Horseradish peroxidase. Complex formation with anions and hydrocyanic acid

Equilibrium binding experiments have been performed with perchlorate, chloride, and acetate in the presence of horseradish peroxidase. The binding of perchlorate and acetate appears to be like that of nitrate, at a site other than the sixth coordination position of the heme iron. Competitive experiments using both nitrate and cyanide demonstrate that two different binding sites are present on the enzyme. Chloride appears to bind at the sixth coordination position as do both fluoride and cyanide. Temperature jump experiments indicate that it is likely the nitrate anion and not undissociated nitric acid which is the binding species. Competitive stopped flow experiments indicate that the bound nitrate slows both the association rate and dissociation rate of cyanide, indicating that nitrate binds close to the sixth coordination position.     

Horseradish peroxidase. XXXIV. Oxidation of compound II to I by periodate and inorganic anion radicals.

The one-electron oxidation of horseradish peroxidase compound II to compound I by sodium periodate was observed. The bimolecular rate constant for the NaIO4--compound II interaction is equal to 9.5 +/- 1 x 10(-3) M-1s-1 at room temperature. Irradiation, using ultraviolet light, of the solution containing compound II and persulfate in the presence of bicarbonate, chloride, or bromide, leads ot the fast accumulation of compound I due to the oxidative action of SO4, CO3, Cl2, and Br2 anion radicals, which are products of the photolysis.     

Horseradish peroxidase. XXXII. pH dependence of the oxidation of L-(-)-tyrosine by compound I.

The rate of oxidation of L-(-)-tyrosine by horseradish peroxidase compound 1 has been studied as a function of pH at 25 degrees C and ionic strength 0.11. Over the pH range of 3.20--11.23 major effects of three ionizations were observed. The pKa values of the phenolic (pKa = 10.10) and amino (pKa = 9.21) dissociations of tyrosine and a single enzyme ionization (pKa = 5.42) were determined from nonlinear least squares analysis of the log rate versus pH profile. It was noted that the less acidic form of the enzyme was most reactive; hence, the reaction is described as base catalyzed. The rate of tyrosine oxidation falls rapidly with the deprotonation of the phenolic group.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

Spectral and kinetic studies on the formation of eosinophil peroxidase compound I and its reaction with halides and thiocyanate

Compound I of peroxidases takes part in both the peroxidation and the halogenation reaction. This study for the first time presents transient kinetic measurements of the formation of compound I of human eosinophil peroxidase (EPO) and its reaction with halides and thiocyanate, using the sequential-mixing stopped-flow technique. Addition of 1 equiv of hydrogen peroxide to native EPO leads to complete formation of compound I. At pH 7 and 15 degrees C, the apparent second-order rate constant is (4.3 +/- 0.4) x 10(7) M(-1) s(-1). The rate for compound I formation by hypochlorous acid is (5.6 +/- 0.7) x 10(7) M(-1) s(-1). EPO compound I is unstable and decays to a stable intermediate with a compound II-like spectrum. At pH 7, the two-electron reduction of compound I to the native enzyme by thiocyanate has a second-order rate constant of (1.0 +/- 0. 5) x 10(8) M(-1) s(-1). Iodide [(9.3 +/- 0.7) x 10(7) M(-1) s(-1)] is shown to be a better electron donor than bromide [(1.9 +/- 0.1) x 10(7) M(-1) s(-1)], whereas chloride oxidation by EPO compound I is extremely slow [(3.1 +/- 0.3) x 10(3) M(-1) s(-1)]. The pH dependence studies suggest that a protonated form of compound I is more competent in oxidizing the anions. The results are discussed in comparison with those of the homologous peroxidases myeloperoxidase and lactoperoxidase and with respect to the role of EPO in host defense and tissue injury.     

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.     

Oxidation of horseradish peroxidase compound II to compound I.

In the reaction between equimolar amounts of horseradish peroxidase and chlorite, the native enzyme is oxidized directly to Compound II (Hewson, W.D., and Hager, L.P. (1979) J. Biol. Chem. 254, 3175-3181). At acidic pH but not at alkaline values, this initial reaction is followed by oxidation of Compound II to Compound I. The highly pH-dependent chemistry of Compound II can be readily demonstrated by the reduction of Compound I, with ferrocyanide at acidic, neutral, and alkaline pH values. Titration at low pH yields very little Compound II, whereas at high pH, the yield is quantitative. Similarly, the reaction of horseradish peroxidase and chlorite at low pH yields Compound I while only Compound II is formed at high pH. At intermediate pH values both the ferrocyanide reduction and the chlorite reaction produce intermediate yields of Compound II. This behavior is explained in terms of acidic and basic forms of Compound II. The acidic form is reactive and unstable relative to the basic form. Compound II can be readily oxidized to Compound I by either chloride or chlorine dioxide in acidic solution. The oxidation does not occur in alkaline solution, nor will hydrogen peroxide cause the oxidation of Compound II, even at low pH.     

