Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

The formation of ferritin from apoferritin. Catalytic action of apoferritin

The iron-storage protein ferritin consists of a protein shell and has an iron content of up to 4500 iron atoms as a microcrystalline ferric oxide hydrate. A study was made of the uptake of ferrous iron by apoferritin in the presence of an oxidizing agent at very low iron:protein ratios. At ratios of less than about 150 iron atoms per apoferritin molecule hyperbolic progress curves were obtained, whereas at higher ratios the curves became sigmoidal under the conditions used. A computer model, developed previously (Macara et al., 1972), was shown to account for this result. The experimental evidence indicates that apoferritin binds ferrous iron and catalyses the initial stage in the formation of the ferric oxide hydrate inside the protein shell. This stage involves the oxidation of sufficient iron within the protein molecule to form a stable nucleus on which the growth of the microcrystalline iron-core particles can proceed. A possible schematic mechanism for the action of apoferritin is suggested.     

The role of peroxidase isoenzyme groups of Nicotiana tabacum in hydrogen peroxide formation

Three peroxidase isoenzyme-groups found in cell walls of tobacco were tested for their capacity to form H₂O₂. Isoenzyme-group G_I, located only in cell walls (G_II and G_III are also found in protoplasts) showed the highest K_app-value for H₂O₂-formation. The lowest K_app-value, i.e., maximal H₂O₂-formation was received for group G_III which is ionically bound to the cell wall. As shown before, G_I yields maximal polymerization rates for coniferyl- and p-coumarylalcohol. These facts indicate that each of the peroxidase isoenzyme groups of the cell wall is involved with different catalytic functions within the same pathways of H₂O₂-formation and succeeding lignification. H₂O₂-formation catalyzed by all 3 groups was increased by very low concentrations of Mn²⁺-ions. The required amount of Mn²⁺ leading to maximal stimulation was in each case dependent on the basic rate of H₂O₂-formation. Maximal stimulation of H₂O₂-formation by phenolic compounds was achieved by coniferylalcohol at a concentration of 10^-4M for all groups. Stimulation by p-coumaryl-and by sinapylalcohol was not as significant.     

Multiple pathways for repair of oxidative DNA damages caused by X rays and hydrogen peroxide in Escherichia coli.

The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.     

Iron incorporation into apoferritin. The role of apoferritin as a ferroxidase

Apoferritin catalyzes the oxidation of Fe(II) to Fe(III). Ferroxidase activity is assayed and characterized by coupling the oxidation with the binding of Fe(III) to transferrin. The initial rate of Fe(II) oxidation is dependent on apoferritin and initial Fe(II) concentration but independent of transferrin concentration. The ferroxidase activity is inhibited by Zn(II). Ferritins with varying loads of iron have the same ferroxidase activity level. It is suggested that the described oxidation process represents the initial step of iron deposition in apoferritin. Since transferrin can intercept Fe(III) before it is deposited in apoferritin, active sites for Fe(II) oxidation must be on or near the surface of apoferritin. This finding is contrary to the current view of apoferritin-catalyzed oxidation of Fe(II) which places active sites in the channels to the core or inside the central core.     

The protective role of polyphenols in cytotoxicity of hydrogen peroxide.

A variety of synthetic and natural polyphenols protect mammalian cells from hydrogen peroxide (H2O2). Cytotoxicity of H2O2 on Chinese hamster V79 cells was assessed with a colony formation assay, and several polyphenols prevented the decrease in the number of colonies caused by H2O2. A study of the structure-activity relationship revealed that affinity of the polyphenols for the cell membrane and the presence of an ortho-dihydroxy moiety in their structure proved essential to this protection.     

Activation of rat liver microsomal glutathione S-transferase by hydrogen peroxide: role for protein-dimer formation.

