Mechanism of the oxidation of 3,5,3',5'-tetramethylbenzidine by myeloperoxidase determined by transient- and steady-state kinetics

Earlier investigations of the oxidation of 3,5,3',5'-tetramethylbenzidine (TMB) using horseradish peroxidase and prostaglandin H-synthase have shown the formation of a cation free radical of TMB in equilibrium with a charge-transfer complex, consistent with either a two- or a one-electron initial oxidation. In this work, we exploited the distinct spectroscopic properties of myeloperoxidase and its oxidized intermediates, compounds I and II, to establish two successive one-electron oxidations of TMB. By employing stopped-flow techniques under transient-state and steady-state conditions, we also determined the rate constants for the elementary steps of the myeloperoxidase-catalyzed oxidation of TMB at pH 5.4 and 20 degrees C. The second-order rate constant for compound I formation from the reaction of native enzyme with H2O2 is 2.6 x 10(7) M-1 s-1. Compound I undergoes a one-electron reduction to compound II in the presence of TMB, and the rate constant for this reaction was determined to be (3.6 +/- 0.1) x 10(6) M-1 s-1. The spectral scans show that compound II accumulates in the steady state. The rate constant for compound II reduction to native enzyme by TMB obtained under steady-state conditions is (9.4 +/- 0.6) x 10(5) M-1 s-1. The results are applied to a new, more accurate assay for myeloperoxidase based upon the formation of the charge-transfer complex between TMB and its diimine final product.     

Mechanism of horseradish peroxidase catalyzed epinephrine oxidation: obligatory role of endogenous O2- and H2O2

Horseradish peroxidase (HRP) catalyzes cyanide sensitive oxidation of epinephrine to adrenochrome at physiological pH in the absence of added H2O2 with concurrent consumption of O2. Both adrenochrome formation and O2 consumption are significantly inhibited by catalase, indicating a peroxidative mechanism as a major part of oxidation due to intermediate formation of H2O2. Sensitivity to superoxide dismutase (SOD) also indicates involvement of O2- in the oxidation. Although SOD-mediated H2O2 formation should continue epinephrine oxidation through a peroxidative mechanism, low catalytic turnover, on the contrary, indicates that O2- takes part in a vital reaction to form an intermediate for adrenochrome formation and O2 consumption. Generation of O2- is evidenced by ferricytochrome c reduction sensitive to SOD. On addition of H2O2, both adrenochrome formation and O2 consumption are further increased due to reaction of molecular oxygen with some intermediate oxidation product. Peroxidative oxidation proceeds by one-electron transfer generating o-semiquinone and similar free radicals which when stabilized with Zn2+ or spin-trap, alpha-phenyl-tert-butylnitrone (PBN), inhibit adrenochrome formation and O2 consumption. The free radicals thus favor reduction of O2 rather than the disproportionation reaction. Spectral studies indicate that, during epinephrine oxidation in the presence of catalase, HRP remains in the ferric state absorbing at 403 nm. This suggests that HRP catalyzes epinephrine oxidation by its oxidase activity through Fe3+/Fe2+ shuttle consuming O2, where the rate of reduction of ferric HRP with epinephrine is slower than subsequent oxidation of ferrous HRP by O2 to form compound III. Compound III was not detected spectrally because of its quick reduction to the ferric state by epinephrine or its subsequent oxidation product. In the absence of catalase, peroxidative cycles predominate when HRP still remains in the ferric state through the transient formation of compounds I and II not detectable spectrally. Among various mono- and dihydroxyl aromatic donors tested, only epinephrine shows the oxidase reaction. Binding studies indicate that epinephrine interferes with the binding of CN-, SCN-, and guaiacol indicating that HRP preferentially binds epinephrine near the heme iron close to the anion or aromatic donor binding site to catalyze electron transfer for oxidation. HRP thus initiates epinephrine oxidation by its oxidase activity generating O2- and H2O2. Once H2O2 is generated, the peroxidative cycle continues with the consumption of O2, through the intermediate formation of O2- and H2O2 which play an obligatory role in subsequent cycles of peroxidation.     