Horseradish peroxidase. XXXVII. Compound I formation from reconstituted enzyme lacking free carboxyl groups as heme side chains

Recombination of apo horseradish peroxidase with 2,4 dimethyldeutero hemin and its mono- and dimethyl esters was performed. The number of free carboxyl side chains in these three hemins is 2, 1 and 0 respectively. Despite such a difference, all of these three reconstituted enzymes can react with H₂O₂ to produce compound I. The second order rate constants for compound I formation are 1.3 × 10⁷ M^?1s^?1, 8.5 × 10⁶ M^?1s^?1 and 5.9 × 10⁶ M^?1s^?1. Therefore the propionate side chain of hemin has no direct role in compound I formation.     

Thermodynamics of the two step formation of horseradish peroxidase compound I

The effects of temperature (20 to -38 degrees C), pressure (normal pressures to 1.2 kbar) and solvent (water, 60% DMSO and 50% methanol) on the reaction of hydrogen peroxide or ethyl peroxide with horseradish peroxidase were studied. The formation of compound I was followed at 403 nm in a stopped flow apparatus adapted for high pressure and low temperature work. As with the alkaline form (Job and Dunford 1978), the neutral form of the peroxidase binds peroxide substrates in two steps. It was the combined use of organic solvents and low temperatures which revealed saturation kinetics: (Formula: see text) compound I, where E = horseradish peroxidase and S peroxide substrate. In water and organic solvents at temperatures above -10 degrees C, K1 was too small and k2 too large to be measured, here K1 X k2 was obtained. k-2 was too small for measurement under all conditions. Whereas K1 was insensitive to the peroxide substrate and solvent composition, k2 was very sensitive. The thermodynamic parameters delta H, delta S and delta V for K1 and k2 were obtained under different experimental conditions and the data are interpreted within the available thermodynamic theories.     

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol–disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV–Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK_a of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 10⁷ M^? 1 s^? 1) and peroxynitrite ((3.7 ± 0.4) × 10⁵ M^? 1 s^? 1) at pH 7.4 and 25 °C.     

Horseradish peroxidase. XXXVI. On the difference between peroxidase and metmyoglobin.

A mechanism for the reaction of hydrogen peroxide with horseradish peroxidase is proposed which involves the catalytic activity of the carboxylate side chain of aspartate residue 43. The corresponding residue in the active site of metmyoglobin is glycine E8, which explains the inability of metmyoglobin to form compound I. Certain aspects of the proposed peroxidase mechanism may be relevant to the catalytic triad for the serine proteases.     

Lignin peroxidase compound III. Mechanism of formation and decomposition

Lignin peroxidase compound III (LiPIII) was prepared via three procedures: (a) ferrous LiP + O2 (LiPIIIa), (b) ferric LiP + O2-. (LiPIIIb), and (c) LiP compound II + excess H2O2 followed by treatment with catalase (LiPIIIc). LiPIIIa, b, and c each have a Soret maximum at approximately 414 nm and visible bands at 543 and 578 nm. LiPIIIa, b, and c each slowly reverted to native ferric LiP, releasing stoichiometric amounts of O2-. in the process. Electronic absorption spectra of LiPIII reversion to the native enzyme displayed isosbestic points in the visible region at 470, 525, and 597 nm, suggesting a single-step reversion with no intermediates. The LiPIII reversion reactions obeyed first-order kinetics with rate constants of approximately 1.0 X 10(-3) s-1. In the presence of excess peroxide, at pH 3.0, native LiP, LiPII, and LiPIIIa, b, and c are all converted to a unique oxidized species (LiPIII*) with a spectrum displaying visible bands at 543 and 578 nm, but with a Soret maximum at 419 nm, red-shifted 5 nm from that of LiPIII. LiPIII* is bleached and inactivated in the presence of excess H2O2 via a biphasic process. The fast first phase of this bleaching reaction obeys second-order kinetics, with a rate constant of 1.7 X 10(1) M-1 s-1. Addition of veratryl alcohol to LiPIII* results in its rapid reversion to the native enzyme, via an apparent one-step reaction that obeys second-order kinetics with a rate constant of 3.5 X 10(1) M-1 s-1. Stoichiometric amounts of O2-. are released during this reaction. When this reaction was run under conditions that prevented further reactions, HPLC analysis of the products demonstrated that veratryl alcohol was not oxidized. These results suggest that the binding of veratryl alcohol to LiPIII* displaces O2-., thus returning the enzyme to its native state. In contrast, the addition of veratryl alcohol to LiPIII did not affect the rate of spontaneous reversion of LiPIII to the native enzyme.     

