A steady-state study on the formation of Compounds II and III of myeloperoxidase

The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a pKa of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5.10(4) M-1.s-1. During the reaction of myeloperoxidase with H2O2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a pKa of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6.10(7) M-1.s-1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III (k = 50 M-1.s-1) proceeded via two different pathways, one of which was inhibitable by tetranitromethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3-11.     

Ceruloplasmin,extracellular-superoxide dismutase,and scavenging of superoxide anion radicals

Ceruloplasmin and extracellular-superoxide dismutase are similar in physical properties. Both are found in extracellular fluids and both are scavengers of the superoxide radical. The relationship between the two proteins was further explored in the present investigation. Ceruloplasmin preparations were found to be commonly contaminated with extracellular-superoxide dismutase. In one preparation, 80% of the superoxide dismutase activity was due to extracellular-superoxide dismutase. Ceruloplasmin, freed from contaminating superoxide dismutase, was found to catalytically dismute the superoxide anion radical with a rate constant of about 1.0 × 10⁴ M⁻ s⁻¹ per copper atom. Under physiological conditions with a low rate of superoxide production, ceruloplasmin preferentially reacts stoichiometrically with the superoxide radical with a rate constant of about 2 × 10⁵ M⁻¹ s⁻¹ per copper atom. Under such conditions, the reaction does not result in hydrogen peroxide formation. From the kinetic data obtained it was calculated that in normal human plasma, extracellular-superoxide dismutase will scavenge about twice as much superoxide as ceruloplasmin. Using immobilized antibodies toward extracellular superoxide dismutase and ceruloplasmin, no antigenic cross-reactivity between the two proteins could be detected.     

Redox properties of heme peroxidases

Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the reduction potentials of three redox couples, namely Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺. In particular, the oxidative power of heme peroxidases is controlled by the (high) reduction potential of the latter two couples. Moreover, the rapid H₂O₂-mediated two-electron oxidation of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on the reduction potential of the Fe³⁺/Fe²⁺ couple. The understanding of the molecular determinants of the reduction potentials of the above redox couples is crucial for the comprehension of the molecular determinants of the catalytic properties of heme peroxidases.This review provides an overview of the data available on the redox properties of Fe³⁺/Fe²⁺, Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺ couples in native and mutated heme peroxidases. The influence of the electron donor properties of the axial histidine and of the polarity of the heme environment is analyzed and the correlation between the redox properties of the heme group with the catalytic activity of this important class of metallo-enzymes is discussed.     

A kinetic analysis of the catalase activity of myeloperoxidase

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.     

On the mechanism of citrate inhibition of ceruloplasmin ferroxidase activity

Ceruloplasmin is a plasma protein, which oxidizes ferrous ions in a catalytic manner. It is considered to function as a ferroxidasein vivo. Citrate was found to inhibit the reaction. The ceruloplasmin catalyzed oxidation ofp-phenylenediamines, however, was not affected by citrate. The inhibitory effect is proposed to be due to formation of Fe²⁺-citrate, which does not react with ceruloplasmin. The stability constant for the Fe²⁺-citrate complex estimated from the present inhibition study is in good agreement with previously published data.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

Abstract

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (k_I 2.1×10⁶ M^-1s^-1), it reacts sluggishly with compound II (k_II 7 M^-1s^-1). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I₅₀) at a concentration of 2 µM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I₅₀ 80 µM) unless superoxide dismutase was present (I₅₀ 5 µM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

The influence of radiotherapy on ceruloplasmin and transferrin in whole blood of breast cancer patients

Ceruloplasmin and transferrin are proteins which play a potential role in the process of breast cancer development. These molecules contain Cu²⁺ (ceruloplasmin) or Fe³⁺ ions (transferrin) and thus constitute paramagnetic centers, which can be studied using electron paramagnetic resonance method. The aim of the study was to determine how paramagnetic centers in whole blood of breast cancer patients change under the influence of radiation therapy. Samples of whole blood were taken from 17 women with breast cancer treated with radiotherapy. The measurements were carried out at 170 K using X-band electron paramagnetic resonance (EPR) spectrometer Bruker EMX-10. Two distinct EPR lines, derived from high-spin Fe³⁺ in transferrin and Cu²⁺ from ceruloplasmin, were revealed in all frozen samples. The amplitude and integrated intensity of the EPR signal from Cu²⁺ in ceruloplasmin significantly decreased in all patients after the delivery of the radiation fraction. When comparing the integral intensity of the signal from Fe³⁺ in transferrin, three different situations were identified which are patient specific: a significant increase, an insignificant change, or a significant decrease after the irradiation. A decreased level of Cu²⁺ from ceruloplasmin in patients after radiotherapy means a low level of ceruloplasmin in the plasma or an increased content of reduced Cu⁺ ions. Differences in the integrated intensity of the EPR signal from transferrin translate directly into the amount of bound iron. The observed changes could indicate how well the organism fights against cancer and how easily it adapts to the situation of biochemical stress.     