Unsteady flow in a rigid 3-D model of the carotid artery bifurcation

Colominic acid (CA), an alpha-(2-->8) N-acetylneuraminic acid (sialic acid) polymer (average molecular weight of 10 kDa) was activated by periodate oxidation of carbon 7 at the non-reducing end of the saccharide. The oxidized CA was then coupled to catalase by reductive amination in the presence of sodium cyanoborohydride. The extent of sialylation of catalase, estimated by ammonium sulfate precipitation as 3.8+/-0.4 (mean+/-S.D.) moles of CA per mole of catalase, did not improve significantly when depolymerized CA was used in the coupling reaction. At the end of the coupling reaction, sialylated catalase exhibited a two-fold (70%) retention of initial activity compared to enzyme controls (29-35%) subjected to the same conditions. Formation of sialylated catalase was confirmed by ammonium sulfate or trichloroacetic acid precipitation, molecular sieve chromatography and SDS-PAGE electrophoresis. Enzyme kinetics studies revealed an increase in the apparent Km of the enzyme from 70.0 (native) to 122.9 mmol l-1 H2O2 (sialylated catalase) indicating a reduction of enzyme affinity for the substrate (hydrogen peroxide) on sialylation. Compared to native enzyme, sialylated catalase was much more stable in the presence of specific proteinases, completely resisting degradation by chymotrypsin and losing only some of its activity in the presence of trypsin. The increased stability conferred to catalase by sialylation agrees with similar observations on enzymes modified by other hydrophilic molecules (e.g., monomethoxypoly(ethyleneglycol)) and suggests that steric stabilization with the biodegradable polysialic acid may prove an alternative means to render therapeutic proteins more effective in vivo.     

Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-alpha

Mammalian tissues express three immunologically distinct peroxiredoxin (Prx) proteins (Prx I, II, and III), which are the products of distinct genes. With the use of recombinant proteins Prx I, II, and III, all have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents for the reduction of H2O2. Prx I and II are cytosolic proteins, whereas Prx III is localized in mitochondria. Transient overexpression of Prx I or II in cultured cells showed that they were able to eliminate the intracellular H2O2 generated in response to growth factors. Moreover, the activation of nuclear factor kappaB (NFkappaB) induced by extracellularly added H2O2 or tumor necrosis factor-alpha was blocked by overproduction of Prx II. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism. In addition, Prx I and II might participate in the signaling cascades of growth factors and tumor necrosis factor-alpha by regulating the intracellular concentration of H2O2.     

Regulation of E2F-1 gene expression in avian cells

Although recent evidence indicated that the production of reactive oxygen species (ROS) by human spermatozoa may be involved in the regulation of capacitation, very little is known about the role of ROS in the acrosome reaction. To address this issue, Percoll-washed spermatozoa were incubated in Ham's F-10 medium in the absence (no capacitation) or presence (capacitation) of fetal cord serum ultrafiltrate (FCSu) or progesterone. The effects of the ROS scavengers, superoxide dismutase (SOD), and catalase were then tested on the acrosome reaction induced by lysophosphatidylcholine (LPC), A23187, and ultrafiltrates from follicular fluid (FFu) and FCSu, as well as on the protein tyrosine phosphorylation associated with this process. 2-Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-a] pyrazin-3-one (MCLA)-amplified chemiluminescence was used to determine the extracellular superoxide (O2.-) production from spermatozoa. The observations that both SOD and catalase reduced (in the case of LPC) or totally prevented (in the other cases) the acrosome reaction of capacitated spermatozoa and that hydrogen peroxide (H2O2) or ROS generated by the combination of xanthine and xanthine oxidase (O2.-, which dismutates to H2O2) triggered the acrosome reaction indicated the involvement of ROS in this process. In fact, capacitated spermatozoa in which the acrosome reaction was induced by LPC, A23187, and FFu produced more O2.- than noncapacitated spermatozoa treated with the same agents. A23187 and LPC had minor effects on protein tyrosine phosphorylation of noncapacitated spermatozoa. However, these inducers caused a decrease in tyrosine phosphorylation of Triton-soluble proteins (mainly those of 37, 42, and 47 kDa) from capacitated spermatozoa, a decrease more pronounced in the presence of SOD. On the other hand, there was a marked increase in tyrosine phosphorylation of few proteins (70 to 105 kDa) from the Triton-insoluble fraction, which was partly reversed by SOD (in the case of LPC and A23187) or catalase (in the case of A23187), or abolished in the presence of the two antioxidants (in the case of A23187). These data indicate that the acrosome reaction is associated with an extracellular O2.- generation by spermatozoa and that both O2.- and H2O2 may be involved in the regulation of this process. The mechanism by which these ROS act is unknown but may involve tyrosine phosphorylation of sperm proteins.     