Studies on compound I formation of the lignin peroxidase from Phanerochaete chrysosporium

Ligninase, isolated from the wood-destroying fungus Phanerochaete chrysosporium, catalyzes the oxidation of lignin and lignin-related compounds. Ligninase reacts with H2O2 to form the classical peroxidase intermediates Compounds I and II. We have determined the activation energy of ligninase Compound I formation to be 5.9 kcal/mol. The effect of pH and ionic strength on the rate of ligninase Compound I formation was studied. In contrast to all other peroxidases, no pH effect was observed. This is despite homology of active-site amino acids residues (Tien, M., and Tu, C.-P. D. (1987) Nature 326, 520-523) which are proposed to affect the pH profile of Compound I formation. Ligninase Compound I formation can also be supported by organic peroxides. The second-order rate constants with the organic peroxides are lower, suggesting that H2O2 is the preferred substrate.     

Oxidation of p-cresol by horseradish peroxidase compound I.

Rate constants for the reaction between horseradish peroxidase compound I and p-cresol have been determined at several values of pH between 2.98 and 10.81. These rate constants were used to construct a log (rate) versus pH profile from which it is readily seen that the most reactive form of the enzyme is its most basic form within this pH range so that base catalysis is occurring. At the maximum rate a second order rate constant of (5.1 +/- 0.3) x 10(-7) M-1 s-1 at 25 degrees is obtained. The activation energy of the reaction at the maximum rate was determined from an Arrhenius plot to be 5.0 +/- 0.5 kcal/mol. Evidence for an exception to the generally accepted enzymatic cycle of horseradish peroxidase is presented. One-half molar equivalent of p-cresol can convert compound I quantitatively to compound II at high pH, whereas usually this step requires 1 molar equivalent of reductant. The stoichiometry of this reaction is pH-dependent.     

EPR studies on compound I of horseradish peroxidase.

Compound I of horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase EC 1.11.1.7) was studied by EPR at low temperatures. An asymmetric signal was found, about 15 Gauss wide and with a g-value of 1.995, which could be detected only at temperatures below 20 K and which had an intensity corresponding to about 1% of the heme content. In a titration with H2O2, the signal intensity was proportional to the concentration of Compound I, reaching a maximum when equivalent amounts of H2O2 were added. This indicates that the signal is not due to an impurity, and it is suggested that a free radical is formed, relaxed by a near-by fast-relaxing iron.     

The kinetics of formation of horseradish peroxidase compound I by reaction with peroxobenzoic acids. pH and peroxo acid substituent effects.

1. The kinetics of formation of horseradish peroxidase Compound I were studied by using peroxobenzoic acid and ten substituted peroxobenzoic acids as substrates. Kinetic data for the formation of Compound I with H2O2 and for the reaction of deuteroferrihaem with H2O2 and peroxobenzoic acids, to form a peroxidatically active intermediate, are included for comparison. 2. The observed second-order rate constants for the formation of Compound I with peroxobenzoic acids decrease with increasing pH, in the range pH 5-10, in contrast with pH-independence of the reaction with H2O2. The results imply that the formation of Compound I involves a reaction between the enzyme and un-ionized hydroperoxide molecules. 3. The maximal rate constants for Compound I formation with unhindered peroxobenzoic acids exceed that for H2O2. Peroxobenzoic acids with bulky ortho substituents show marked adverse steric effects. The pattern of substituent effects does not agree with expectations for an electrophilic oxidation of the enzyme by peroxoacid molecules in aqueous solution, but is in agreement with that expected for a reaction involving nucleophilic attack by peroxo anions. 4. Possible reaction mechanisms are considered by which the apparent conflict between the pH-effect and substituent-effect data may be resolved. A model in which it is postulated that a negatively charged 'electrostatic gate' controls access of substrate to the active site and may also activate substrate within the active site, provides the most satisfactory explanation for both the present results and data from the literature.     

Kinetic evidence of horseradish peroxidase oxidation by compound I.

The kinetics of compound II formation, obtained upon mixing a highly purified horseradish peroxidase and hydrogen peroxide, was spectrophotometrically studied at three wavelengths in the absence of an added reducing agent. Our experiments confirm George's finding that more than one mole of compound II is formed per mole of hydrogen peroxide added. The new mechanism that we propose, contrary to the mechanism of George, is only valid when compound II is obtained in the absence of an added donor. Moreover, it is not inconsistent with the classical Chance mechanism of oxidation of an added donor by the system peroxidase -- hydrogen peroxide. According to this new mechanism, in the absence of an added donor, compound II formation involved two pathways. The first pathway is the monomolecular reduction of compound I by the endogenous donor, and the second pathway is the formation of two moles of compound II through the oxidoreduction reaction between one mole of peroxidase and one mole of compound I.