Enhanced chemiluminescence in the peroxidase-luminol-H2O2 system: Anomalous reactivity of enhancer phenols with enzyme intermediates

Phenols which markedly enhance chemiluminescence in the horseradish peroxidase catalysed oxidation of luminol by hydrogen peroxide show anomalously high reactivity (by factors of ～10² compared with published Hammett correlations) in the reduction of the enzyme intermediates, Compound I and Compound II. The results support the hypothesis that efficient production of phenoxy radicals from phenols is a necessary criterion for chemiluminescence enhancer action.     

Mechanism of Copper Uptake from Blood Plasma Ceruloplasmin by Mammalian Cells

Ceruloplasmin, the main copper binding protein in blood plasma, has been of particular interest for its role in efflux of iron from cells, but has additional functions. Here we tested the hypothesis that it releases its copper for cell uptake by interacting with a cell surface reductase and transporters, producing apoceruloplasmin. Uptake and transepithelial transport of copper from ceruloplasmin was demonstrated with mammary epithelial cell monolayers (PMC42) with tight junctions grown in bicameral chambers, and purified human ⁶⁴Cu-labeled ceruloplasmin secreted by HepG2 cells. Monolayers took up virtually all the ⁶⁴Cu over 16h and secreted half into the apical (milk) fluid. This was partly inhibited by Ag(I). The ⁶⁴Cu in ceruloplasmin purified from plasma of ⁶⁴Cu-injected mice accumulated linearly in mouse embryonic fibroblasts (MEFs) over 3-6h. Rates were somewhat higher in Ctr1+/+ versus Ctr1-/- cells, and 3-fold lower at 2°C. The ceruloplasmin-derived ⁶⁴Cu could not be removed by extensive washing or trypsin treatment, and most was recovered in the cytosol. Actual cell copper (determined by furnace atomic absorption) increased markedly upon 24h exposure to holoceruloplasmin. This was accompanied by a conversion of holo to apoceruloplasmin in the culture medium and did not occur during incubation in the absence of cells. Four different endocytosis inhibitors failed to prevent ⁶⁴Cu uptake from ceruloplasmin. High concentrations of non-radioactive Cu(II)- or Fe(III)-NTA (substrates for cell surface reductases), or Cu(I)-NTA (to compete for transporter uptake) almost eliminated uptake of ⁶⁴Cu from ceruloplasmin. MEFs had cell surface reductase activity and expressed Steap 2 (but not Steaps 3 and 4 or dCytB). However, six-day siRNA treatment was insufficient to reduce activity or uptake. We conclude that ceruloplasmin is a circulating copper transport protein that may interact with Steap2 on the cell surface, forming apoceruloplasmin, and Cu(I) that enters cells through CTR1 and an unknown copper uptake transporter.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (kI 2.1 x 10(6) M(-1)s(-1)), it reacts sluggishly with compound II (kII 7 M(-1)s(-1)). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I50) at a concentration of 2 microM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I50 80 microM) unless superoxide dismutase was present (I50 5 microM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

Characterization of the oxidized states of bromoperoxidase

Bromoperoxidase Compound I has been formed in reactions between bromoperoxidase and organic peroxide substrates. The absorbance spectrum of bromoperoxidase Compound I closely resembles the Compound I spectra of other peroxidases. The pH dependence of the second order rate constant for the formation of Compound I with hydrogen peroxide demonstrates the presence of an ionizable group at the enzyme active site having a pKa of 5.3. Protonation of this acidic group inhibits the rate of Compound I formation. This pKa value is higher than that determined for other peroxidases but the overall pH rate profiles for Compound I formation are similar. The one-electron reduction of bromoperoxidase Compound I yields Compound II and a second reduction yields native enzyme. Bromoperoxidase Compound II readily forms Compound III in the presence of an excess of hydrogen peroxide. Compound III passes through an as yet uncharacterized intermediate (III) in its decay to native enzyme. Compound III is produced and accumulates in enzymatic bromination reactions to become the predominate steady state form of the enzyme. Since Compound III is inactive as catalyst for enzymatic bromination, its accumulation leads to an idling reaction pathway which displays an unusual kinetic pattern for the bromination of monochlorodimedone.     