Investigation of the mechanism of non-turnover-dependent inactivation of purified human 5-lipoxygenase. Inactivation by H2O2 and inhibition by metal ions

Human 5-lipoxygenase is a non-heme iron protein which is reported to be highly unstable in the presence of oxygen. The results of this investigation demonstrate that H2O2 generated during air oxidation of thiols is the main factor in non-turnover-dependent inactivation of purified recombinant human 5-lipoxygenase for the following reasons: catalase protects against oxygen-dependent inactivation of the enzyme in the presence of dithiothreitol; the active, stable enzyme can be prepared under aerobic conditions with the exclusion of dithiothreitol and contaminating metal ions; 10 microM H2O2 causes the rapid inactivation of the enzyme. The native (ferrous) enzyme is approximately seven times more sensitive to inactivation by H2O2 than the ferric enzyme, suggesting that the mechanism of inactivation involves a Fenton-type reaction of the ferrous enzyme with H2O2, resulting in the formation of an activated oxygen species. Purification of 5-lipoxygenase under aerobic conditions (no dithiothreitol) results in an increase in both the specific activity of the purified protein [up to 70 mumol 5(S)-hydroperoxy-6-trans-8, 11, 14-cis-icosatetraenoic acid (5-HPETE)/mg protein] and in the ratio of specific activity to enzyme iron content compared to enzyme purified under anaerobic conditions in the presence of dithiothreitol. The reaction of the highly active 5-lipoxygenase enzyme shows a dependence on physiological intracellular calcium concentrations, half-maximal product formation being obtained at 0.9 microM free Ca2+. The maximal enzyme activity is also dependent on EDTA and dithiothreitol and low amounts of carrier protein, as well as the known activators PtdCho and ATP. Ca2+ can be substituted by Mn2+, Ba2+ and Sr2+, although lower levels of stimulation are obtained. 5-Lipoxygenase is strongly inhibited by low concentrations (< or = 10 microM) of Zn2+ and Cu2+. The inhibition by Cu2+ is apparently irreversible, whereas that by Zn2+ is slowly reversed (t1/2 = 2 min) in the presence of excess EDTA. These observations on the mechanism of non-turnover-dependent inactivation of 5-lipoxygenase, and the optimisation of assay conditions, have facilitated the purification of large quantities of relatively stable enzyme that will be useful for further kinetic and physical studies.     

Kinetic studies on the removal of extracellular hydrogen peroxide by cultured fibroblasts

To investigate the function of antioxidant enzymes in intact cells, we examined the removal of extracellular H2O2 by cultured fibroblasts (IMR-90). H2O2 concentration dependence of the reaction rate was interpreted as that the process involves two kinetically different reactions (referred to as reactions 1 and 2). Reaction 1 was characterized by a relatively low Km value (about 40 microM), and reaction 2 by linear dependence of the rate up to 500 microM H2O2. The magnitude of reaction 1 was reduced by treatment of the cells with diethyl maleate or 6-amino-nicotinamide, while reaction 2 was inhibited by 3-amino-1,2,4-triazole treatment. It was concluded that reactions 1 and 2 are principally due to GSH peroxidase and catalase, respectively. The values of kinetic parameters were estimated by curve-fitting, and it was inferred that 80 to 90% of H2O2 is decomposed by GSH peroxidase at H2O2 concentrations lower than 10 microM. The contribution of catalase increases with the increase in H2O2 concentration. The intact cells showed a low catalase activity (about 15%), as compared with the activity found in the solubilized cells. The low catalase activity was ascribed to the latency of the enzyme caused by localization in peroxisomes. Fibroblasts also removed intracellular H2O2 generated by menadione. Treatment with diethyl maleate greatly impaired the H2O2-removing capability and caused H2O2 efflux into the medium.     

