Peroxidase Compound Research Articles

Symmetry states of metalloporphyrin π-cation radicals, models for peroxidase compound I

Oxoferryl porphyrin π-cation radical active sites of compound I intermediates which are found in enzymes such as peroxidases and catalases have been extensively modeled by oxidized synthetic metalloporphyrins. The electronic symmetry states of these compounds were initially assigned on the basis of electronic absorption data. In recent years new experimental and theoretical results have become available which have led to a re-evaluation and modification of the original assignments. A historical perspective of these developments is provided in the context of recent NMR, resonance Raman, and other spectroscopic data and theoretical calculations for the synthetic models and enzymatic systems.

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 k1=2.0×107 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 pKa 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 pKa 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, k2=(2.5±0.1)×104 M−1 s−1 and k3=(2.2±0.1)×104 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.

To the Mechanism of Horseradish Peroxidase-Mediated Degradation of a Recalcitrant Dye Remazol Brilliant Blue R

Thein vitroenzymatic metabolism of a recalcitrant dye Remazol Brilliant Blue R (RBBR) was investigated using horseradish peroxidase (HRP). At optimum pH (4.5), the apparent Michaelis constant (KM) value for the oxidation of RBBR catalyzed by HRP is 14.8 μmol l-1. HRP-mediated conversion of RBBR proceedsviaa conventional peroxidase reaction, by a sequential one-electron oxidation of two molecules of RBBR by the peroxidase Compounds I and II. The oxidation is inhibited by radical trapping agents (nicotinamide adenine dinucleotide reduced (NADH), ascorbate, glutathione). This confirms that the peroxidase-mediated oxidation of RBBR proceedsviaradical mechanism. Gel permeation profile of the RBBR oxidation products shows that the pattern of molecular weight distribution was shifted to the higher molecular weight region indicating formation of RBBR oligomers. In addition to HRP, the RBBR dye is also oxidized by another peroxidase, the mammalian lactoperoxidase.

Violacein transformation by peroxidases and oxidases: implications on its biological properties

1,3-Dihydro-2H-indol-2-one derivative (violacein) is extracted from Chromobacterium violaceum and presents several biological activities as antibiotic, antitumoral, antichagasic and antioxidant. In order to increase its biological activities and decrease its toxicity, one strategy is to slightly transform the molecules through a specific group transformation. Peroxidases, which are hemoproteins, are known as excellent oxidants producing hydroxylation and ring cleavage. Laccases are phenol oxidases produced by fungi as plants and belong to the oxidase group which complexes copper. This enzyme generates phenolates, quinones and also ring cleavage even in the presence of a mediator as 1-hydroxybenzotriazol (HBT). Lignin peroxidase and manganese peroxidase (LiP/MnP) pool from Phanerochaete chrysosporium, Horseradish peroxidase (HRP-VI) from horseradish, Lactoperoxidase (LPO) from bovine milk and Laccase (Lac) from Trametes versicolor acting on violacein were studied. Kinetics parameters and products distribution indicated a fast and efficient violacein transformation with all of them. HRP-VI acting on violacein was studied in details and spectral evidence indicated that Horseradish peroxidase compound II was formed during the oxidation reaction. Similar behavior with LiP/MnP pool was observed. Laccase, in the presence and absence of mediator (HBT), also showed a rapid violacein transformation. In a more detailed study with the HRP-VI reaction, a hydroxilation in the indol unit and a ring contraction of the pyrrol moiety of violacein molecule occurred.

Kinetics and Mechanism of the H2O2 Electroreduction on the Peroxidase-Promoted Electrodes

The regularities of the bioelectrocatalytic reduction of H2O2 in the presence of peroxidase (POD) or a POD–Nafion composite adsorbed on the surface of carbon black or isotropic pyrocarbon are considered. The effect of the surface coverage with the enzyme, the H2O2 concentration, and the H2O2 supply rate to the electrode on the system activity is studied. Specific activities of three electrode types (POD adsorbed on carbon black or pyrocarbon, and the composite deposited on pyrocarbon) are close to one another. A mechanism for the bioelectrocatalytic reduction of H2O2 in a wide potential range is proposed. At potentials near the steady-state value, the reaction rate is limited by the electrochemical kinetics. At high polarizations, the formation of a POD compound with H2O2 is the limiting stage.

