Reaction of hydrogen peroxide with ferrylhemoglobin: superoxide production and heme degradation

The reaction of Fe(II) hemoglobin (Hb) but not Fe(III) hemoglobin (metHb) with hydrogen peroxide results in degradation of the heme moiety. The observation that heme degradation was inhibited by compounds, which react with ferrylHb such as sodium sulfide, and peroxidase substrates (ABTS and o-dianisidine), demonstrates that ferrylHb formation is required for heme degradation. A reaction involving hydrogen peroxide and ferrylHb was demonstrated by the finding that heme degradation was inihibited by the addition of catalase which removed hydrogen peroxide even after the maximal level of ferrylHb was reached. The reaction of hydrogen peroxide with ferrylHb to produce heme degradation products was shown by electron paramagnetic resonance to involve the one-electron oxidation of hydrogen peroxide to the oxygen free radical, superoxide. The inhibition by sodium sulfide of both superoxide production and the formation of fluorescent heme degradation products links superoxide production with heme degradation. The inability to produce heme degradation products by the reaction of metHb with hydrogen peroxide was explained by the fact that hydrogen peroxide reacting with oxoferrylHb undergoes a two-electron oxidation, producing oxygen instead of superoxide. This reaction does not produce heme degradation, but is responsible for the catalytic removal of hydrogen peroxide. The rapid consumption of hydrogen peroxide as a result of the metHb formed as an intermediate during the reaction of reduced hemoglobin with hydrogen peroxide was shown to limit the extent of heme degradation.     

A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin

We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases.     

Heme degradation during autoxidation of oxyhemoglobin

Two fluorescent heme degradation compounds are detected during autoxidation of oxyhemoglobin. These fluorescent compounds are similar to fluorescent compounds formed when hydrogen peroxide reacts with hemoglobin [E. Nagababu and J. M. Rifkind, Biochem. Biophys. Res. Commun. 247, 592-596 (1998)]. Low levels of heme degradation in the presence of superoxide and catalase are attributed to a reaction involving the superoxide produced during autoxidation. The inhibition of most of the degradation by catalase suggests that the hydrogen peroxide generated during autoxidation of oxyhemoglobin produces heme degradation by the same mechanism as the direct addition of hydrogen peroxide to hemoglobin. The formation of the fluorescent degradation products was inhibited by the peroxidase substrate, ABTS, which reduces ferrylhemoglobin to methemoglobin, indicating that ferrylhemoglobin is produced during the autoxidation of hemoglobin. It is the transient formation of this highly reactive Fe(IV) hemoglobin, which is responsible for most of the heme degradation during autoxidation.     

Contribution of protein conformation to stereochemistry and reactivity of the active center of heme proteins and enzymes. The existence of horseradish peroxidase conformations and their possible role in the catalysis mechanism

In the spectral region 350-800 nm at 4.2 K we measured magnetic circular dichroism (MCD) spectra of the pentacoordinated complex of protcheme with 2-methylimidazole, deoxyleghemoglobin, neutral and alkaline forms of reduced horseradish peroxidase in the equilibrium states, as well as in non-equilibrium states produced by low-temperature photolysis of their carbon monoxide derivatives. Earlier the corresponding results have been obtained for myoglobin, hemoglobin and cytochromes P-450 and P-420. The energies of Fe-N (proximal His) and Fe-N(pyrroles) bonds and their changes upon ligand binding in heme proteins and enzymes were compared with those in the model heme complex thus providing conformational contribution into stereochemistry of the active site. The examples of weak and strong conformational "pressure" on stereochemistry were analysed and observed. If conformational energy contribution into stereochemistry prevails the electronic one the heme stereochemistry remains unchanged on ligand binding as it was observed for leghemoglobin and alkaline horseradish peroxidase. The change of bond energies in myoglobin and hemoglobin on ligand binding are comparable with those in protein free pentacoordinated protoheme, giving an example of weak conformational contribution to heme stereochemistry. The role of protein conformation energy in the modulation of ligand binding properties of heme in leghemoglobin relative to those in myoglobins is discussed. The most striking result were obtained in the study of reduced horseradish peroxidase in the pH region of 6.0-10.2. It was found that such different perturbations as ligand binding and heme-linked ionization of the distal amino acid residue induce identical changes in heme stereochemistry. Neither heme-linked ionization in the carbon monoxide complex nor the geometry of Fe-Co bond affect the heme local structure of photoproducts. These and other findings suggest a very low conformation mobility of horseradish peroxidase whose protein constraints appear to allow only two preferable geometries of specific amino acid residues that form the heme pocket. The role of the two tertiary structure constraints on the heme in the mechanism of horseradish peroxidase function is discussed. It is supposed that one conformation produces a heme environment suitable for two-electron oxidation of the native enzyme to compound I by hydrogen peroxide while another conformation changes the heme stereochemistry in the direction favourable for back reduction of compound I by the substrate to the resting enzyme through two one-electron steps. The switch from one tertiary structure to another is expected to be induced by substrate bind     