Effect of heavy metal ions on the release of reactive oxygen intermediates by bovine alveolar macrophages

Short-term incubations of bovine alveolar macrophages (BAM) with metal-containing dusts induce the release of reactive oxygen intermediates (ROI). Incubations of BAM (90 min) with dissolved metal compounds (0.1-100 microM) combined with quartz dusts were performed to investigate the effects of single elements on BAM stimulation. As(III), as well as the calcium antagonists, Ni(II) and Ce(III), inhibited the secretion of superoxide anions (O2-) and hydrogen peroxide (H2O2). O2- concentrations were lowered by Mn(II) and Fe(II). Increased ROI concentrations were observed with V(IV) (O2- and H2O2) and Fe(III) (O2-). The addition of Cd(II), Cr(III) and V(V) showed no effect on the dust-induced respiratory burst. The influence of insoluble heavy metal compounds on ROI secretion by BAM were studied with metal oxide-coated silica particles. In most cases the release of ROI was not affected by the chemical modification of the particle surface. Coating with CuO markedly lowered the concentrations of O2- and H2O2, whereas vanadium(IV) oxide considerably increased both ROIs. Although most of the investigated metal compounds did not alter ROI secretion our present results with V(IV) and Fe(III) confirm our recent statistical evaluation of the effects of heavy metal-containing dusts on ROI secretion (Berg et al., 1993, J. Toxicol. Environ. Health 39, 341).     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-1-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (.OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the .OH generation. UV and O2 consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H2O2 did not generate any significant amount of .OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H2O2-->Co(II) + .OH + OH-] seems responsible for .OH generation. H2O2 is produced from O2.- via dismutation, O2.- is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or beta-ananyl-3-methyl-L-histidine alters, its oxidation-reduction potential and makes Co(II) capable of generating .OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H2O2-->Co(III) + .OH + OH-]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and beta-ananyl-3-methyl-L-histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Site-specific DNA damage induced by NADH in the presence of copper(II): role of active oxygen species

Oxidative DNA damage by NAD(P)H in the presence of metal ions has been characterized by using 32P 5' end-labeled DNA fragments obtained from human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. NADH, as well as other endogenous reductants, induced DNA damage in the presence of Cu(II). The order of inducing effect on Cu(II)-dependent DNA damage was ascorbate > reduced glutathione (GSH) > NADH > NADPH. Although NADH caused no or little DNA damage in the presence of Fe(III)-EDTA, the addition of H2O2 induced the DNA damage. The Cu(II)-mediated DNA damage induced by NADH was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator; but not by scavengers of hydroxyl free radical (.OH), suggesting the involvement of active species derived from hydrogen peroxide (H2O2) and Cu(I) rather than .OH. The predominant cleavage sites were thymine residues located 5' and/or 3' to guanine. The cleavage pattern was similar to that induced by Cu(II) plus GSH, Cu(II) plus ascorbate, or Cu(I) plus H2O2. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by NADH increased with its concentration in the presence of Cu(II). UV-visible spectroscopy indicated the facilitation of reduction of Cu(II) by NADH under some conditions. ESR spin-trapping experiments and mass spectrometry showed that the carbon-centered radical was formed during the reaction of NADH with Cu(II). These results suggest that optimal molar ratios of DNA/metal ion yield copper with a high redox potential which catalyzes NADH autoxidation to NAD. being further oxidized to NAD+ with generation of superoxide radical and that H2O2 reacts with Cu(I) to form active oxygen species such as copper(I)-peroxide complex causing DNA damage.     