Peroxidases

The family of human peroxidases described includes myeloperoxidase, eosinophil peroxidase, uterine peroxidase, lactoperoxidase, salivary peroxidase, thyroid peroxidase and prostaglandin H1/2 synthases. The chemical identity of the peroxidase compound I and II oxidation states for the different peroxidases are compared. The identities of the distal and proximal amino acids of the catalytic site of each peroxidase are also compared. The gene characteristics and chromosomal location of the human peroxidase family have been tabulated and their molecular evolution discussed. Myeloperoxidase polymorphism and the mutations identified so far that affect myeloperoxidase activity and modulate their susceptibility to disease is described. The mechanisms for hypohalous and hypothiocyanate formation by the various peroxidases have been compared. The cellular function of the peroxidases and their hypohalites have been described as well as their inflammatory effects. The peroxidase catalysed cooxidation of drugs and xenobiotics that results in oxygen activation by redox cycling has been included. Low-density lipoprotein oxidation (initiation of atherosclerosis), chemical carcinogenesis, idiosyncratic drug reactions (e.g. agranulocytosis), liver necrosis or teratogenicity initiated by the cooxidation of endogenous substrates, plasma amino acids, drugs and xenobiotics catalysed by peroxidases or peroxidase containing cells have also been compared. Finally, peroxidase inhibitors currently in use for treating various diseases are described.

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.

Application of Marcus theory of electron transfer for the reactions between HRP compound I and II and 2,4-disubstituted phenols

Reactions between horseradish peroxidase (HRP) compound I and II and some natural phenolic antioxidants were studied at pH 7. The bimolecular rate constants for these reactions were determined using a sequential mixing stopped-flow spectrometer. The rate constants for the reactions of compound I were found to be two orders of magnitude higher than those for compound II. The phenols under study showed a significant difference in their one-electron reduction potential values. As the rate constants also changed systematically with their one-electron potentials, the Marcus theory of electron transfer was applied to the above determined rate constants and the thermodynamic driving force (Δ G°), from which the reorganization energy (λ) for the electron transfer from phenols to both compound I and II was estimated.

Reactivity of horseradish peroxidase compound II toward substrates: kinetic evidence for a two-step mechanism.

Transient kinetic analysis of biphasic, single turnover data for the reaction of 2,2'-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS) with horseradish peroxidase (HRPC) compound II demonstrated preequilibrium binding of ABTS (k(+5) = 7.82 x 10(4) M(-)(1) s(-)(1)) prior to rate-limiting electron transfer (k(+6) = 42.1 s(-)(1)). These data were obtained using a stopped-flow method, which included ascorbate in the reaction medium to maintain a low steady-state concentration of ABTS (pseudo-first-order conditions) and to minimize absorbance changes in the Soret region due to the accumulation of ABTS cation radicals. A steady-state kinetic analysis of the reaction confirmed that the reduction of HRPC compound II by this substrate is rate-limiting in the complete peroxidase cycle. The reaction of HRPC with o-diphenols has been investigated using a chronometric method that also included ascorbate in the assay medium to minimize the effects of nonenzymic reactions involving phenol-derived radical products. This enabled the initial rates of o-diphenol oxidation at different hydrogen peroxide and o-diphenol concentrations to be determined from the lag period induced by the presence of ascorbate. The kinetic analysis resolved the reaction of HRPC compound II with o-diphenols into two steps, initial formation of an enzyme-substrate complex followed by electron transfer from the substrate to the heme. With o-diphenols that are rapidly oxidized, the heterolytic cleavage of the O-O bond of the heme-bound hydrogen peroxide (k(+2) = 2.17 x 10(3) s(-)(1)) is rate-limiting. The size and hydrophobicity of the o-diphenol substrates are correlated with their rate of binding to HRPC, while the electron density at the C-4 hydroxyl group predominantly influences the rate of electron transfer to the heme.

Nicotinamide adenine dinucleotide species in the horseradish peroxidase-oxidase oscillator.

NADH chemistry ancillary to the oscillatory peroxidase-oxidase (PO) reaction has been reexamined. Previously, (NAD)2 has been thought of as a terminal, inert product of the PO reaction. We now show that (NAD)2 is a central reactant in this system. Although we found traces of the dimer after several hours of the PO reaction, no accumulation of the dimer occurred, regardless of the reaction time or the number of oscillations. (NAD)2 can convert horseradish peroxidase (HRP) compound I (CpI) to compound II (CpII) with apparent rate constant (2.7 +/- 0.2) x 105 M-1.s-1 and CpII to HRP at 1 x 105 M-1.s-1. Moreover, a reduction of HRP compound III (CpIII) to CpI by (NAD)2 occurs with a rate constant faster than 5 x 106 M-1.s-1. The (NAD)2 reduction of CpIII provides an alternative to the reduction by NAD radical suggested by Yokota and Yamazaki. HRP catalyzes oxidation of alpha-NADH, not only the beta anomer as previously assumed. Rate constants of alpha- and beta-NADH reactions with CpI are (7.4 +/- 0.4) x 105 M-1.s-1, and (1.7 +/- 0.2) x 105 M-1.s-1, and with CpII are estimated as 5 x 104 M-1.s-1, and 4 x 104 M-1.s-1. Apparent rate constants of reduction of methylene blue (MB) to leuco-methylene blue (MBH) are 3.8 x 104 M-1.s-1 for NADH and 6.4 x 104 M-1.s-1 for NAD dimer, (NAD)2, while reoxidation of MBH proceeds at (2.1 +/- 0.2) x 103 M-1.s-1 All the rates were measured in 0.1 M acetate buffer, pH 5.1.