The mechanism of oxygen radical-dependent activation of hepatic microsomal glutathione S-transferase by hydrogen peroxide was studied. Glutathione S-transferase activity in liver microsomes was increased 1.5-fold by incubation with 0.75 mM hydrogen peroxide at 37 degrees C for 10 min, and the increase in activity was reversed by incubation with dithiothreitol. Purified glutathione S-transferase was also activated by hydrogen peroxide after incubation at room temperature, and the increase in the activity was also reversed by dithiothreitol. Immunoblotting with anti-microsomal glutathione S-transferase antibodies after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hydrogen peroxide-treated microsomes or purified glutathione S-transferase revealed the presence of a glutathione S-transferase dimer. These results indicate that the hydrogen peroxide-dependent activation of the microsomal glutathione S-transferase is associated with the formation of a protein dimer.     

Lipid peroxide formation in microsomes. The role of non-haem iron

1. Metal ion-chelating agents such as EDTA, o-phenanthroline or desferrioxamine inhibit lipid peroxide formation when rat liver microsomes prepared from homogenates made in pure sucrose are incubated with ascorbate or NADPH. 2. Microsomes treated with metal ion-chelating agents do not form peroxide on incubation unless inorganic iron (Fe²⁺ or Fe³⁺) in a low concentration is added subsequently. No other metal ion can replace inorganic iron adequately. 3. Microsomes prepared from sucrose homogenates containing EDTA (1mm) do not form lipid peroxide on incubation with ascorbate or NADPH unless Fe²⁺ is added. Washing the microsomes with sucrose after preparation restores most of the capacity to form lipid peroxide. 4. Lipid peroxide formation in microsomes prepared from sucrose is stimulated to a small extent by inorganic iron but to a greater extent if adenine nucleotides, containing iron compounds as a contaminant, are added. 5. The iron contained in normal microsome preparations exists in haem and in non-haem forms. One non-haem component in which the iron may be linked to phosphate is considered to be essential for both the ascorbate system and NADPH system that catalyse lipid peroxidation in microsomes.     

The oxygen-evolving complex requires chloride to prevent hydrogen peroxide formation.

Illumination of PSII core preparations can cause the production of H2O2 at rates which approach 60 mumol of H2O2 (mg of Chl.h)-1. The rate of peroxide production is maximal at pH 7.2 at low sucrose concentrations and at concentrations of Cl- (1.5-3.0 mM) that limit the rate of the oxidation of water to O2. The rate of H2O2 production increased with pH from pH 6.8 to 7.2 and was inversely proportional to the oxidation of water to O2 from pH 6.8 to 7.5. While EDTA does not inhibit H2O2 production, this reaction is abolished by 5 mM NH2OH and inhibited by the same concentrations of NH3 that affect water oxidation which indicates that the oxygen-evolving complex is responsible for the production of peroxide generated upon illumination of PSII core preparations. These results support a mechanism in which bound Cl- in the S2 state is displaced by OH- ions which are then oxidized by the OEC to form H2O2. Thus, the OEC requires Cl- to prevent access to the active site of the OEC until four oxidizing equivalents can be generated to allow the oxidation of water to O2.     

Investigation of hydrogen peroxide formation in plants

A convenient and simple electrophoretic procedure was used to study the NAD(P)H-dependent generation of the hydrogen peroxide needed for the polymerization of coniferyl alcohol by peroxidases from the wood of Ailanthus glandulosa. The results showed that an NAD(P)H-dependent generation of hydrogen peroxide could be brought about by either: a FMN or riboflavin-dependent system; or a Mn²⁺ -dependent system. The most active system was the one incorporating Mn²⁺, followed closely by that incorporating riboflavin. In nature it appears that the method of hydrogen peroxide formation is determined by the amounts of cofactors present in the lignifying tissue. Because no quantitative data are available in the literature, further studies of the concentrations of these cofactors in the plant cell-wall are needed.     

The insulin-like effect of hydrogen peroxide on pathways of lipid synthesis in rat adipocytes.