Oxidation of phenolic compounds by lactoperoxidase. Evidence for the presence of a low-potential compound II during catalytic turnover

The lactoperoxidase (LPO)-catalyzed oxidation of p-phenols by hydrogen peroxide has been studied. The behavior of the enzyme differs from that of other peroxidases in this reaction. In particular LPO shows several catalytic intermediates during the catalytic cycle because of its capability to delocalize an oxidizing equivalent on a protein amino acid residue. In the phenol oxidation the enzyme Compound I species, containing an iron-oxo and a protein radical, uses the iron-oxo group at acidic pH and the protein radical in neutral or basic medium. Kinetic and spectroscopic studies indicate that the ionization state of an amino acid residue with pKa 5.8 +/- 0.2, probably the distal histidine, controls the enzyme intermediate forms at different pH. LPO undergoes inactivation during the oxidation of phenols. The inactivation is reversible and depends on the easy formation of Compound III even at low oxidant concentration. The inactivation is due to the substrate redox potential since the best substrate is that with lowest redox potential, while the worst substrate has the highest potential. This strongly indicates that Compound II, formed during catalytic turnover, has a low redox potential, making easier its oxidation by hydrogen peroxide to Compound III. The dependence of LPO activity on the phenols redox potential suggests that the protein radical where an oxidizing equivalent can be localized is a tyrosyl residue.     

Hydrogen peroxide production by monoamine oxidase during ischemia-reperfusion in the rat brain

Monoamine oxidase (MAO) as a source of hydrogen peroxide (H2O2) was evaluated during ischemia-reperfusion in vivo in the rat brain. H2O2 production was assessed with and without inhibition of MAO during and after 15 min of ischemia. Metabolism of H2O2 by catalase during ischemia and reperfusion was measured in forebrain homogenates using aminotriazole (ATZ), an irreversible H2O2-dependent inhibitor of catalase. Catecholamine and glutathione concentrations in forebrain were measured with and without MAO inhibitors. During ischemia, forebrain blood flow was reduced to 8% of baseline and H2O2 production decreased as measured at the microperoxisome. During reperfusion, a rapid increase in H2O2 generation occurred within 5 min as measured by a threefold increase in oxidized glutathione (GSSG). The H2O2-dependent rates of ATZ inactivation of catalase between control and ischemia-reperfusion were similar, indicating that H2O2 was more available to glutathione peroxidase than to catalase in this model. MAO inhibitors eliminated the biochemical indications of increased H2O2 production and increased the catecholamine concentrations. Mortality was 67% at 48 h after ischemia-reperfusion, and there was no improvement in survival after inhibition of MAO. We conclude that MAO is an important source of H2O2 generation early in brain reperfusion, but inhibition of the enzyme does not improve survival in this model despite ablating H2O2 production.     

Phenylhydrazones as new good substrates for the dioxygenase and peroxidase reactions of prostaglandin synthase: formation of iron(III)-sigma-phenyl complexes

Phenylhydrazones of various aromatic and aliphatic aldehydes or ketones act as good substrates of the dioxygenase reaction of prostaglandin synthase (PGHS). Corresponding alpha-azo hydroperoxides are formed as intermediates with maximum initial rates of O2 consumption between 8 and 230 mol (mol of PGHS)-1 s-1 for benzophenone and hexanal phenylhydrazone, respectively. The Km values for these reactions vary from 100 to 300 microM. These alpha-azo hydroperoxides are then converted to the corresponding alpha-azo alcohols by the peroxidase reaction of PGHS. During such oxidations of phenylhydrazones by PGHS, a new complex of this hemeprotein characterized by peaks at 438 and 556 nm is formed. This complex was obtained both by direct reaction of PGHS Fe(III) with phenyldiazene and by reaction of PGHS Fe(III) with phenylhydrazine in the presence of O2. By analogy to results previously reported for hemoglobin, myoglobin, catalase, and cytochrome P450, this species should be a sigma-phenyl PGHS FeIII-Ph complex. The PGHS FeIII-Ph complex should derive from an oxidation of the intermediate alpha-azo alcohol by PGHS Fe(III), cleavage of the resulting alkoxy radical with formation of a ketone (or aldehyde) and Ph*, and combination of PGHS Fe(II) with Ph*. Such an oxidation of alpha-azo alcohols by lipoxygenase-FeIII with formation of Ph* was reported previously. The formation of Ph* and of PGHS FeIII-Ph is likely the cause of the inhibitory effects previously reported for arylhydrazones toward PGHS.     