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.

Nucleotide sequence analysis, overexpression in Escherichia coli and kinetic characterization of Anacystis nidulans catalase-peroxidase

Bifunctional catalase-peroxidases are the least understood type of peroxidases. A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Anacystis nidulans ( Synechococcus PCC 6301) is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the examination of both the spectral characteristics and the reactivity of its redox intermediates by using the multi-mixing stopped-flow technique. The homodimeric acidic protein (pI = 4.6) contained high catalase activity (apparent K m = 4.8 mM and apparent k cat = 8850 s –1). Cyanide is shown to be an effective inhibitor of the catalase reaction. The second-order rate constant for cyanide binding to the ferric protein is (6.9 ± 0.2) × 10 5 M –1 s –1 at pH 7.0 and 15 °C and the dissociation constant of the cyanide complex is 17 μM. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. The apparent second-order rate constant for formation of compound I from the ferric enzyme and peroxoacetic acid is (1.3 ± 0.3) × 10 4 M –1 s –1 at pH 7.0 and 15 °C. The spectrum of compound I is characterized by about 40% hypochromicity, a Soret region at 406 nm, and isosbestic points between the native enzyme and compound I at 355 and 428 nm. Rate constants for reduction of KatG compound I by o-dianisidine, pyrogallol, aniline and isoniazid are shown to be (7.3 ± 0.4) × 10 6 M –1 s –1, (5.4 ± 0.3) × 10 5 M –1 s –1, (1.6 ± 0.3) × 10 5 M –1 s –1 and (4.3 ± 0.2) × 10 4 M –1 s –1, respectively. The redox intermediate formed upon reduction of compound I did not exhibit the classical red-shifted peroxidase compound II spectrum which characterizes the presence of a ferryl oxygen species. Its spectral features indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.

On the role of methylene blue in the oscillating peroxidase–oxidase reaction

The role of methylene blue (MB) and its interaction with various enzymic and nonenzymic intermediates in the oscillating peroxidase–oxidase reaction were investigated. Time dependent spectral changes were deconvoluted into time series of the different intermediates. Ferric peroxidase, ferrous peroxidase, and peroxidase compound III account for most of the spectral changes, while the two remaining oxidation states of the enzyme (compound I and II) are only present in very low amounts during any phase of the oscillations. The presence of MB is required for the generation of sustained oscillations and complex dynamics. The amount of ferrous peroxidase formed during an oscillatory cycle decreases with increasing concentrations of MB. At ‘‘high ’’ concentrations of MB only damped oscillations are observed. These findings are interpreted in terms of either a competition between MB and other reactants for NAD• radicals, or of a reaction between MB and ferrous peroxidase. In the absence of MB only damped oscillations can be observed due to slow and irreversible degradation of the enzyme. This suggests that MB also protects the enzyme against degradation.

Role of Protein Environment in Horseradish Peroxidase Compound I Formation: Molecular Dynamics Simulations of Horseradish Peroxidase−HOOH Complex

The signature feature of the enzymatic cycle of the peroxidase family of metabolizing heme proteins is formation of the catalytically active compound I species from the inactive ferric resting form, via a putative transient peroxide bound intermediate. While there is some evidence for this intermediate, the mechanism of formation of compound I from it and the role of nearby amino acids in facilitating it are still unresolved. To further probe this mechanism and investigate the possible role of the protein in compound I formation, molecular dynamics simulations of the peroxide bound complex of horseradish peroxidase isoenzyme C (HRP-C−HOOH) were performed. For such a typical peroxidase, a role of two conserved amino acids in the distal binding pocket, histidine and arginine, has been suggested in facilitating the peroxide O−O bond cleavage necessary for compound I formation. Since HRP functions cover a wide range of pH values, protein simulations were carried out for two models differing only in the state of protonation of the conserved histidine. The neutral histidine corresponds to a high-pH model, and the cationic histidine corresponds to a low-pH model. The unique robust H bonds identified in the molecular dynamics simulations of the two models suggest two different modes of binding of the peroxide to the heme iron, different mechanisms of compound I formation, and a different role for the key HRP residues involved in its formation in the two models.

