Superoxide peroxidase activity of ovoperoxidase, the cross-linking enzyme of fertilization

Ovoperoxidase, an enzyme secreted by the eggs of the sea urchin Stronglycocentrotus purpuratus upon activation, catalyzes the formation of dityrosine residues in the fertilization envelope. This cross-linking reaction requires extracellular H2O2, which is produced by the egg during the cyanide-insensitive "respiratory burst" of fertilization. While investigating the possibility that the sea urchin oxidase might generate O2- as a precursor to H2O2, we discovered that ovoperoxidase possessed O2- degrading activity. Ovoperoxidase catalyzed the breakdown of O2- in a reaction that was sensitive to inhibition by catalase, indicating a requirement for H2O2. High concentrations of either O2- or H2O2 inhibited the O2- degrading activity of ovoperoxidase, as did the peroxidase inhibitors aminotriazole, azide, and phenylhydrazine. When ovoperoxidase was heated at 56 degrees C, it lost O2- degrading activity in parallel with peroxidase activity. In contrast, the copper-chelating agent diethyldithiocarbamate, which completely inactivated CuZn superoxide dismutase, failed to affect ovoperoxidase. The requirement for H2O2 and the inhibition by aminotriazole, azide, and phenylhydrazine support the hypothesis that ovoperoxidase catalyzes the breakdown of O2- by a peroxidative mechanism. Ovoperoxidase may play a role in protecting the developing embryo from oxidants derived from O2-.     

The mode of decomposition of Angeli's salt (Na2N2O3) and the effects thereon of oxygen,nitrite, superoxide dismutase,and glutathione

The classical view of the aerobic decomposition of Angeli's salt is that it releases NO(2)(-) + NO(-)/HNO the latter then reacting with O(2) to yield ONOO(-). An alternative that has recently been proposed envisions electron transfer to O(2) followed by decomposition to NO(2)(-) + NO. The classical view is now strongly supported by the observation that the rates of decomposition of Angeli's salt under 20% O(2) or 100% O(2) were equal. Moreover, NO(2)(-), which inhibits this decomposition by favoring the back reaction, was more effective in the absence of agents that scavenge NO(-)/HNO. It is thus clear that Angeli's salt is a useful source of NO(-)/HNO for use in defined aqueous systems. The measurements made in the course of this work allowed approximation of the rate constants for the reactions of NO(-)/HNO with NO(2)(-), O(2), glutathione, or Cu, Zn superoxide dismutase. The likelihood of the formation of NO(-)/HNO in vivo is also discussed.     

Effect of cisplatin, lipopolysaccharide, muramyl dipeptide and recombinant interferon-gamma on murine macrophages in vitro. II. Production of H2O2, O2- and interleukin-1

Murine macrophage monolayers treated with cisplatin, lipopolysaccharide (LPS), muramyl dipeptide (MDP) or recombinant interferon-gamma (rIFN gamma) for 2-48 h showed significant increases in the release of H2O2, O2- and interleukin-1 (IL-1) as compared to untreated macrophages. The treatment of macrophages with different combinations of the above agents did not induce synergistic or additive effects on the production of H2O2, O2- and IL-1. The priming of macrophages with rIFN gamma had a significant effect in the additional increased production of H2O2, O2- and IL-1 when subsequently treated with cisplatin, LPS or MDP.     

Respiration in the burrowing brittlestar, Hemipholis elongata say (Echinodermata, Ophiuroidea): a study of the effects of environmental variables on oxygen uptake

The burrowing brittlestar Hemipholis elongata (Say) maintains a constant M(O2)of 3.79+/-1.47 micromol O(2) g(-1) h(-1) (for 0.2-0.3 g animals, mean+/-S.D., n=7), measured in the burrow, over a broad range of PO(2). Below the critical PO(2) of 37 mm Hg, M(O(2)) becomes dependent on the oxygen tension. M(O2) is a function of the size of H. elongata; the scaling exponent is 0. 83 and is similar to those reported for other echinoderms. The M(O2) of H. elongata is unaffected by removal from the burrow, by hypercapnia, by exposure to hydrogen sulfide, or by temperature change in the range from 20 to 32 degrees C. The relative insensitivity of H. elongata to these factors may be an adaptation to life in the highly variable estuarine and tidal creek environments where the animals are frequently found.     