Highly purified ceruloplasmin messenger RNA from rat liver

Summary Highly purified ceruloplasmin mRNA was isolated from rat liver polyribosomes. The molecular weight of ceruloplasmin mRNA is in a range from 1.05 to 1.25 · 10⁶ daltons which is large enough to code for a putative precursor of ceruloplasmin (∼700 amino acids). Ceruloplasmin mRNA contains 3′-terminal poly(A) the length of which varies from 38 to 165 nucleotides. The 5′-end of ceruloplasmin mRNA is blocked with confronting m⁷G residue which is a component of cap I (m⁷G^5′ppp^5′X^mpAp). The addition of ceruloplasmin mRNA to wheat-germ cell-free system programmed the synthesis of a product that was largely precipitated by anti-ceruloplasmin immunoglobulins. The translation product was homogeneous in polyacrylamide gel-sodium dodecylsulfate electrophoresis. Cell-free translation of ceruloplasmin mRNA was sensitive to inhibition by cap analogue.     

The catalytic mechanism of Pseudomonas cytochrome c peroxidase

The catalytic mechanism of Pseudomonas cytochrome c peroxidase has been studied using rapid-scan spectrometry and stopped-flow measurements. The reaction of the totally ferric form of the enzyme with H₂O₂ was slow and the complex formed was inactive in the peroxidatic cycle, whereas partially reduced enzyme formed highly reactive intermediates with hydrogen peroxide. Rapid-scan spectrometry revealed two different spectral forms, one assignable to Compound I and the other to Compound II as found in the reaction cycle of other peroxidases. The formation of Compound I was rapid approaching that of diffusion control. The stoichiometry of the peroxidation reaction, deduced from the formation of oxidized electron donor, indicates that both the reduction of Compound I to Compound II and the conversion of Compound II to resting (partially reduced) enzyme are one-electron steps. It is concluded that the reaction mechanism generally accepted for peroxidases is applicable also to Pseudomonas cytochrome c peroxidase, the intramolecular source of one electron in Compound I formation, however, being reduced heme c.     

Inactivation of human myeloperoxidase by hydrogen peroxide

Human myeloperoxidase (MPO) uses hydrogen peroxide generated by the oxidative burst of neutrophils to produce an array of antimicrobial oxidants. During this process MPO is irreversibly inactivated. This study focused on the unknown role of hydrogen peroxide in this process. When treated with low concentrations of H₂O₂ in the absence of reducing substrates, there was a rapid loss of up to 35% of its peroxidase activity. Inactivation is proposed to occur via oxidation reactions of Compound I with the prosthetic group or amino acid residues. At higher concentrations hydrogen peroxide acts as a suicide substrate with a rate constant of inactivation of 3.9 × 10⁻³ s⁻¹. Treatment of MPO with high H₂O₂ concentrations resulted in complete inactivation, Compound III formation, destruction of the heme groups, release of their iron, and detachment of the small polypeptide chain of MPO. Ten of the protein’s methionine residues were oxidized and the thermal stability of the protein decreased. Inactivation by high concentrations of H₂O₂ is proposed to occur via the generation of reactive oxidants when H₂O₂ reacts with Compound III. These mechanisms of inactivation may occur inside neutrophil phagosomes when reducing substrates for MPO become limiting and could be exploited when designing pharmacological inhibitors.     

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20–40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 ± 5 s⁻¹; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s⁻¹ can be estimated at 5°C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10⁻⁴ to 10⁻⁵-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:! covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.     

Structural and spectroscopic characterisation of a heme peroxidase from sorghum

A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV–visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A Fe^III/Fe^II reduction potential of ?266 mV vs NHE was determined. Stopped-flow experiments with H₂O₂ showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na⁺ ion) and proximal (assigned as a Ca²⁺) sides of the heme, which is consistent with the Ca²⁺-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions.     

Purification of undegraded ceruloplasmin from outdated human plasma

A method for the rapid isolation of homogeneous undegraded ceruloplasmin from outdated human plasma is reported. The procedure consists of a precipitation step with polyethylene glycol 4000, batchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin was purified 1740-fold and the yield from outdated plasma was 67%. The purified ceruloplasmin was found to be homogeneous on anionic polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-PAGE, isoelectric focusing, and low-speed equilibrium centrifugation. The isoelectric point as determined by isoelectric focusing was 4.4. The purified enzyme was sensitive to storage; when a sample was resubmitted to PAGE after 4 months of storage at 4 degrees C, two bands were obtained and the fast-moving band showed no oxidase activity. The molecular weight estimated by gel electrophoresis and sedimentation equilibrium centrifugation was 130,000.     

