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.     

An enzymatic determination of hydrogen peroxide by using spectrophotometry.

A new spectrophotometric method for determining low hydrogen peroxide concentrations by using horseradish peroxidase in the presence of NADH at pH 7.5 has been described. Both total NADH consumption and initial reaction rate may be used for the determination. Using the NADH consumption, a linear response with respect to hydrogen peroxide was observed in the concentration range 7 x 10(-8)-2.5 x 10(-6) M. Due to the presence of superoxide dismutase, hydrogen peroxide is partly regenerated and an amplification of the signal results, which explains the sensitivity.     

Reaction of phenylaminoethyl selenides with peroxynitrite and hydrogen peroxide

Peroxynitrite, a reactive cytotoxic species generated by the reaction of superoxide with nitric oxide, rapidly oxidizes phenylaminoethyl selenide (PAESe) and its para-substituted derivatives with second-order rate constants ranging from 900 to 3000 M(-1) s(-1) at neutral pH (pH 7.0) and 25 degrees C. These values are approximately 3 x 10(4) times greater than the corresponding rate constants for the reactions of selenides with hydrogen peroxide. The peroxynitrite reaction was also studied at alkaline pH. HPLC analysis confirms that both the peroxynitrite and hydrogen peroxide reactions produced the corresponding phenylaminoethyl selenoxide (PAESeO) as the sole selenium-containing product, with a stoichiometry of 1 mol of PAESe oxidized per 1 mol of PAESeO formed per 1 mol of oxidant reacted. The influence of para-substituents on the rate constants was investigated using Hammett plots; in both cases the data are consistent with an S(N)2-type mechanism, wherein the selenium atom acts as the nucleophile. Our results provide further evidence that organoselenium compounds may play a protective role in the defense against the many reactive oxidizing species produced in cellular metabolism.     

Importance of hydroxyl radical in the vanadium-stimulated oxidation of NADH

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.     

Reactions of soybean peroxidase and hydrogen peroxide pH 2.4-12.0, and veratryl alcohol at pH 2.4

Peroxidase from soybean seed coat (SBP) has properties that makes it particularly suited for practical applications. Therefore, it is essential to know its fundamental enzymatic properties. Stopped-flow techniques were used to investigate the pH dependence of the reaction of SBP and hydrogen peroxide. The reaction is linearly dependent on hydrogen peroxide concentration at acidic and neutral pH with the second order rate constant k(1)=2.0x10(7) M(-1) s(-1), pH 4-8. From pH 9.3 to 10.2 the reaction is biphasic, a novel observation for a peroxidase at alkaline pH. A fast reaction has the characteristics of the reaction at neutral pH, and a slow reaction shows hyperbolic dependence on hydrogen peroxide concentration. At pH >10.5 only the slow reaction is seen. The shift in mechanism is coincident with the change in haem iron co-ordination to a six-coordinate low spin hydroxy ligated alkaline form. The pK(a) value for the alkaline transition was observed at 9.7+/-0.1, 9.6+/-0.1 and 9.9+/-0.2 by spectrophotometric titration, the fast phase amplitude, and decrease in the apparent second order rate constant, respectively. An acidic pK(a) at 3.2+/-0.3 was also determined from the apparent second order rate constant. The reactions of soybean peroxidase compounds I and II with veratryl alcohol at pH 2.44 give very similar second order rate constants, k(2)=(2.5+/-0.1)x10(4) M(-1) s(-1) and k(3)=(2.2+/-0.1)x10(4) M(-1) s(-1), respectively, which is unusual. The electronic absorption spectra of compounds I, II and III at pH 7.07 show characteristic bands at 400 and 651 nm (compound I), 416, 527 and 555 nm (compound II), and 414, 541 and 576 nm (compound III). No additional intermediates were observed.     

Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.     

Reactions of yeast thioredoxin peroxidases I and II with hydrogen peroxide and peroxynitrite: rate constants by competitive kinetics