Dose-dependent regulation of superoxide anion on the proliferation, differentiation, apoptosis and necrosis of human hepatoma cells: the role of intracellular Ca 2+

Dose-dependent regulation of cellular processes is one important characteristic of signaling molecules. Although recent studies suggest that reactive oxygen species (ROS) may act as in vivo signaling molecules, the dose-dependent regulation of ROS on cellular processes together in one system needs to be evaluated. After treating cells with gradually increased O(2)(-), generated by the hypoxanthine-xanthine oxidase system, it was found that: (i) the proliferation of hepatoma cells firstly increased at 1-2 microM O(2)(-), then decreased markedly as the concentration increased; (2) at 8 or 16 microM O(2)(-), re-differentiation of hepatoma cells was induced, as indicated by the indices relating to cell malignancy or differentiation, such as cell surface charge, alpha-fetoprotein, gamma-glutamyltranspeptidase, tyrosine-alpha-ketoglutarate transaminase, cAMP, and the tumor's clonogenic potential; (iii) at 16 microM O(2)(-), accompanied by the re-differentiation of cells, cell apoptosis was also simultaneously induced as indicated by the appearance of apoptotic bodies, detached cells, and other apoptotic morphological features, as well as specific DNA fragmentation; (iv) at the highest concentration of O(2)(-) (32 microM) in this study, cell necrosis was dramatically induced as shown by Trypan blue exclusion; (v), an increase of intracellular Ca(2+) ([Ca(2+)](i)) was observed at all concentrations of O(2)(-) treatment, and this [Ca(2+)](i) increase was found to be involved in the regulation of O(2)(-) on the cellular processes. In conclusion, these results indicate that O(2)(-) could dose-dependently regulate the processes of cells, where Ca(2+) is one of its molecular targets, and hence provide a direct support for the hypothesis that ROS themselves are important signaling molecules.     

Peroxynitrite targets the epidermal growth factor receptor, Raf-1, and MEK independently to activate MAPK

Activation of ERK-1 and -2 by H(2)O(2) in a variety of cell types requires epidermal growth factor receptor (EGFR) phosphorylation. In this study, we investigated the activation of ERK by ONOO(-) in cultured rat lung myofibroblasts. Western blot analysis using anti-phospho-ERK antibodies along with an ERK kinase assay using the phosphorylated heat- and acid-stable protein (PHAS-1) substrate demonstrated that ERK activation peaked within 15 min after ONOO(-) treatment and was maximally activated with 100 micrometer ONOO(-). Activation of ERK by ONOO(-) and H(2)O(2) was blocked by the antioxidant N-acetyl-l-cysteine. Catalase blocked ERK activation by H(2)O(2), but not by ONOO(-), demonstrating that the effect of ONOO(-) was not due to the generation of H(2)O(2). Both H(2)O(2) and ONOO(-) induced phosphorylation of EGFR in Western blot experiments using an anti-phospho-EGFR antibody. However, the EGFR tyrosine kinase inhibitor AG1478 abolished ERK activation by H(2)O(2), but not by ONOO(-). Both H(2)O(2) and ONOO(-) activated Raf-1. However, the Raf inhibitor forskolin blocked ERK activation by H(2)O(2), but not by ONOO(-). The MEK inhibitor PD98059 inhibited ERK activation by both H(2)O(2) and ONOO(-). Moreover, ONOO(-) or H(2)O(2) caused a cytotoxic response of myofibroblasts that was prevented by preincubation with PD98059. In a cell-free kinase assay, ONOO(-) (but not H(2)O(2)) induced autophosphorylation and nitration of a glutathione S-transferase-MEK-1 fusion protein. Collectively, these data indicate that ONOO(-) activates EGFR and Raf-1, but these signaling intermediates are not required for ONOO(-)-induced ERK activation. However, MEK-1 activation is required for ONOO(-)-induced ERK activation in myofibroblasts. In contrast, H(2)O(2)-induced ERK activation is dependent on EGFR activation, which then leads to downstream Raf-1 and MEK-1 activation.     

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.     