Effect of covalent links on the structure, spectra, and redox properties of myeloperoxidase--a density functional study

The enzyme myeloperoxidase shows several unusual properties compared to other peroxidases, e.g. a red-shifted absorption spectrum and a peroxidase activity towards chloride. It has been suggested that this is caused by the unusual covalent links between the heme group and the surrounding protein, but whether it is caused by the two ester links to Glu-242 and Asp-94 or the sulfonium ion linkage to Met-243 is unclear. To investigate these suggestions, we have used density functional theory to study the structure, spectra, and reduction potential of 25 models of myeloperoxidase in the reduced (Fe^II) and oxidized (Fe^III) states, as well as in the compound I (formally Fe^VO) and II (Fe^IVO or Fe^IVOH) states, using appropriate models of the linkages to the Asp, Glu, and Met residues (including the back-bone connection between Glu-242 and Met-243) in varying combinations. The calculated spectral shifts indicate that both the ester and sulfonium linkages play a role in the spectral shift. On the other hand, the sulfonium linkage seems to be mainly responsible for the high positive reduction potential for the both ferric/ferrous and compound I/II couples of myeloperoxidase.     

Modeling the reactions of superoxide and myeloperoxidase in the neutrophil phagosome: implications for microbial killing

Neutrophils kill bacteria by ingesting them into phagosomes where superoxide and cytoplasmic granule constituents, including myeloperoxidase, are released. Myeloperoxidase converts chloride and hydrogen peroxide to hypochlorous acid (HOCl), which is strongly microbicidal. However, the role of oxidants in killing and the species responsible are poorly understood and the subject of current debate. To assess what oxidative mechanisms are likely to operate in the narrow confines of the phagosome, we have used a kinetic model to examine the fate of superoxide and its interactions with myeloperoxidase. Known rate constants for reactions of myeloperoxidase have been used and substrate concentrations estimated from neutrophil morphology. In the model, superoxide is generated at several mm/s. Most react with myeloperoxidase, which is present at millimolar concentrations, and rapidly convert the enzyme to compound III. Compound III turnover by superoxide is essential to maintain enzyme activity. Superoxide stabilizes at approximately 25 microM and hydrogen peroxide in the low micromolar range. HOCl production is efficient if there is adequate chloride supply, but further knowledge on chloride concentrations and transport mechanisms is needed to assess whether this is the case. Low myeloperoxidase concentrations also limit HOCl production by allowing more hydrogen peroxide to escape from the phagosome. In the absence of myeloperoxidase, superoxide increases to >100 microM but hydrogen peroxide to only approximately 30 microM. Most of the HOCl reacts with released granule proteins before reaching the bacterium, and chloramine products may be effectors of its antimicrobial activity. Hydroxyl radicals should form only after all susceptible protein targets are consumed.     

A rapid-scan spectrometric and stopped-flow study of compound I and compound II of Pseudomonas cytochrome c peroxidase

A quantitative yield of half-reduced (ferrous-ferric) cytochrome c peroxidase from Pseudomonas aeruginosa has been obtained by using either ascorbate or NADH as reductant of the resting (ferric-ferric) enzyme along with phenazine methosulfate as mediator. The formation of Compounds I and II from the half-reduced enzyme and hydrogen peroxide has been studied at 25 degrees C using rapid-scan spectrometry and stopped-flow measurements. The spectra of Compound I in the Soret and visible regions were recorded within 5 ms after mixing the half-reduced enzyme with H2O2. The spectrum of the primary compound at the Soret region had a maximum at 414 nm, and in the visible region at 528 and 556 nm. The spectrum of Compound I showed no bands in the 650-nm region, excluding the possibility of a pi-cation radical being part of the catalytic mechanism. Compound I was stable for at least 12 s when no reducing equivalents were present. In the presence of reduced azurin, half-reduced enzyme reacted with H2O2 to form Compound II within 50 ms. The spectrum of Compound II had a Soret maximum at 411 nm. In the visible region the Compound II spectrum was close to that of the totally oxidized, resting enzyme form. In the presence of excess azurin, Compound II was converted rapidly to the half-reduced enzyme form. The kinetics of Compound I formation was also followed with peracetic acid, ethylhydroperoxide, and m-chloroperbenzoic acid as electron acceptors. The rate constants of these reactions are diminished compared to that of hydrogen peroxide, indicating a closed structure for the heme pocket of the enzyme.