Peroxiredoxins are receiving increasing attention as defenders against oxidative damage and sensors of hydrogen peroxide-mediated signaling events. Likely to be critical for both functions is a rapid reaction with hydrogen peroxide, typically with second-order rate constants higher than 10(5) M(-1) s(-1). Until recently, however, the values reported for these rate constants have been in the range of 10(4)-10(5) M(-1) s(-1), including those for cytosolic thioredoxin peroxidases I (Tsa1) and II (Tsa2) from Saccharomyces cerevisiae. To resolve this apparent paradox, we developed a competitive kinetic approach with horseradish peroxidase to determine the second-order rate constant of the reaction of peroxiredoxins with peroxynitrite and hydrogen peroxide. This method was validated and allowed for the determination of the second-order rate constant of the reaction of Tsa1 and Tsa2 with peroxynitrite (k approximately 10(5) M(-1) s(-1)) and hydrogen peroxide (k approximately 10(7) M(-1) s(-1)) at pH 7.4, 25 degrees C. It also permitted the determination of the pKa of the peroxidatic cysteine of Tsa1 and Tsa2 (Cys47) as 5.4 and 6.3, respectively. In addition to providing a useful method for studying thiol protein kinetics, our studies add to recent reports challenging the popular belief that peroxiredoxins are poor enzymes toward hydrogen peroxide, as compared with heme and selenium proteins.     

Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest

Hydrogen sulfide (H(2)S) is an endogenously generated gas that can also be administered exogenously. It modulates physiological functions and has reported cytoprotective effects. To evaluate a possible antioxidant role, we investigated the reactivity of hydrogen sulfide with several one- and two-electron oxidants. The rate constant of the direct reaction with peroxynitrite was (4.8±1.4)×10(3)M(-1) s(-1) (pH 7.4, 37°C). At low hydrogen sulfide concentrations, oxidation by peroxynitrite led to oxygen consumption, consistent with a one-electron oxidation that initiated a radical chain reaction. Accordingly, pulse radiolysis studies indicated that hydrogen sulfide reacted with nitrogen dioxide at (3.0±0.3)×10(6)M(-1) s(-1) at pH 6 and (1.2±0.1)×10(7)M(-1) s(-1) at pH 7.5 (25°C). The reactions of hydrogen sulfide with hydrogen peroxide, hypochlorite, and taurine chloramine had rate constants of 0.73±0.03, (8±3)×10(7), and 303±27M(-1) s(-1), respectively (pH 7.4, 37°C). The reactivity of hydrogen sulfide was compared to that of low-molecular-weight thiols such as cysteine and glutathione. Considering the low tissue concentrations of endogenous hydrogen sulfide, direct reactions with oxidants probably cannot completely account for its protective effects.     

Kinetics of formation of the primary compound (compound I) from hydrogen peroxide and turnip peroxidases.

A kinetic study of the reaction of two turnip peroxidases (P1 and P7) with hydrogen peroxide to form the primary oxidized compound (compound I) has been carried out over the pH range from 2.4 to 10.8. In the neutral and acidic pH regions, the rates depend linearly on hydrogen peroxide concentration whereas at alkaline pH values the rates display saturation kinetics. A compound is made with the cyanide binding reaction to peroxidases since the two reactions are influenced in the same manner by ionization of groups on the native enzymes. Two different ionization processes of peroxidase P1 with pKa values of 3.9 and 10 are required to explain the rate pH profile for the reaction with H2O2. Protonation of the former group and ionization of the latter causes a decrease in the rate of reaction of the enzyme with H2O2. In the case of peroxidase P7 a minimum model involves three ionizable groups with pKa values of 2.5, 4 and 9. Protonation of the former two groups and ionization of the latter lowers the reaction rate. In the pH-independent region, the rate of formation of compound I was measured as a function of temperature. From the Arhenius plots the activation energy for the reaction was calculated to be 2.9 +/- 0.1 kcal/mol for P1 and 5.4 +/- 0.3 kcal/mol for P7. However, the rates are independent of viscosity in glycerol-water mixtures up to 30% glycerol.     

Hydroxylating activity of tyrosinase and its dependence on hydrogen peroxide

The aim of this work was to study the hydroxylation of N, N-dimethyltyramine (DMTA) by tyrosinase in the presence of hydrogen peroxide, a reaction that does not take place without the addition of the hydrogen peroxide. Some properties of this hydroxylating activity are analyzed. The kinetic parameters of mushroom tyrosinase toward hydrogen peroxide (K(m) = 0.5 mM, V(m) = 11 microM/min, V(m)/K(m) = 2.2 x 10(-2) min(-1)) and toward DMTA (K(m) = 0.3 mM, V(m) = 4.8 microM/min, V(m)/K(m) = 16 x 10(-2) min(-1)) were evaluated. There was a lag period, which was similar to the characteristic lag of monophenolase activity at the expense of molecular oxygen. The length of this lag phase decreased with increasing hydrogen peroxide concentration, and disappeared at approximately 0.5 mM H(2)O(2). However, the lag was longer with higher DMTA concentrations. The pH optimum range for this hydroxylating activity was 6.0 to 7.0. The lag also varied with pH, increasing at pH values higher than 6.7. The presence of hydrogen peroxide is necessary for the oxidation of DMTA, as is the presence of active enzyme since the reaction was completely inhibited when selective tyrosinase inhibitors were added.     