Singlet oxygen production from the peroxidase-catalyzed oxidation of indole-3-acetic acid

The aerobic oxidation of indole-3-acetic acid catalyzed by horseradish peroxidase produces 1268 nm emission characteristic of singlet oxygen. Lactoperoxidase also oxidizes indole-3-acetic acid to produce singlet oxygen, but in contrast to horseradish peroxidase, this enzyme system requires hydrogen peroxide. In both of these systems, the intensity of the 1268 nm emission is small due to quenching of the singlet oxygen by indole-3-acetic acid and by reaction products derived from indole-3-acetic acid. The biomolecular reaction of peroxyl radicals via a Russell mechanism is a plausible mechanism for the singlet oxygen generation in these systems. Under typical conditions of p2H 4.0, 1 microM horseradish peroxidase, 1 mM indole-3-acetic acid, and 240 microM oxygen, the singlet oxygen yield was 15 +/- 1 microM or 13% of the amount predicted by the Russell mechanism.     

Identification of a decarboxylation product of retinoic acid

The oxidative decarboxylation of retinoic acid was investigated utilizing a model system concisting of all-trans-retinoic acid, H₂O₂ and horseradish peroxidase. The decarboxylation products were purified by high-performance liquid chromatography on bonded, octadecylsilane columns. Based on mass spectral, nuclear magnetic resonance, ultraviolet and Fourier transform infrared analyses, the major decarboxylation product was identified as a 4-oxo-C₁₉ aldehyde with a hyrdoxyl group on the side chain at C₉, specifically 8-(2,6,6,-trimethyl-3-oxocyclohex-1-enyl)2,6-dimethyl-6-hydroxyoctatrienal.     

Kinetic studies on oxidation of aromatic donor molecules by horseradish peroxidase and lactoperoxidase

Based on kinetic evidence, it has been shown for the first time that the mode of binding of aromatic donor molecules is similar in horseradish peroxidase and lactoperoxidase; also that the nature of the heme plays an important role in the reaction with hydrogen peroxide, and has no effect on the reaction of the intermediate compound II with aromatic substrates.     

Oxyferryl heme and not tyrosyl radical is the likely culprit in prostaglandin H synthase-1 peroxidase inactivation

Prostaglandin H synthase-1 (PGHS-1) is a bifunctional heme protein catalyzing both a peroxidase reaction, in which peroxides are converted to alcohols, and a cyclooxygenase reaction, in which arachidonic acid is converted into prostaglandin G2. Reaction of PGHS-1 with peroxide forms Intermediate I, which has an oxyferryl heme and a porphyrin radical. An intramolecular electron transfer from Tyr385 to Intermediate I forms Intermediate II, which contains two oxidants: an oxyferryl heme and the Tyr385 radical required for cyclooxygenase catalysis. Self-inactivation of the peroxidase begins with Intermediate II, but it has been unclear which of the two oxidants is involved. The kinetics of tyrosyl radical, oxyferryl heme, and peroxidase inactivation were examined in reactions of PGHS-1 reconstituted with heme or mangano protoporphyrin IX with a lipid hydroperoxide, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and ethyl hydrogen peroxide (EtOOH). Tyrosyl radical formation was significantly faster with 15-HPETE than with EtOOH and roughly paralleled oxyferryl heme formation at low peroxide levels. However, the oxyferryl heme intensity decayed much more rapidly than the tyrosyl radical intensity at high peroxide levels. The rates of reactions for PGHS-1 reconstituted with MnPPIX were approximately an order of magnitude slower, and the initial species formed displayed a wide singlet (WS) radical, rather than the wide doublet radical observed with PGHS-1 reconstituted with heme. Inactivation of the peroxidase activity during the reaction of PGHS-1 with EtOOH or 15-HPETE correlated with oxyferryl heme decay, but not with changes in tyrosyl radical intensity or EPR line shape, indicating that the oxyferryl heme, and not the tyrosyl radical, is responsible for the self-destructive peroxidase side reactions. Computer modeling to a minimal mechanism was consistent with oxyferryl heme being the source of peroxidase inactivation.     

Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla

Vitreoscilla is a gram-negative bacterium that contains a unique bacterial hemoglobin that is relatively autoxidizable. It also contains a catalase whose primary function may be to remove hydrogen peroxide produced by this autoxidation. This enzyme was purified and partially characterized. It is a protein of 272,000 Da with a probable A2B2 subunit structure, in which the estimated molecular size of A is 68,000 Da and that of B, 64,000 Da, and an average of 1.6 molecules of protoheme IX per tetramer. The turnover number for its catalase activity was 27,000 s-1 and the Km for hydrogen peroxide was 16 mM. The peroxidase activity measured using o-dianisidine was 0.6% that of the catalase activity. Cyanide, which inhibited both catalase and peroxidase activities, bound the heme in a noncooperative manner. Azide inhibited the catalase activity but stimulated the peroxidase activity. An apparent compound II was formed by the reaction of the enzyme with ethyl hydrogen peroxide. The enzyme was reducible by dithionite, and the ferrous enzyme reacted with CO. The cellular content of Vitreoscilla hemoglobin varies during the growth cycle and in cells grown under different conditions, but the ratio of hemoglobin to catalase activity remained relatively constant, indicating possible coordinated biosynthesis and supporting the putative role of Vitreoscilla catalase as a scavenger of peroxide generated by Vitreoscilla hemoglobin.     

Hydrogen-peroxide-induced heme degradation in red blood cells: the protective roles of catalase and glutathione peroxidase

Catalase and glutathione peroxidase (GSHPX) react with red cell hydrogen peroxide. A number of recent studies indicate that catalase is the primary enzyme responsible for protecting the red cell from hydrogen peroxide. We have used flow cytometry in intact cells as a sensitive measure of the hydrogen-peroxide-induced formation of fluorescent heme degradation products. Using this method, we have been able to delineate a unique role for GSHPX in protecting the red cell from hydrogen peroxide. For extracellular hydrogen peroxide, catalase completely protected the cells, while the ability of GSHPX to protect the cells was limited by the availability of glutathione. The effect of endogenously generated hydrogen peroxide in conjunction with hemoglobin autoxidation was investigated by in vitro incubation studies. These studies indicate that fluorescent products are not formed during incubation unless the glutathione is reduced to at least 40% of its initial value as a result of incubation or by reacting the glutathione with iodoacetamide. Reactive catalase only slows down the depletion of glutathione, but does not directly prevent the formation of these fluorescent products. The unique role of GSHPX is attributed to its ability to react with hydrogen peroxide generated in close proximity to the red cell membrane in conjunction with the autoxidation of membrane-bound hemoglobin.     

The origin of red cell fluorescence caused by hydrogen peroxide treatment

Fluorescence in red cells following hydrogen peroxide treatment has been attributed to lipid peroxidation of the membrane. The putative relationship between lipid peroxidation and fluorescence was questioned by the finding that BHT and alpha-tocopherol, which are thought to inhibit lipid peroxidation, do not inhibit the fluorescence detected by flow cytometry. Furthermore, lipid peroxidation induced in red cells by the Fe(III)-ADP-ascorbate system did not produce fluorescence. These results require an alternative explanation for the hydrogen peroxide-induced fluorescence. A role for reduced hemoglobin is indicated by the inhibition of fluorescence by pretreatment of cells with CO that binds strongly to ferrohemoglobin and nitrite that oxidizes ferrohemoglobin. Our earlier studies have shown the formation of fluorescent heme degradation products during the reaction of purified hemoglobin with hydrogen peroxide, which was also inhibited by CO and nitrite pretreatment. The fluorescence produced in red cells after the addition of hydrogen peroxide can, therefore, be attributed to fluorescent heme degradation products.     