A superoxide anion generator, pyrogallol induces apoptosis in As4.1 cells through the depletion of intracellular GSH content

We investigated the involvement of ROS such as H2O2 and O2*-, and GSH in As4.1 cell death induced by pyrogallol. The intracellular H2O2 levels were decreased or increased depending on the concentration and incubation time of pyrogallol. The levels of O2*- were significantly increased. Pyrogallol reduced the intracellular GSH content. And ROS scavengers, Tempol, Tiron, Trimetazidine and NAC could not significantly down-regulate the production of H2O2 and O2*-. However, these ROS scavengers slightly inhibited apoptosis. Interestingly, Tempol showing the recovery of GSH depletion induced by pyrogallol significantly decreased apoptosis without the significant reduction of intracellular O2*- levels. SOD and catalase did not change the level of H2O2 but decreased the level of O2*-. The inhibition of GSH depletion by these was accompanied with the decrease of apoptosis, as evidenced by sub-G1 DNA content, annexin V staining, mitochondria membrane potential (DeltaPsi(m)) and Western data. In addition, ROS scavengers and SOD did not alter a G2 phase accumulation of the cell cycle induced by pyrogallol. However, catalase changed the cell cycle distributions of pyrogallol-treated cells to those of pyrogallol-untreated cells. In summary, we have demonstrated that pyrogallol potently generates ROS, especially O2*-, in As4.1 JG cells, and Tempol, SOD and catalase could rescue to a lesser or greater extent cells from pyrogallol-induced apoptosis through the up-regulation of intracellular GSH content.     

Polymorphonuclear leukocyte-mediated, antibody-dependent, cellular cytotoxicity against tumor cells: dependence on oxygen and the respiratory burst.

Experiments were done to determine 1) whether the respiratory burst of superoxide anion (O2-) production in polymorphonuclear leukocytes (PMN) is triggered during antibody-dependent killing of tumor cells and 2) whether O2- production is essential for cytotoxicity. Three parameters of the respiratory burst (1-14C-glucose oxidation, oxygen consumption, and O2- release) were increased 2.5- to 7.3-fold during killing of antibody-primed tumor cells by human PMN. Added catalase and superoxide dismutase did not inhibit lysis, possibly because these enzymes were unable to diffuse into the inter-plasma-membrane space between killer and target cells. Evidence for an O2- requirement for cytotoxicity was the fact that concentrations of amobarbital or phenylbutazone sufficient to inhibit the cyanide-insensitive respiration of PMN also inhibited cytotoxicity. Also, hypoxic conditions inhibited cytotoxicity from 29 to 73%. The requirement for oxygen was most likely related to O2- generation and not mitochondrial respiration since cyanide and azide, which inhibit mitochondrial respiration, increased cytotoxicity.     

Unique oxidative mechanisms for the reactive nitrogen oxide species, nitroxyl anion

The nitroxyl anion (NO-) is a highly reactive molecule that may be involved in pathophysiological actions associated with increased formation of reactive nitrogen oxide species. Angeli's salt (Na2N2O3; AS) is a NO- donor that has been shown to exert marked cytotoxicity. However, its decomposition intermediates have not been well characterized. In this study, the chemical reactivity of AS was examined and compared with that of peroxynitrite (ONOO-) and NO/N2O3. Under aerobic conditions, AS and ONOO- exhibited similar and considerably higher affinities for dihydrorhodamine (DHR) than NO/N2O3. Quenching of DHR oxidation by azide and nitrosation of diaminonaphthalene were exclusively observed with NO/N2O3. Additional comparison of ONOO- and AS chemistry demonstrated that ONOO- was a far more potent one-electron oxidant and nitrating agent of hydroxyphenylacetic acid than was AS. However, AS was more effective at hydroxylating benzoic acid than was ONOO-. Taken together, these data indicate that neither NO/N2O3 nor ONOO- is an intermediate of AS decomposition. Evaluation of the stoichiometry of AS decomposition and O2 consumption revealed a 1:1 molar ratio. Indeed, oxidation of DHR mediated by AS proved to be oxygen-dependent. Analysis of the end products of AS decomposition demonstrated formation of NO2- and NO3- in approximately stoichiometric ratios. Several mechanisms are proposed for O2 adduct formation followed by decomposition to NO3- or by oxidation of an HN2O3- molecule to form NO2-. Given that the cytotoxicity of AS is far greater than that of either NO/N2O3 or NO + O2, this study provides important new insights into the implications of the potential endogenous formation of NO- under inflammatory conditions in vivo.     