Formation of aminoxyl radicals in the reaction between penicillins and hydrogen peroxide.

Aminoxyl radicals are formed in high yield in the reaction between penicillins and hydrogen peroxide in water solutions in the pH range between 7 and 8. The nine-line EPR spectrum, 3 x 3 (1:2:1), indicated an interaction of the unpaired electron with one 14N nucleus (aN = 1.44 mT) and two equivalent hydrogen nuclei (aH = 2.00 mT). The reaction involves an oxidative cleavage of the beta-lactam ring of the penicillins with the formation of a cyclic aminoxyl radical, in which the thiazolidine ring carries the nitroxide group (= N-O.). It is suggested that the reaction with the formation of aminoxyl radicals can also take place in vivo in the deactivation of penicillins by metabolically formed hydrogen peroxide.     

Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide.

The reactivities of glutathione, cysteine, cysteamine, penicillamine, N-acetylcysteine, dithiothreitol and captopril with superoxide generated from xanthine oxidase and hypoxanthine, and with reagent hydrogen peroxide, have been investigated. Rates of thiol loss on adding hydrogen peroxide, and superoxide-dependent thiol loss and oxygen uptake were measured. The relative reactivities of the different thiols with both oxidants were inversely related to the pK of the thiol group, such that at pH 7.4, penicillamine was the most reactive. N-acetylcysteine weakly reactive and no reaction was seen with captopril. For hydrogen peroxide, the calculated rate constants for the reaction with the thiolate anion all fell within the range 18-26 M(-1) s(-1). With superoxide, our results are consistent with each thiol reacting via a short chain that consumes oxygen and regenerates superoxide. Only with some of the thiols, was the consumed oxygen recovered as hydrogen peroxide. Reported values for the rate constant for the reaction of thiols with superoxide vary over four orders of magnitude, with the highest being > 10(5) M(-1) s(-1). Due to the complexity of the chain reaction, no study so far has been able to obtain accurate values and we consider the best estimates to be in the 30 to 1000 M(-1) s(-1) range.     

Reaction of ferrous lactoperoxidase with hydrogen peroxide and dioxygen: an anaerobic stopped-flow study

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears, and saliva and has physiological significance in antimicrobial defense which involves (pseudo-)halide oxidation. LPO compound III (a ferrous-dioxygen complex) is known to be formed rapidly by an excess of hydrogen peroxide and could participate in the observed catalase-like activity of LPO. The present anaerobic stopped-flow kinetic analysis was performed in order to elucidate the catalytic mechanism of LPO and the kinetics of compound III formation by probing the reactivity of ferrous LPO with hydrogen peroxide and molecular oxygen. It is shown that ferrous LPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl LPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (7.2+/-0.3) x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C. The H2O2-mediated conversion of compound II to compound III follows also second-order kinetics (220 M(-1) s(-1) at pH 7.0 and 25 degrees C). Alternatively, compound III is also formed by dioxygen binding to ferrous LPO at an apparent bimolecular rate constant of (1.8+/-0.2) x 10(5) M(-1) s(-1). Dioxygen binding is reversible and at pH 7.0 the dissociation constant (K(D)) of the oxyferrous form is 6 microM. The rate constant of dioxygen dissociation from compound III is higher than conversion of compound III to ferric LPO, which is not affected by the oxygen concentration and follows a biphasic kinetics. A reaction cycle including the redox intermediates compound II, compound III, and ferrous LPO is proposed, which explains the observed (pseudo-)catalase activity of LPO in the absence of one-electron donors. The relevance of these findings in LPO catalysis is discussed.     

Enhanced polysaccharide recovery from agricultural residues and perennial grasses treated with alkaline hydrogen peroxide

Pretreatment of lignocellulosic materials with alkaline hydrogen peroxide greatly increases their susceptibility to enzymatic cellulose hydrolysis. During the course of the pretreatment reaction (18 h), the pH rises slowly, increasing from pH 11.5 to a final pH > 12. As a result, most of the hemicellulose in the lignocellulosic substrate becomes solubilized. Maintaining the reaction pH near the optimum of 11.5 prevents hemicellulose solubilization and decreases the time required for effective pretreatment to about 6 h. Alkaline peroxide pretreatment is most effective on lignocellulose from monocotyledonous plants, especially members of the family Gramineae. Enzymatic saccharification efficiencies > 90% of theoretical were attained from high yielding perennial grasses such as big bluestem (Andropogon gerardi) and Indian grass (Sorghastrum nutans) after alkaline peroxide pretreatment.     