Effect of radical scavengers on the inactivation of papain by ascorbic acid in the presence of cupric ions

Incubation of papain (EC 3.4.22.2) with ascorbic acid (AsA) and Cu2+ in acetate buffer (pH 5.6) results in an irreversible loss of enzyme activity by site-specific generation of free radicals [H. Kanazawa, S. Fujimoto, A. Ohara, Biol. Pharm.Bull., 16, 11 (1993)]. In this study, the effect of some compounds, known free radical scavengers, on the relationship between the inactivation of papain by the Cu(2+)-AsA system and the oxidation of AsA was investigated. Catalase completely protected the enzyme from inactivation by the Cu(2+)-AsA system, although hydrogen peroxide (H2O2) by itself, known to be generated during the autoxidation of AsA, did not inactivate the enzyme. The oxidation of AsA was unaffected by catalase. Both thiourea and sodium thiocyanate completely protected the enzyme from inactivation, while AsA was partially oxidized only in the initial stage. In the presence of potassium iodide, both the inactivation of the enzyme and the oxidation of AsA were characterized by a rapid initial phase followed by a stable phase where no reaction took place and, subsequently, a slower phase. Histidine partially prevented the inactivation of the enzyme and the oxidation of AsA. The present results suggest that H2O2 serves as a source of secondary, highly reactive species, probably hydroxyl radicals, which are responsible for the inactivation, and that the protection from inactivation by some radical scavengers, such as thiourea, sodium thiocyanate, potassium iodide, and histidine, is based on the removal of metal ions (Cu2+ or Cu+) at the specific site of inactivation.     

A GTP-binding protein inhibits a gastric housekeeping chloride channel via intracellular production of superoxide

A housekeeping basolateral Cl- channel of rabbit gastric parietal cells, the single channel conductance of which is about 0.3 picosiemens, is opened by prostaglandin E2 and closed by intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S). In the present patch clamp study, we found a novel GTP gamma S-dependent regulatory mechanism of the Cl- channel. GTP gamma S significantly decreased the open probability of the single Cl- channel without altering unit conductance. An intracellular application of superoxide dismutase (SOD; 100 units/ml) inhibited the GTP gamma S (50 microM)-induced closure of the Cl- channel. SOD plus catalase (100 units/ml) also inhibited the GTP gamma S-induced effect, while catalase alone did not inhibit it. In the absence of GTP gamma S, an intracellular application of hydrogen peroxide (H2O2; 30 microM) did not affect the Cl- channel current. Desferrioxamine (50 microM) which inhibits hydroxyl radical (.OH) production was without effect on the GTP gamma S-induced closure. These results suggest that the GTP gamma S-induced closure of the Cl- channel was due to intracellular production of superoxide (O2.-), but not due to .OH or H2O2. Furthermore, an artificial production of O2.- inside the cell by lumazine (50-100 microM) plus xanthine oxidase (0.5-1 milliunit/ml) in the absence of GTP gamma S also closed the channel. The lumazine/xanthine oxidase-induced closure of the channel was inhibited by SOD, but not by catalase or desferrioxamine. We conclude from these results that GTP-binding protein-coupled production of O2.- leads to closure of the Cl- channel in rabbit gastric parietal cells.     