The Reaction Rates of NO with Horseradish Peroxidase Compounds I and II

In this study the reactions between nitric oxide (NO) and horseradish peroxidase (HRP) compounds I and II were investigated. The reaction between compound I and NO has biphasic kinetics with a clearly dominant initial fast phase and an apparent second-order rate constant of (7.0 ± 0.3) × 105 M−1 s−1 for the fast phase. The reaction of compound II and NO was found to have an apparent second-order rate constant of kapp = (1.3 ± 0.1) × 106 M−1 s−1 or (7.4 ± 0.7) × 105 M−1 s−1 when measured at 409 nm (the isosbestic point between HRP and HRP–NO) and 419 nm (λmax of compound II and HRP–NO), respectively. Interestingly, the reaction of compound II with NO is unusually high relative to that of compound I, which is usually the much faster reaction. Since horseradish peroxidase is prototypical of mammalian peroxidases with respect to the oxidation of small substrates, these results may have important implications regarding the lifetime and biochemistry of NO in vivo after inflammation where both NO and H2O2 generation are increased several fold.

A First-Principles Quantum Chemical Analysis of the Factors Controlling Ruffling Deformations of Porphyrins: Insights from the Molecular Structures and Potential Energy Surfaces of Silicon, Phosphorus, Germanium, and Arsenic Porphyrins and of a Peroxidase Compound I Model

Using nonlocal density functional theory calculations, we have examined several factors influencing ruffling deformations of porphyrin and porphyrazine complexes. Because a ruffling distortion is often a direct result of a small macrocycle core size, which, in turn, is brought about by complexation of a central ion with a small ionic radius, this study focuses on the conformations and potential energy surfaces of porphyrin complexes with small central ions (herafter symbolized M) such as SiIV, PV, GeIV, and AsV. The optimized geometries exhibit ruffling torsion angles ranging from 0° for (P)GeIVF2 and (P)SiIV(C⋮CPh)2 to about 55° for [(P)PVF2]+. For relatively substantial ruffling distortions, a good linear correlation has been found between the ruffling torsion angle and the M−N distance for a wide variety of central ions including transition metals, for sterically unhindered porphyrins, and for a database including both experimental and optimized structures. The threshold between ruffling and planar structures is at M−N bond distances of 2.00−2.02 Å for sterically unhindered porphyrins and at 1.85−1.87 Å for porphyrazines. The calculations confirm an experimental observation that electron-withdrawing axial ligands lead to increased ruffling, especially for phosophorous and silicon porphyrins. The ortho hydrogens of axial phenyl ligands and the 2- and 6- hydrogens of axial pyridine ligands can sterically interfere with the porphyrin and contribute to ruffling. In addition to the M−N distance, a number of other geometrical parameters also vary systematically with the ruffling distortion. Thus, the Cα−Cmeso−Cα angle decreases with ruffling and the CβCβ distance and Cα−N−Cα angle increase with ruffling. These structural variations are reflected in a number of ruffling-sensitive vibrational frequencies.. The ruffled D2d geometries of [(P)PVF2]+ and [(P)PVCl2]+ are stabilized by 9.25 and 5.26 kcal/mol, respectively, relative to planar D4h symmetry-constrained optimized geometries. In contrast, the ruffled D2d geometries of [(Pz)PVF2]+ and of all the silicon complexes studied are more stable than the corresponding D4h symmetry-constrained optimized geometries by less than 0.01 kcal/mol. This underscores the extreme softness of ruffling deformations and shows that even fairly large distortions, where the ruffling torsion angle changes by up to 25°, can occur with almost no expenditure of energy. Finally, through a reinvestigation of a recent study of a peroxidase compound I model, we have uncovered a heretofore unsuspected role of Fe(3dxy)-porphyrin(atu) orbital interactions in macrocycle ruffling. Also seen for certain low-spin ferrihemes, this orbital interaction is not important for manganese porphyrins.