Tropolone as a substrate for horseradish peroxidase

Tropolone (2,4,6-cycloheptatrien-1-one), in the presence of hydrogen peroxide but not in its absence, can serve as a donor for the horseradish peroxidase catalysed reaction. The product formed is yellow and is characterized by a new peak at 418 nm. The relationship between the rate of oxidation of tropolone (ΔA at 418 nm/min) and various concentrations of horseradish peroxidase, tropolone and hydrogen peroxide is described. The yellow product obtained by the oxidation of tropolone by horseradish peroxidase in the presence of hydrogen peroxide was purified by chromatography on Sephadex G-10 and its spectral properties at different pHs are presented. The M, of the yellow product was estimated to be ca 500, suggesting that tropolone, in the presence of horseradish peroxidase and hydrogen peroxide is converted to a tetratropolone.     

Catalase-peroxidase (Mycobacterium tuberculosis KatG) catalysis and isoniazid activation

Resonance Raman spectra of native, overexpressed M. tuberculosis catalase-peroxidase (KatG), the enzyme responsible for activation of the antituberculosis antibiotic isoniazid (isonicotinic acid hydrazide), have confirmed that the heme iron in the resting (ferric) enzyme is high-spin five-coordinate. Difference Raman spectra did not reveal a change in coordination number upon binding of isoniazid to KatG. Stopped-flow spectrophotometric studies of the reaction of KatG with stoichiometric equivalents or small excesses of hydrogen peroxide revealed only the optical spectrum of the ferric enzyme with no hypervalent iron intermediates detected. Large excesses of hydrogen peroxide generated oxyferrous KatG, which was unstable and rapidly decayed to the ferric enzyme. Formation of a pseudo-stable intermediate sharing optical characteristics with the porphyrin pi-cation radical-ferryl iron species (Compound I) of horseradish peroxidase was observed upon reaction of KatG with excess 3-chloroperoxybenzoic acid, peroxyacetic acid, or tert-butylhydroperoxide (apparent second-order rate constants of 3.1 x 10(4), 1.2 x 10(4), and 25 M(-1) s(-1), respectively). Identification of the intermediate as KatG Compound I was confirmed using low-temperature electron paramagnetic resonance spectroscopy. Isoniazid, as well as ascorbate and potassium ferrocyanide, reduced KatG Compound I to the ferric enzyme without detectable formation of Compound II in stopped-flow measurements. This result differed from the reaction of horseradish peroxidase Compound I with isoniazid, during which Compound II was stably generated. These results demonstrate important mechanistic differences between a bacterial catalase-peroxidase and the homologous plant peroxidases and yeast cytochrome c peroxidase, in its reactions with peroxides as well as substrates.     

Proton nuclear magnetic resonance characterization of the oxidized intermediates of cytochrome c peroxidase

Oxidation of cytochrome c peroxidase with hydrogen peroxide to form the initial oxidized intermediate, cytochrome c peroxidase compound I, drastically alters the proton hyperfine nmr spectrum. In contrast to studies of horseradish peroxidase, where the spectrum of horseradish peroxidase compound I is similar to that of the native protein, cytochrome c peroxidase compound I exhibits only broad resonances near 17 and 30 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate. No unique resonances attributable to cytochrome c peroxidase compound II could be identified. These results define the molecular conditions for which resolved hyperfine resonances of the iron(IV) states of heme proteins may be observed when the data presented here are compared with the data from horseradish peroxidase. Oxidation of cytochrome c peroxidase while it is complexed to ferricytochrome c reveals that the heme resonances of cytochrome c are not influenced by the oxidation state of cytochrome c peroxidase.     