Catalase-free photosystem II: the O2-evolving complex does not dismutate hydrogen peroxide

A photosystem II (PSII) membrane-associated heme catalase has been identified as a major source of the dark H2O2-dismutation reaction in PSII membrane samples [Sheptovitsky, Y. G., and Brudvig, G. W. (1996) Biochemistry 35, 16255-16263]. Based on this finding, a catalase-free PSII membrane sample was prepared by using mild heat treatment to deplete most of the PSII membrane-associated heme catalase followed by inhibition of the residual catalase with 50 mM 3-amino-1,2,4-triazole, a specific heme catalase inhibitor that binds covalently to compound I. After these treatments, the PSII membrane sample exhibited only 0.02% of the original H2O2-dismutation activity when assayed in the presence of 20 mM 3-amino-1,2,4-triazole. This small residual H2O2-dismutation activity is attributed to adventitious metal ions or the non-heme iron in PSII because the activity was still present in a Mn-depleted PSII sample but was completely suppressed by adding 5 mM ferricyanide to the assay buffer; the effect of ferricyanide is attributed to oxidation of H2O2-dismutating cations. Although the H2O2-dismutation activity was completely eliminated by these treatments, the light-induced O2-evolution activity was retained. A single saturating flash given to catalase-free PSII membranes did not induce any H2O2-dismutation activity. These results demonstrate that the S1/S-1 and S2/S0 cycles of the O2-evolving complex of PSII do not occur in the presence of H2O2, as proposed by Velthuys, B., and Kok, B. [(1978) Biochim. Biophys. Acta 502, 211-221]. The light-induced O2-evolution activity in catalase-free PSII was found to be irreversibly impaired by micromolar concentrations of H2O2. Thus, it is possible that the PSII membrane-associated heme catalase plays an important role in protection of the O2-evolving complex from damage by H2O2.     

Zn-exchange and M?ssbauer studies on the [Fe-Fe] derivatives of the purple acid Fe(III)-Zn(II)-phosphatase from kidney beans

In order to perform M?ssbauer studies, Zn(II) in the Fe(III)-Zn(II) purple acid phosphatase of the red kidney bean has been exchanged by incubating the semiapoenzyme with 57Fe(II). The resulting Fe(III)-57Fe(II) enzyme has 125% activity, compared with that of the Zn(II) enzyme. It can be oxidized by H2O2 or peroxydisulfate to the Fe(III)-57Fe(III) species with a 30-times lower activity. Incubation of the metal-free apoenzyme with 57Fe(II) in the presence of O2 leads to the 57Fe(III)-57Fe(II) species which is stable in dilute solutions, but partially oxidized during the concentration procedure to the 57Fe(III)-57Fe(III) enzyme. Limited reduction of the oxidized enzyme with ascorbate delivers a mixture of the Fe(II)-Fe(II)/Fe(III)-Fe(III) species, but not the mixed valent Fe(III)-Fe(II) species, indicating that after the transfer of the first electron the second electron of the ascorbate radical is immediately transferred to the second Fe(III). The M?ssbauer spectra of the oxidized species show at 4.2 K two quadrupole doublets with delta of 0.51 mm/s and 0.53 mm/s and delta E of 1.46 and 0.96 mm/s indicating high spin Fe(III) in two different binding sites, obviously with a higher asymmetry in the chromophoric Fe(III) site. The values are too low for a mu-oxo bridge. The mixed-valent Fe(III)-Fe(II) species shows two quadrupole doublets with delta values of 0.55 mm/s and 1.14 mm/s and delta E values of 1.43 mm/s and 3.01 mm/s at 70 K for high spin Fe(II) and Fe(III), but the signal of the Fe(II) component shows magnetic patterns at 4.2 K indicating a half-integer spin system with antiferromagnetic coupling. The Fe(II)-Fe(II) system exhibits two quadrupole doublets with delta values of 1.18 mm/s and 1.22 mm/s and with delta E values of 3.69 mm/s and 2.68 mm/s again indicating a higher asymmetry in the originally chromophoric Fe(III)-binding site. Addition of phosphate shows only minor differences in the oxidized enzyme and in the mixed valent Fe(III)-Fe(II) system. Interaction with O2 is discussed.     