In addition to the well known insulin-like effects of certain concentrations of H2O2 on glucose transport and oxidation in isolated rat adipocytes, the present work demonstrates that lipid synthesis from glucose is also enhanced over a narrow range of H2O2 concentrations (0.15 to 0.5 mM) added to the incubation medium. As in the case of insulin, H2O2 was found to stimulate greater glucose incorporation into glyceride-fatty acid than incorporation into glyceride-glycerol. As part of a multifaceted regulation of lipogenesis, H2O2, like insulin, increased the amount of pyruvate dehydrogenase in the active form without increasing the total amount of pyruvate dehydrogenase. Pyruvate dehydrogenase activity increased within 5 min of H2O2 incubation, reached a maximum at 15 min and declined thereafter as the H2O2 disappeared from the incubation medium. While medium glucose per se was found to activate the enzyme, it is unlikely that the effect of H2O2 was mediated by the known enhancement of glucose transport since the effects on the enzyme were maximal in the absence of glucose in the incubation medium. These findings add to the growing list of insulin effects that are reproduced by H2O2, and strengthen the hypothesis that assigns H2O2 the role of "second messenger" of insulin.     

The role of peroxide in haem degradation. A study of the oxidation of ferrihaems by hydrogen peroxide.

The oxidation of ferrihaems by H2O2 was studied as a model for haem catabolism. Rates of ferrihaem oxidation were evaluated by using a new computer-based method that measures the loss in catalytic activity of the ferrihaem during oxidation. For protoferrihaem, deuteroferrihaem, coproferrihaem and mesoferrihaem, oxidation proceeded via the monomeric species and no dimer contribution was detectable. The pH-dependence of oxidation was studied in the range 6.5--11. Within experimental error, the data were compatible with an inverse linear dependence on [H+]. This was interpreted in terms of attack by HO2- on monomeric ferrihaem. The specific second-order rate constants for oxidation of monomeric species by HO2- were of the same order of magnitude for all the ferrihaems, and were in the sequence coproferrihaem greater than protoferrihaem greater than mesoferrihaem congruent to deuteroferrihaem. A model is suggested involving formation of a ferrihaem monomerperoxide complex, which may either dissociate with the formation of a peroxidatic intermediate or be involved in an intramolecular oxidation of the ferrihaem. Haem catabolism may occur via the same or a similar intermediate.     

Haemichrome formation from haemoglobin subunits by hydrogen peroxide.

The effect of H2O2 on ferrous human haemoglobin subunits (alphash-, betash-, alphapmb- and betapmb-chains) was studied. These chains were easily transformed to haemichrome by the addition of H2O2 or H2O2-generating systems, including glucose oxidase (EC 1.1.3.4) AND XANTHINE OXIDASE (EC 1.2.3.2), and this was ascertained by e.p.r. measurements and by absorption spectra. The changes in these haemoglobin subunits were not inhibited by superoxide dismutase (EC 1.15.1.1), but were decreased by catalase (EC 1.11.1.6). The rate of oxidation of alphapmb-chains was higher than that of alphash-chains, and the rate of oxidation of betapmb-chains was higher than that of betash-chains. Haemichrome was demonstrated to be formed directly from these ferrous chains by the attack by H2O2, and this process did not involve formation of methaemoglobin. On the basis of these findings the kinetics of the reaction between the haemoglobin subunits and H2O2 was studied, and the pathological significance of H2O2 in disorders of erythrocytes such as thalassaemia was discussed.     