On the role of bicarbonate in peroxidations catalyzed by Cu,Zn superoxide dismutase

The interaction of Cu,ZnSOD with H2O2 generates an oxidant at the active site that can then cause either the inactivation of this enzyme or the oxidation of a variety of exogenous substrates. We show that the rate of inactivation, imposed by 10-mM H2O2 at 25 degrees C and pH 7.2, is not influenced by 10-mM HCO3-; whereas the oxidation of 2,2'-azino-bis-[3-ethylbenzothiazoline sulfonate] (ABTS=) is virtually completely dependent upon HCO3-. The reduction of the active site Cu(II) by H2O2, which precedes inactivation of the enzyme, occurred at the same rate in phosphate buffer with or without bicarbonate added. These results indicate that HCO3- does not play a role in facilitating the interaction of H2O2 with the active site copper, but they can be accommodated by the proposal that HCO3- is oxidized to HCO3*, which then diffuses from that site and causes the oxidation of substrates, such as ABTS=, that are too large to traverse the solvent access channel to the Cu(II).     

Reactive oxygen species and oocyte aging: role of superoxide, hydrogen peroxide, and hypochlorous acid

Aging of the unfertilized oocyte inevitably occurs following ovulation, limiting its fertilizable life span. However, the mechanisms that regulate oocyte aging are still unclear. We hypothesize that reactive oxygen species such as superoxide (O2-), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) are likely candidates that may initiate these changes in the oocyte. In order to test this hypothesis, we investigated direct effects of O2- [hypoxanthine/xanthine oxidase system generating 0.12 (n=42) and 0.25 (n=45) microM O2-/min], H2O2 (20 or 100 microM, n=60), and HOCl, (1, 10, and 100 microM, n=50) on freshly ovulated or relatively old mouse oocytes, while their sibling oocytes were fixed immediately or cultured under physiological conditions (n=96). The aging process was assessed by the zona pellucida dissolution time (ZPDT), ooplasm microtubule dynamics (OMD), and cortical granule (CG) status. The ZPDT increased 2-fold in relatively old, compared to young, untreated oocytes (P<0.0001). Exposure to O2- increased it even further (P<0.0001). Similarly, more O2- exposed oocytes exhibited increased OMD and major CG loss, with fewer having normal OMD and intact CG compared to untreated controls. Interestingly, young oocytes resisted "aging," when exposed to 20 microM H2O2, while the same enhanced the aging phenomena in relatively old oocytes (P<0.05). Exposure to even very low levels of HOCl induced the aging phenomena in young and relatively old oocytes, and higher concentrations of HOCl compromised oocyte viability. Overall, O2-, H2O2, and HOCl each augment oocyte aging, more so in relatively old oocytes, suggesting compromised antioxidant capacity in aging oocytes.     

Metabolism of [17-2H]pregnenolone into 5-[17 beta-2H, 17 alpha-18O]androstene-3 beta, 17 alpha-diol and other products by incubation with the microsomal fraction of boar testis under 18O2 atmosphere.

[17-2H]Pregnenolone was incubated with the microsomal fraction of boar testis under an 18O2 atmosphere. The metabolites were analyzed by gas chromatography-mass spectrometry, and the following six metabolites labeled with 2H or 18O (or both) were identified: 17 alpha-[17-18O]hydroxypregnenolone, [17-18O]dehydroepiandrosterone, 5-[17-18O]androstene-3 beta, 17 beta-diol, 16 alpha-[16-18O]hydroxy[17-2H]pregnenolone, 5-[17 beta-2H, 17-18O]androstene-3 beta,17 alpha-diol, and 5,16-[17-2H]androstadien-3 beta-ol. The time course of the formation of these metabolites from pregnenolone was also studied using 14C-labeled substrate. The results obtained from these experiments suggest that the first three metabolites were synthesized by a well-documented pathway--pregnenolone yields 17 alpha-hydroxypregnenolone yields dehydroepiandrosterone yields 5-androstene-3 beta,17 beta-diol--, and that 16 alpha-hydroxypregnenolone, 5-androstene-3 beta,17 alpha-diol and 5,16-androstadien-3 beta-ol were synthesized from [17-2H]pregnenolone with retention of 17-2H.     