Fungal peroxidase: its structure,function, and application

Arthromyces ramosus, a novel hyphomycete, extracellularly produces a single species of a heme-containing peroxidase. The A. ramosus peroxidase, ARP, shows a broad specificity for hydrogen donors and high catalytic efficiency as does the well-known peroxidase from horseradish roots (HRP). However, it also exhibits unique catalytic properties. These features permit a wide range of applications for ARP, including high-sensitivity chemiluminescent determination of biological materials, protein cross-linking, and dye-transfer inhibition during laundering. The primary and tertiary structures of ARP are very similar to those of the class (II) lignin and manganese peroxidases of the plant peroxidase superfamily. Mechanistic studies of the ARP-catalyzed reaction revealed that it also proceeds with the classical peroxidase cycle; the native ferric ARP undergoes two-electron oxidation by hydrogen peroxide to yield compound (I), followed by two successive one-electron reductions by the hydrogen donor. X-ray crystallography, site-directed mutagenesis, and spectral analyses of ARP have afforded detailed information on the molecular mechanism of the ARP catalysis, and revealed the roles of active site amino acid residues and dynamic features of coordination as well as spin states of heme iron during catalysis.     

4-phenylylboronic acid: A new type of enhancer for the horseradish peroxidase catalysed chemiluminescent oxidation of luminol

4-Phenylylboronic acid enhances the light emission from the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide. Optimization studies showed that the greatest enhancement was obtained using micromolar concentrations of the new enhancer. The largest degree of enhancement was found with the basic isoenzyme of horseradish peroxidase (Type VIA), and lesser degrees of enhancement were obtained with Type VII and Type IX horseradish peroxidase. The enhancer was also effective in the peroxidase catalysed oxidation of isoluminol by peroxide.     

Glutathione-dependent oxidative modification of protoporphyrin and other dicarboxylic porphyrins by mammalian and plant peroxidases.

Protoporphyrin, an intermediate in heme and chlorophyll biosynthesis, can accumulate in human and plant tissues under certain pathological conditions and is a photosensitizer used in cancer phototherapy. We previously showed that protoporphyrin and the related non-natural dicarboxylic porphyrin deuteroporphyrin are rapidly oxidized by horseradish peroxidase in the presence of some thiols, especially glutathione. This study reports that bovine lactoperoxidase, but not leucocyte myeloperoxidase, can also catalyze this reaction and that Tween and ascorbic acid are inhibitors. Exogenous hydrogen peroxide is not required and cannot replace glutathione. Deuteroporphyrin was oxidized to a unique green chlorin product with two oxygen functions added directly to the characteristic reduced pyrrole ring of the chlorin. Spectroscopic and chromatographic results suggest that protoporphyrin was oxidized not to a green chlorin, but to a much more polar red porphyrin modified by oxidative addition to the two vinyl side chains. Two related nonnatural dicarboxylic porphyrins, with ethyl or hydroxyethyl instead of vinyl side chains, are not substrates or products for this enzymatic conversion.     

Structural change and catalytic activity of horseradish peroxidase in oxidative polymerization of phenol

The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.     

NADH peroxidase activity of rubrerythrin

P. S. Alban et al. (J. Appl. Microbiol. (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function. They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity. Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase. The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase. These results are consistent with the phenotype of the H2O2-resistant strain of S. volutans. The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states. Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea.     

The reaction of hydrogen peroxide with the dimethyl ester heme derivative of cytochrome c peroxidase

A modified cytochrome c peroxidase was prepared by reconstituting apocytochrome c peroxidase with protoheme in which both heme propionic acid groups were converted to the methyl ester derivatives. The modified enzyme reacted with hydrogen peroxide with a rate constant of (1.3 +/- 0.2) x 10(7) M-1 s-1, which is 28% that of the native enzyme. The reaction between the modified enzyme and hydrogen peroxide was pH-dependent with an apparent pK of 5.1 +/- 0.1 compared to a value of 5.4 +/- 0.1 for the native enzyme. These observations support the conclusion that the apparent ionization near pH 5.4, which influences the hydrogen peroxide-cytochrome c peroxidase reaction is not due to the ionization of the propionate side chains of the heme group in the native enzyme. A second apparent ionization, with pK of 6.1 +/- 0.1, influences the spectrum of the modified enzyme which changes from a high spin type at low pH to a low spin type at high pH.