Determination of hydrogen peroxide generated by reduced nicotinamide adenine dinucleotide oxidase

Hydrogen peroxide can be conveniently determined using horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). However, interference occurs among assay components in the presence of reduced nicotinamide adenine dinucleotide (NADH) that is also a substrate of NADH oxidase. So, depletion of NADH is required before using the HRP method. Here, we report simple and rapid procedures to accurately determine hydrogen peroxide generated by NADH oxidase. All procedures developed were based on the extreme acid lability of NADH and the stability of hydrogen peroxide, because NADH was decomposed at pH 2.0 or 3.0 for 10 min, while hydrogen peroxide was stable at pH 2.0 or 3.0 for at least 60 min. Acidification and neutralization were carried out by adjusting sample containing NADH up to 30 microM to pH 2.0 for 10 min before neutralizing it back to pH 7.0. Then, hydrogen peroxide in the sample was measured using the HRP method and its determination limit was found to be about 0.3 microM. Alternatively, hydrogen peroxide in samples containing NADH up to 100 microM could be quantitated using a modified HRP method that required an acidification step only, which was found to have a determination limit of about 3 microM hydrogen peroxide in original samples.     

Inhibition of porcine kidney betaine aldehyde dehydrogenase by hydrogen peroxide

Renal hyperosmotic conditions may produce reactive oxygen species, which could have a deleterious effect on the enzymes involved in osmoregulation. Hydrogen peroxide was used to provoke oxidative stress in the environment of betaine aldehyde dehydrogenase in vitro. Enzyme activity was reduced as hydrogen peroxide concentration was increased. Over 50% of the enzyme activity was lost at 100 μM hydrogen peroxide at two temperatures tested. At pH 8.0, under physiological ionic strength conditions, peroxide inhibited the enzyme. Initial velocity assays of betaine aldehyde dehydrogenase in the presence of hydrogen peroxide (0-200 μM) showed noncompetitive inhibition with respect to NAD(+) or to betaine aldehyde at saturating concentrations of the other substrate at pH 7.0 or 8.0. Inhibition data showed that apparent V(max) decreased 40% and 26% under betaine aldehyde and NAD(+) saturating concentrations at pH 8.0, while at pH 7.0 V(max) decreased 40% and 29% at betaine aldehyde and NAD(+) saturating concentrations. There was little change in apparent Km(NAD) at either pH, while Km(BA) increased at pH 7.0. K(i) values at pH 8 and 7 were calculated. Our results suggest that porcine kidney betaine aldehyde dehydrogenase could be inhibited by hydrogen peroxide in vivo, thus compromising the synthesis of glycine betaine.     

Low-temperature bleaching of cotton induced by glucose oxidase enzymes and hydrogen peroxide activators

Glucose oxidase enzymes were used to produce hydrogen peroxide from glucose and oxygen in aqueous solutions. Different working conditions, that is, temperature, aeration with liquefied air, presence of cotton fibre and time of enzyme activity, were tested in order to obtain a solution with the highest possible concentration of hydrogen peroxide. The hydrogen peroxide produced was transformed into different peracids which could bleach the cotton fabric under mild conditions, at a pH between 7 and 8 and at a temperature of around 60°C. The conversion or activation of hydrogen peroxide was conducted with the bleach activators TAED, NOBS and TBBC. The concentrations of hydrogen peroxide and peracids in the solutions were measured with sodium thiosulphate titrations.

The results indicated that the formation of hydrogen peroxide with glucose oxidase was effective under optimal conditions, which are 50°C, pH 4.6 and aeration. Convenient activators for the conversion of hydrogen peroxide into peracids were TAED and TBBC, which enabled attainment of a relatively high degree of whiteness at pH 7.5 and temperature 50°C. Using the activator NOBS under these conditions did not provide enough peracid to markedly improve whiteness.     