Peroxidase Activity of Myoglobin Is Enhanced by Chemical Mutation of Heme-Propionates

Peroxidase activity of a myoglobin reconstituted with a chemically modified heme 1 is reported. The heme 1 bearing a total of eight carboxylates bound to the terminal of propionate side chains is incorporated into apomyoglobin from horse heart to obtain a new reconstituted myoglobin, rMb(1), with a unique binding domain structure. The UV−vis, CD, and NMR spectra of rMb(1) are comparable with those of native myoglobin, nMb. The mixing of rMb(1) with hydrogen peroxide yields a peroxidase compound II-like species, rMb(1)-II, since the spectrum of rMb(1)-II is identical with that observed for nMb. Stoichiometric oxidation of several small molecules by rMb(1)-II, demonstrates the significant reactivity. (i) The oxidation of cationic substrate such as [Ru(NH3)6]2+ by rMb(1)-II is faster than that observed for oxoferryl species of nMb, nMb-II. (ii) Anionic substrates such as ferrocyanide are unsuitable for the oxidation by rMb(1)-II. (iii) Oxidations of catechol, hydroquinone, and guaiacol are dramatically enhanced by rMb(1)-II (14−32-fold) compared to those observed for nMb-II. Thus, the chemical modification of heme-propionates can alter substrate specificity. Steady-state kinetic measurements indicate that both the reactivity and substrate affinity toward guaiacol oxidation by rMb(1) are improved, so that the specificity, kcat/Km, is 13-fold higher than that in nMb. This result strongly suggests that the artificially modified heme-propionates may increase the accessibility of neutral aromatic substrates to the heme active site. The present work demonstrates that the chemical mutation of prosthetic group is a new strategy to create proteins with engineered function.

Spectral and kinetic studies of the oxidation of monosubstituted phenols and anilines by recombinant Synechocystis catalase-peroxidase compound I.

A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Synechocystis PCC 6803 is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the first examination of second-order rate constants for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by a bifunctional catalase-peroxidase compound I using the sequential-mixing stopped-flow technique. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. A >/=50-fold excess of peroxoacetic acid is required to obtain a spectrum of relatively pure and stable compound I which is characterized by about 40% hypochromicity, a Soret maximum at 406 nm, and isosbestic points between the native enzyme and compound I at 357 and 430 nm. The apparent second-order rate constant for formation of compound I from ferric enzyme and peroxoacetic acid is (8.74 +/- 0.26) x 10(3) M(-)(1) s(-)(1) at pH 7. 0. Reduction of compound I by aromatic donor molecules is dependent upon the substituent effect on the benzene ring. The apparent second-order rate constants varied from (3.6 +/- 0.1) x 10(6) M(-)(1) s(-)(1) for p-hydroxyaniline to (5.0 +/- 0.1) x 10(2) M(-)(1) s(-)(1) for p-hydroxybenzenesulfonic acid. They are shown to correlate with the substituent constants in the Hammett equation, which suggests that in bifunctional catalase-peroxidases the aromatic donor molecule donates an electron to compound I and loses a proton simultaneously. The value of rho, the susceptibility factor in the Hammett equation, is -3.4 +/- 0.4 for the phenols and -5.1 +/- 0.8 for the anilines. The pH dependence of compound I reduction by aniline exhibits a relatively sharp maximum at pH 5. The redox intermediate formed upon reduction of compound I has spectral features which indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.

Saddle Distortions of Ferryl-Porphyrin Models for Peroxidase Compound I: A Density Functional Study

Saddle Distortions of Ferryl-Porphyrin Models for Peroxidase Compound I: A Density Functional Study

Kinetic evidence for the formation of a Michaelis‒Menten-like complex between horseradish peroxidase compound II and di-(N-acetyl-L-tyrosine)

The formation of a reversible adsorption complex between a dimer of N-acetyl-L-tyrosine [di-(N-acetyl-ʟ-tyrosine), (NAT)2] and horseradish peroxidase (HRP) compound II (CII) was demonstrated using a kinetic approach. A specific K value (0.58 mM) was deduced for this step from stopped-flow measurements. The dimerization of the dipeptide Gly-Tyr was analysed at the steady state and compared with (NAT)2 dimerization [(NAT)2 → (NAT)4]. A saturation of the enzyme was observed for both substrates within their range of solubility. In each case the rate of dimerization reflected the rate-limiting step of compound II reduction to the native HRP (E) (k/K≈ kII → E). The k values for (Gly-Tyr)2 and (NAT)4 formation were 254 s-1 and 3.6 s-1 respectively. The K value of Gly-Tyr was 24 mM. It was observed that the value (0.7 mM) for (NAT)2 was close both to its specific K value for the second step of reduction (CII → E) and to its thermodynamic dissociation constant (Kd = 0.7 mM) with the resting form of the enzyme. As (NAT)2 was a tighter ligand but a poorer substrate than Gly-Tyr, a steady-state kinetic study was performed in the presence of both substrates. A kinetic model which includes an enzyme-substrate adsorption prior to each of the two steps of reduction was derived. This one agreed reasonably well with the experimental data.