least two distinct pathways, one of which involves the production of superoxide radicals and hence is inhibitable by superoxide dismutase. It is possible that the free radicals so generated could induce oxidative injury which may be central to the MPTP/MPDP(+)-induced neuropathy.     

The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical

We have studied the time course of the absorption of bovine liver catalase after pulse radiolysis with oxygen saturation in the presence and absence of superoxide dismutase. In the absence of superoxide dismutase, catalase produced Compound I and another species. The formation of Compound I is due to the reaction of ferric catalase with hydrogen peroxide, which is generated by the disproportionation of the superoxide anion (O-2). The kinetic difference spectrum showed that the other species was neither Compound I nor II. In the presence of superoxide dismutase, the formation of this species was found to be inhibited, whereas that of Compound I was little affected. This suggests that this species is formed by the reaction of ferric catalase with O-2 and is probably the oxy form of this enzyme (Compound III). The rate constant for the reaction of O-2 and ferric catalase increased with a decrease in pH (cf. 4.5 X 10(4) M-1 s-1 at pH 9 and 4.6 X 10(6) M-1 s-1 at pH 5.). The pH dependence of the rate constant can be explained by assuming that HO2 reacts with this enzyme more rapidly than O-2.     

Chelated iron-catalyzed OH. formation from paraquat radicals and H2O2: mechanism of formate oxidation

Traces of iron, when complexed with either EDTA or diethylenetriaminepentaacetic acid (DTPA), catalyze an OH.-producing reaction between H2O2 and paraquat radical (PQ+.): H2O2 + PQ+.----PQ++ + OH. + OH-.[1]. Kinetic studies show that oxidation of formate induced by this reaction occurs by a Fenton-type mechanism, analagous to that assumed in the metal-catalyzed Haber-Weiss reaction, in which the rate determining step is H2O2 + Fe2+ (chelator)----Fe3+(chelator) + OH. + OH-,[7]; with k7 = 7 X 10(3) M-1 s-1 for EDTA and 8 X 10(2) M-1 s-1 for DTPA at pH 7.4. PQ+. rapidly reduces both Fe3+ (EDTA) and Fe3+ (DTPA), and hence allows both agents to catalyze [1] with comparable efficiency, in contrast to the much lower efficiency reported for the latter as a catalyst for the Haber-Weiss reaction. The catalytic properties of these chelating agents is attributed to their lowering of E0 (Fe3+/Fe2+) by 0.65 V, thus making [7] thermodynamically possible at pH 7. Approximately 2.5% of the OH. produced is consumed by internal or "cage" reactions, which decompose the chelator and produce CO2; however, the majority (97%) diffuses into the bulk solution and participates in competitive reactions with OH. scavengers.     

The influence of temperature and osmolyte on the catalytic cycle of cytochrome c oxidase.

The influence of temperature on cytochrome c oxidase (CCO) catalytic activity was studied in the temperature range 240-308 K. Temperatures below 273 K required the inclusion of the osmolyte ethylene glycol. For steady-state activity between 278 and 308 K the activation energy was 12 kcal x mol-1; the molecular activity or turnover number was 12 s-1 at 280 K in the absence of ethylene glycol. CCO activity was studied between 240 and 277 K in the presence of ethylene glycol. The activation energy was 30 kcal x mol-1; the molecular activity was 1 s-1 at 280 K. Ethylene glycol inhibits CCO by lowering the activity of water. The rate limitation in electron transfer (ET) was not associated with ET into the CCO as cytochrome a was predominantly reduced in the aerobic steady state. The activity of CCO in flash-induced oxidation experiments was studied in the low temperature range in the presence of ethylene glycol. Flash photolysis of the reduced CO complex in the presence of oxygen resulted in three discernable processes. At 273 K the rate constants were 1500 s-1, 150 s-1 and 30 s-1 and these dropped to 220 s-1, 27 s-1 and 3 s-1 at 240 K. The activation energies were 5 kcal.mol-1, 7 kcal.mol-1, and 8 kcal.mol-1, respectively. The fastest rate we ascribe to the oxidation of cytochrome a3, the intermediate rate to cytochrome a oxidation and the slowest rate to the re-reduction of cytochrome a followed by its oxidation. There are two comparisons that are important: (a). with vs. without ethylene glycol and (b). steady state vs. flash-induced oxidation. When one makes these two comparisons it is clear that the CCO only senses the presence of osmolyte during the reductive portion of the catalytic cycle. In the present work that would mean after a flash-induced oxidation and the start of the next reduction/oxidation cycle.     

Ferricytochrome c oxidation of cobaltocytochrome c. Comparison of experiments with electron-transfer theories.

Electron transfer from cobaltocytochrome c to ferricytochrome c has been studied by stopped-flow kinetics. The second-order rate constant at pH 7.0, 0.1 ionic strenght, 0.2 M phosphate, and 25 degrees C is 8.3 x 103 M-1 s-1. The activation parameters obtained from measurements made between 20 and 50 degrees C are deltaHnot equal to = 2.3 kcal mol-1 and deltaSnot equal to = -33 eu. The rate constant is not significantly dependent on ionic strength; it is also relatively independent of pH between the pK values for conformation transitions. The rate diminishes at pH greater than 12. The self-exchange reaction of cobalt cytochrome c was investigated with pulsed Fourier transform 1H NMR. The rate is too slow on the 1H NMR scale; it is estimated to be less than 133 M-1 s-1. These results together with the self-exchange rates of iron cytochrome c [Gupta, R.K., Koenig, S. H., and Redfield, A. G. (1972), J. Magn. Reson. 7, 66] were analyzed by theories of Jortner and Hopfield. The theories predict the self-exchange of Cocyt c to be too slow for 1H NMR determination. The rate constant calculated by the nonadiabatic multiphonon electron-tunneling theory for the Fecyt c-Fecyt c+ and Cocyt c-Fecyt c+ electron transfers are in good agreement with experiments.