Salicylic acid is a modulator of tobacco and mammalian catalases

Salicylic acid (SA) plays a key role in the establishment of resistance to microbial pathogens in many plants. The discovery that SA inhibits catalase from tobacco led us to suggest that H2O2 acts as second messenger to activate plant defenses. Detailed analyses of SA's interaction with tobacco and mammalian catalases indicate that SA acts as an electron donor for the peroxidative cycle of catalase. When H2O2 fluxes were relatively low (1 microM/min or less), SA inhibited catalase, consistent with its suggested signaling function via H2O2. However, significant inhibition was only observed at 100 microM SA or more, a level reached in infected, but not in uninfected, leaves. This inhibition was probably due to siphoning catalase into the slow peroxidative reaction. Surprisingly, SA was also able to protect catalase from inactivation by damaging levels of H2O2 (lower millimolar range), which is generally assumed to reflect accumulation of inactive ferro-oxy intermediates. SA did so by supporting or substituting for the protective function of catalase-bound NADPH. These results add new features to SA's interaction with heme enzymes and its in vivo redox properties. Thus, SA, in addition to its proposed signaling function, may also have an important antioxidant role in containing oxidative processes associated with plant defense responses.     

Prostate cancer in the African American: is this a different disease?

Reactive oxygen species such as superoxides, hydrogen peroxide (H2O2) and hydroxyl radicals have been suggested to be involved in the catalytic action of nitric oxide synthase (NOS) to produce NO from L-arginine. An examination was conducted on the effects of oxygen radical scavengers and oxygen radical-generating systems on the activity of neuronal NOS and guanylate cyclase (GC) in rat brains and NOS from the activated murine macrophage cell line J774. Catalase and superoxide dismutase (SOD) showed no significant effects on NOS or GC activity. Nitroblue tetrazolium (NBT, known as a superoxide radical scavenger) and peroxidase (POD) inhibited NOS, but their inhibitory actions were removed by increasing the concentration of arginine or NADPH respectively, in the reaction mixture. NOS and NO-dependent GC were inactivated by ascorbate/FeSO4 (a metal-catalyzed oxidation system), 2'2'-azobis-amidinopropane (a peroxy radical producer), and xanthine/xanthine oxidase (a superoxide generating system). The effects of oxygen radicals or antioxidants on the two isoforms of NOS were almost similar. However, H2O2 activated GC in a dose-dependent manner from 100 microM to 1 mM without significant effects on NOS. H2O2-induced GC activation was blocked by catalase. These results suggested that oxygen radicals inhibited NOS and GC, but H2O2 could activate GC directly.     

Heme oxygenase-1, intermediates in verdoheme formation and the requirement for reduction equivalents

Conversion of heme to verdoheme by heme oxygenase-1 (HO-1) is thought to involve alpha-meso-hydroxylation and elimination of the meso-carbon as CO, a reaction supported by both H2O2 and NADPH-cytochrome P450 reductase/O2. Anaerobic reaction of the heme-HO-1 complex with 1 eq of H2O2 produces an enzyme-bound intermediate identified by spectroscopic methods as alpha-meso-hydroxyheme. This is the first direct evidence for HO-1-catalyzed formation of alpha-meso-hydroxyheme. alpha-meso-Hydroxyheme exists as a mixture of Fe(III) phenolate, Fe(III) keto anion, and Fe(II) keto pi neutral radical resonance structures. EPR shows that complexation with CO enhances the Fe(II) pi neutral radical component. Reaction of the alpha-meso-hydroxyheme-HO-1 complex with O2 generates Fe(III) verdoheme, which can be reduced in the presence of CO to the Fe(II) verdoheme-CO complex. Thus, conversion of alpha-meso-hydroxyheme to Fe(III) verdoheme, in contrast to a previous report (Matera, K. M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and Ikeda-Saito, M. (1996) J. Biol. Chem. 271, 6618-6624), does not require a reducing equivalent. An electron is only required to reduce ferric to ferrous verdoheme in the first step of its conversion to biliverdin.