Purification and properties of NADH oxidase from Bacillus megaterium

NADH oxidase, which catalyzes the oxidation of NADH, with the consumption of a stoichiometric amount of oxygen, to NAD+ and hydrogen peroxide was purified from Bacillus megaterium by 5'-AMP Sepharose affinity chromatography to homogeneity. The enzyme is a dimeric protein containing 1 mol of FAD per mol of subunit, Mr = 52,000. The absorption maxima of the native enzyme (oxidized form) were found at 270, 383, and 450 with a shoulder at 475 nm in 50 mM KPi buffer, pH 7.0. The visible absorption bands at 383 and 450 nm disappeared on the addition of NADH under anaerobic conditions and reappeared upon the introduction of air. Thus, the non-covalently bound FAD functioned as a prosthetic group for the enzyme. We tentatively named this new enzyme NADH oxidase (NADH:oxygen oxidoreductase, hydrogen peroxide forming). This enzyme stereospecifically oxidizes the pro-S hydrogen at C-4 of the pyridine ring of NADH.     

Spectrofluorometric analysis of hydrogen peroxide

The pH of maximum fluorescence (above pH 7) and the optimal excitation and emission wavelengths (468 nm and 519 nm, respectively) were determined for 2′,7′-dichlorofluorescein (DCF). The stoichiometry after hydrolysis of the oxidation of the stable nonfluorescent compound 2′,7′-dichlorofluorescin diacetate (LDADCF) was determined and found to be 2 moles of DCF produced per mole of hydrogen peroxide used.     

Examination of the reaction of fully reduced cytochrome oxidase with hydrogen peroxide by flow-flash spectroscopy

The reaction of cytochrome c oxidase with hydrogen peroxide has been of great value in generating and characterizing oxygenated species of the enzyme that are identical or similar to those formed during turnover of the enzyme with dioxygen. Most previous studies have utilized relatively low peroxide concentrations (millimolar range). In the current work, these studies have been extended to the examination of the kinetics of the single turnover of the fully reduced enzyme using much higher concentrations of peroxide to avoid limitations by the bimolecular reaction. The flow-flash method is used, in which laser photolysis of the CO adduct of the fully reduced enzyme initiates the reaction following rapid mixing of the enzyme with peroxide, and the reaction is monitored by observing the absorbance changes due to the heme components of the enzyme. The following reaction sequence is deduced from the data. (1) The initial product of the reaction appears to be heme a(3) oxoferryl (Fe(4+)=O(2)(-) + H(2)O). Since the conversion of ferrous to ferryl heme a(3) (Fe(2+) to Fe(4+)) is sufficient for this reaction, presumably Cu(B) remains reduced in the product, along with Cu(A) and heme a. (2) The second phase of the reaction is an internal rearrangement of electrons and protons in which the heme a(3) oxoferryl is reduced to ferric hydroxide (Fe(3+)OH(-)). In about 40% of the population, the electron comes from heme a, and in the remaining 60% of the population, Cu(B) is oxidized. This step has a time constant of about 65 micros. (3) The third apparent phase of the reaction includes two parallel reactions. The population of the enzyme with an electron in the binuclear center reacts with a second molecule of peroxide, forming compound F. The population of the enzyme with the two electrons on heme a and Cu(A) must first transfer an electron to the binuclear center, followed by reaction with a second molecule of peroxide, also yielding compound F. In each of these reaction pathways, the reaction time is 100-200 micros, i.e., much faster than the rate of reaction of peroxide with the fully oxidized enzyme. Thus, hydrogen peroxide is an efficient trap for a single electron in the binuclear center. (4) Compound F is then reduced by the final available electron, again from heme a, at the same rate as observed for the reduction of compound F formed during the reaction of the fully reduced oxidase with dioxygen. The product is the fully oxidized enzyme (heme a(3) Fe(3+)OH(-)), which reacts with a third molecule of hydrogen peroxide, forming compound P. The rate of this final reaction step saturates at high concentrations of peroxide (V(max) = 250 s(-)(1), K(m) = 350 mM). The data indicate a reaction mechanism for the steady-state peroxidase activity of the enzyme which, at pH 7.5, proceeds via the single-electron reduction of the binuclear center followed by reaction with peroxide to form compound F directly, without forming compound P. Peroxide is an efficient trap for the one-electron-reduced state of the binuclear center. The results also suggest that the reaction of hydrogen peroxide to the fully oxidized enzyme may be limited by the presence of hydroxide associated with the heme a(3) ferric species. The reaction of hydrogen peroxide with heme a(3) is very substantially accelerated by the availability of an electron on heme a, which is presumably transferred to the binuclear center concomitant with a proton that can convert the hydroxide to water, which is readily displaced.