Non-oxygen-forming pathways of hydrogen peroxide degradation by bovine liver catalase at low hydrogen peroxide fluxes

Heme catalases are considered to degrade two molecules of H₂O₂ to two molecules of H₂O and one molecule of O₂ employing the catalatic cycle. We here studied the catalytic behaviour of bovine liver catalase at low fluxes of H₂O₂ (relative to catalase concentration), adjusted by H₂O₂-generating systems. At a ratio of a H₂O₂ flux (given in μM/min^- 1) to catalase concentration (given in μM) of 10 min^- 1 and above, H₂O₂ degradation occurred via the catalatic cycle. At lower ratios, however, H₂O₂ degradation proceeded with increasingly diminished production of O₂. At a ratio of 1 min^- 1, O₂ formation could no longer be observed, although the enzyme still degraded H₂O₂. These results strongly suggest that at low physiological H₂O₂ fluxes H₂O₂ is preferentially metabolised reductively to H₂O, without release of O₂. The pathways involved in the reductive metabolism of H₂O₂ are presumably those previously reported as inactivation and reactivation pathways. They start from compound I and are operative at low and high H₂O₂ fluxes but kinetically outcompete the reaction of compound I with H₂O₂ at low H₂O₂ production rates. In the absence of NADPH, the reducing equivalents for the reductive metabolism of H₂O₂ are most likely provided by the protein moiety of the enzyme. In the presence of NADPH, they are at least in part provided by the coenzyme.     

An assessment of proposed mechanisms for sensing hydrogen peroxide in mammalian systems

Despite much recent interest in the biochemistry of reactive oxygen species, the mechanisms by which hydrogen peroxide (H2O2) functions in mammalian cells remain poorly defined. Proposed mechanisms for sensing H2O2 in mammalian cells include inactivation of protein tyrosine phosphatases and dual specificity phosphatases as well as inactivation of peroxiredoxins. In this critical review, proteins proposed to serve as sensors for H2O2 in mammals will be compared to peroxidases, catalases, and the bacterial H2O2 sensor OxyR for their ability to react with H2O2, in the context of our current knowledge concerning the concentrations of H2O2 present in cells.     

Multiple pathways for repair of hydrogen peroxide-induced DNA damage in Escherichia coli.

The repair response of Escherichia coli to hydrogen peroxide has been examined in mutants which show increased sensitivity to this agent. Four mutants were found to show increased in vivo sensitivity to hydrogen peroxide compared with wild type. These mutants, in order of increasing sensitivity, were recA, polC, xthA, and polA. The polA mutants were the most sensitive, implying that DNA polymerase I is required for any repair of hydrogen peroxide damage. Measurement of repair synthesis after hydrogen peroxide treatment demonstrated normal levels for recA mutants, a small amount for xthA mutants, and none for polA mutants. This is consistent with exonuclease III being required for part of the repair synthesis seen, while DNA polymerase I is strictly required for all repair synthesis. Sedimentation analysis of cellular DNA after hydrogen peroxide treatment showed that reformation was absent in xthA, polA, and polC(Ts) strains but normal in a recA cell line. By use of a lambda phage carrying a recA-lacZ fusion, we found hydrogen peroxide does not induce the recA promoter. Our findings indicate two pathways of repair for hydrogen peroxide-induced DNA damage. One of these pathways would utilize exonuclease III, DNA polymerase III, and DNA polymerase I, while the other would be DNA polymerase I dependent. The RecA protein seems to have little or no direct function in either repair pathway.     

The role of catalase in hydrogen peroxide resistance in fission yeast Schizosaccharomyces pombe.

The role of catalase in hydrogen peroxide resistance in Schizosaccharomyces pombe was investigated. A catalase gene disruptant completely lacking catalase activity is more sensitive to hydrogen peroxide than the parent strain. The mutant does not acquire hydrogen peroxide resistance by osmotic stress, a treatment that induces catalase activity in the wild-type cells. The growth rate of the disruptant is not different from that of the parent strain. Additionally, transformed cells that overexpress the catalase activity are more resistant to hydrogen peroxide than wildtype cells with normal catalase activity. These results indicate that the catalase of S. pombe plays an important role in resistance to high concentrations of hydrogen peroxide but offers little in the way of protection from the hydrogen peroxide generated in small amounts under normal growth conditions.