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP

The cooperative O(2)-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O(2)-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O(2)-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O(2)-affinities (K(T)=0.0085mmHg(-1) and K(R)=0.011mmHg(-1)) and thus a very low cooperativity (K(R)/K(T)=1.3 and L(0)=2.4). (1)H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O(2)-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, (1)H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14ppm, originated from inter- dimeric α(1)β(2)- (or α(2)β(1)-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15-25ppm, caused by high-spin heme-Fe(II)s. Upon addition of O(2), the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O(2), but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O(2) (P(O2)) of 760mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P(O2) of 760mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α(1)G- and β(1)G-helices, and the other weaker binding site causes the R→T transition without release of O(2). The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α(1)β(1) (or α(2)β(2)) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α(1)α(2)- (or β(1)β(2)-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O(2)-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb - [the deoxy-state]=[the T-quaternary structure]=[the low O(2)-affinity state] and [the oxy-state]=[the R-quaternary structure]=[the high O(2)-affinity state] and the O(2)-affiny of Hb being regulated by the T/R-quaternary structural transition - are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.     

Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4

The slow-reacting substance (SRS) bioactivity of leukotrienes C4 (LTC4) and D4 (LTD4) was rapidly decreased by incubation with eosinophil peroxidase (EPO), H2O2, and iodide, bromide, or to a lesser degree, chloride, LTB4 chemotactic activity was also decreased by the EPO-H2-H2-halide system, although at a slower rate. Myeloperoxidase could substitute for EPO in these reactions. Leukotriene inactivation was greatly decreased or abolished by deletion of any of the components of the system or by the addition of the hemeprotein inhibitors, azide, cyanide, or aminotriazole, indicating a requirement for peroxidase. The H2O2 concentration employed in the above studies was 10(-4) M. H2O2 at higher concentrations (5 x 10(-4) to 10(-2) M) inactivated LTC4 and LTD4 in the absence of EPO and a halide but had no effect on the chemotactic activity of LTB4. We have previously shown that horse eosinophils stimulated with the calcium ionophore A23187 generate SRS. In the present study, eosinophils stimulated in this way were found to release extracellularly both H2O2 and EPO. Incubation of eosinophils with azide that inhibits EPO, and catalase that degrades H2O2, significantly increased the amount of SRS activity detected in the extracellular medium after A23187 stimulation. These findings suggests eosinophils may play an important modulating role in hypersensitivity reactions both by the production of leukotrienes and by their inactivation through the release of H2O2 and EPO.     

Nitric oxide, complex I, and the modulation of mitochondrial reactive species in biology and disease

Mitochondria are the specialized organelles for energy metabolism but also participate in the production of O(2) active species, cell cycle regulation, apoptosis and thermogenesis. Classically, regulation of mitochondrial energy functions was based on the ADP/ATP ratio, which dynamically stimulates the transition between resting and maximal O(2) uptake. However, in the last years, NO was identified as a physiologic regulator of electron transfer and ATP synthesis by inhibiting cytochrome oxidase. Additionally, NO stimulates the mitochondrial production of O(2) active species, primarily O(2)(-) and H(2)O(2), and, depending on NO matrix concentration, of ONOO(-), which is responsible for the nitrosylation and nitration of mitochondrial components. By this means, alteration in mitochondrial complexes restricts energy output, further increases O(2) active species and changes cell signaling for proliferation and apoptosis through redox effects on specific pathways. These mechanisms are prototypically operating in prevalent generalized diseases like sepsis with multiorgan failure or limited neurodegenerative disorders like Parkinson's disease. Complex I appears to be highly susceptible to ONOO(-) effects and nitration, which defines an acquired group of mitochondrial disorders, in addition to the genetically induced syndromes. Increase of mitochondrial NO may follow over-expression of nNOS, induction and translocation of iNOS, and activation and/or increased content of the newly described mtNOS. Likewise, mtNOS is important in the modulation of O(2) uptake and cell signaling, and in mitochondrial pathology, including the effects of aging, dystrophin deficiency, hypoxia, inflammation and cancer.     

Copper(I)-phenolate complexes as models of the reduced active site of galactose oxidase: synthesis, characterization, and O2 reactivity

The Cu(I)-phenolate complexes (1)LCu and (2)LCu and the Cu(I)-phenol complex [H(2)LCu(CNC(6)H(3)Me(2))]BArF(4) were prepared and structurally characterized by X-ray crystallography, where (1)L(-) and (2)L(-) are ligands comprised of a 2,4-di- tert-butylphenolate linked to 1-isopropyl-1,5-diazacyclooctane or 1,4-diisopropyl-1,4,7-triazacyclononane, respectively. The reduced galactose oxidase (GAO) structural models (1)LCu and (2)LCu were found to be highly reactive with O(2), and through combined stopped-flow kinetic and EPR, UV-vis, and resonance Raman spectroscopic studies of the oxygenation of (2)LCu at low temperature, new intermediates relevant to those postulated for the active site oxidation step of the GAO catalytic cycle were identified. The oxygenation was shown by kinetics experiments to proceed via initial binding of O(2) to yield a green, unusually thermodynamically stable 1:1 adduct, (2)LCu(O(2)). Symmetric (eta(2)) binding of a superoxo ligand was indicated by oxygen-isotope-sensitive features in resonance Raman spectra obtained in batch experiments; peaks at nu((16)O(2))=1120 cm(-1), nu((18)O(16)O)=1093 cm(-1), and nu((18)O(2))=1058 cm(-1) were assigned as O-O stretching vibrations. These data represent the first experimental evidence for such superoxide coordination in complexes of tetradentate tripodal ligands and provide new precedent for how O(2) may bind at the reduced GAO active site. The 1:1 Cu/O(2) adduct subsequently evolves into a metastable purple species that is only observable under conditions of substoichiometric O(2). The kinetics of formation of this transient species are second order overall (rate= k'(2)[(2)LCu(O(2))][(2)LCu]). It exhibits an absorption band with lambda(max)=565 nm (epsilon=17900 M(-1) cm(-1)) and multiple oxygen-isotope-sensitive nu(Cu-O) and nu(O-O) features in the respective regions 500-550 cm(-1) and 700-850 cm(-1) in Raman spectra, with excitation-wavelength-dependent intensities that correlate with the 565 nm absorption feature. On the basis of the combined data available, the presence of multiple isomeric peroxodicopper species in the transient purple solution is postulated.     

Lignin peroxidase oxidation of Mn2+ in the presence of veratryl alcohol, malonic or oxalic acid, and oxygen

Veratryl alcohol (3,4-dimethoxybenzyl alcohol) appears to have multiple roles in lignin degradation by Phanerochaete chrysosporium. It is synthesized de novo by the fungus. It apparently induces expression of lignin peroxidase (LiP), and it protects LiP from inactivation by H2O2. In addition, veratryl alcohol has been shown to potentiate LiP oxidation of compounds that are not good LiP substrates. We have now observed the formation of Mn3+ in reaction mixtures containing LiP, Mn2+, veratryl alcohol, malonate buffer, H2O2, and O2. No Mn3+ was formed if veratryl alcohol or H2O2 was omitted. Mn3+ formation also showed an absolute requirement for oxygen, and oxygen consumption was observed in the reactions. This suggests involvement of active oxygen species. In experiments using oxalate (a metabolite of P. chrysosporium) instead of malonate, similar results were obtained. However, in this case, we detected (by ESR spin-trapping) the production of carbon dioxide anion radical (CO2.-) and perhydroxyl radical (.OOH) in reaction mixtures containing LiP, oxalate, veratryl alcohol, H2O2, and O2. Our data indicate the formation of oxalate radical, which decays to CO2 and CO2.-. The latter reacts with O2 to form O2.-, which then oxidizes Mn2+ to Mn3+. No radicals were detected in the absence of veratryl alcohol. These results indicate that LiP can indirectly oxidize Mn2+ and that veratryl alcohol is probably a radical mediator in this system.     

Oxygen profiles in, and in the agar beneath, colonies of Bacillus cereus, Staphylococcus albus and Escherichia coli

The paper reports the use of microelectrodes to measure O2 penetration in different aged colonies of Bacillus cereus, Escherichia coli and Staphylococcus albus. In young (18 h) colonies of B. cereus and E. coli O2 disappeared at depths of 25-30 micron and 35-40 micron respectively. In young S. albus colonies, O2 reached a minimum but was never completely absent. As colonies aged (24-168 h) the depth to which O2 penetrated increased.