Preliminary studies on quinoprotein glucose dehydrogenase under extreme conditions of temperature and pressure.

The kinetics of the reduction of the quinoprotein glucose dehydrogenase by substrate were studied as a function of 3 parameters: pressure (1-1000 bar), temperature (down to -25 degrees C) and solvent (water and 40% dimethyl sulfoxide, DMSO) using a high-pressure low-temperature stopped-flow apparatus. A 2-step formation of the reduced enzyme by its substrate (xylose), was observed. A rapid equilibrium described by the constant K1 was followed by a slower process described by the constants k2 and k-2. By using the transition state theory, the thermodynamic quantities delta V (activation volumes) were determined for these various kinetics constants under different experimental conditions. The results are discussed in terms of conformational change and solvation effect on the protein shell, and compared with results obtained for other systems as the 2-step formation of horseradish peroxidase compound I.     

Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism

The catalytic constant (k(cat)) and the second-order association constant of compound II with reducing substrate (k(5)) of horseradish peroxidase C (HRPC) acting on phenols and anilines have been determined from studies of the steady-state reaction velocities (V(0) vs. [S(0)]). Since k(cat)=k(2)k(6)/k(2)+k(6), and k(2) (the first-order rate constant for heterolytic cleavage of the oxygen-oxygen bond of hydrogen peroxide during compound I formation) is known, it has been possible to calculate the first-order rate constant for the transformation of each phenol or aniline by HRPC compound II (k(6)). The values of k(6) are quantitatively correlated to the sigma values (Hammett equation) and can be rationalized by an aromatic substrate oxidation mechanism in which the substrate donates an electron to the oxyferryl group in HRPC compound II, accompanied by two proton additions to the ferryl oxygen atom, one from the substrate and the other the protein or solvent. k(6) is also quantitatively correlated to the experimentally determined (13)C-NMR chemical shifts (delta(1)) and the calculated ionization potentials, E (HOMO), of the substrates. Similar dependencies were observed for k(cat) and k(5). From the kinetic analysis, the absolute values of the Michaelis constants for hydrogen peroxide and the reducing substrates (K(M)(H(2)O(2)) and K(M)(S)), respectively, were obtained.     

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation thermodynamics of the binding of carbon monoxide to horseradish peroxidase. Role of pressure, temperature and solvent

The kinetics at 423 nm of the binding of carbon monoxide to ferrous horseradish peroxidase were studied as a function of three parameters: pressure (1-1200 bar), temperature (34 to -20 degrees C) and solvent (water, 40% ethylene glycol, 50% methanol) using a high-pressure stopped-flow apparatus. By using transition state theory the thermodynamic quantities delta V, delta S and delta H were determined under these different experimental conditions and were found to be greatly modulated by the physico-chemical parameters of the media. The results suggest that the macroscopic thermodynamic response is mainly controlled by the solvent. By adjusting two variables (among T, P, solvent), it is possible either to amplify or to cancel out the effect of the third.     

Effects of pressure and temperature on the reactions of horseradish peroxidase with hydrogen cyanide and hydrogen peroxide.

Reactions of ferric horseradish peroxidase with hydrogen cyanide and hydrogen peroxide were studied as a function of pressure. Activation volumes are small and differ in sign (delta V = 1.7 +/- 0.5 ml/mol for peroxidase + HCN and -1.5 +/- 0.5 ml/mol for peroxidase + H2O2). The temperature dependence of cyanide binding to horseradish peroxidase was also determined. A comparison is made of relevant parameters for cyanide binding and compound I formation.     

Redox intermediates of plant and mammalian peroxidases: a comparative transient-kinetic study of their reactivity toward indole derivatives.

A comparative study on the reactivity of five indole derivatives (tryptamine, N-acetyltryptamine, tryptophan, melatonin, and serotonin), with the redox intermediates compound I (k2) and compound II (k3) of the plant enzyme horseradish peroxidase (HRP) and the two mammalian enzymes lactoperoxidase (LPO) and myeloperoxidase (MPO), was performed using the sequential-mixing stopped-flow technique. The calculated bimolecular rate constants (k2, k3) revealed substantial differences regarding the oxidazibility of the substrates by redox intermediates at pH 7.0 and 25 degrees C. With HRP it was shown that k2 and k3 are mainly determined by the reduction potential (Eo') of the substrate with k2 being 7-45 times higher than k3. Compound I of mammalian peroxidases was a much better oxidant than HRP compound I with the consequence that the influence of the indole structure on k2 of LPO and MPO was small varying by a factor of only 88 and 38, respectively, which is in strong contrast to a factor of 160,000 determined for k2 of HRP. Interestingly, the k3 values for all three enzymes were very similar. Oxidation of substrates by mammalian peroxidase compound II is strongly constrained by the nature of the substrate. The k3 values for the five indoles varied by a factor of 3,570 (LPO) and 200,000 (MPO), suggesting that the reduction potential of compound II of mammalian peroxidase is less positive than that of compound I, which is in contrast to the plant enzyme.     

Formation of a bioconjugate composed of hemin, smectite, and quaternary ammonium chloride that is soluble and active in hydrophobic media

Hemin (Fe(3+)) was adsorbed onto synthetic smectite (clay mineral) intercalated with a quaternary alkenylammonium compound, dioleyldimethylammonium chloride (DOA), to form a hemin-smectite-DOA conjugate. The hemin-smectite-DOA conjugate was soluble in organic solvents such as benzene and toluene to form a transparent colloidal solution with a light yellow color. Its absorption spectrum in benzene showed two bands, 600 and 568 nm, in the visible region and a sharp Soret band at 400 nm with the molar extinction coefficient of 7.5 x 10(4) M(-1) cm(-1). The formation of the conjugate of smectite and DOA was confirmed by X-ray diffraction analysis: the basal spacing, d(001), of hemin-smectite-DOA conjugate was 19 A which is an expansion of the interlayer space by 5 A based upon the basal spacing of smectite of 14 A. Hemin-smectite-DOA conjugate catalyzed the peroxidase-like reaction in organic solvents using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The temperature-dependent peroxidase-like activity of the conjugate was compared with peroxidase activity of horseradish peroxidase. The hemin-smectite-DOA conjugate exhibited higher activity as the temperature was increased from 30 to 70 degrees C, while horseradish peroxidase activity was reduced as the temperature was increased.     

Solvent effect on lipase enantioselectivity. Evidence for the presence of two thermodynamic states

The enantioselectivity of lipase-catalyzed kinetic resolutions has been measured at various temperatures in binary mixtures of solvents. Varying the solvent composition and temperature had a profound effect on the enantiomeric ratio. The values for delta delta H(R-S)(#) and delta delta S(R-S)(#), calculated from the E values measured at various temperatures, were estimated as a function of the solvent composition. By plotting delta delta H(R-S)(#) versus delta delta S(R-S)(#) as a function of the solvent composition, an extreme was observed. The resulting "hairpin-type" enthalpy-entropy compensation plots can be described by assuming the presence of two thermodynamically distinct physical states, displaying different enantioselectivities, that are in equilibrium with one another. Changing the solvent composition results in a change in the equilibrium constant K(eq) for the two states. The intriguing bell-shaped curves of the enantioselectivity versus solvent composition observed for lipase-catalyzed kinetic resolutions can be described assuming a linear correlation for the logarithm of K(eq) and the solvent composition. Thus, a simulation of the two-state model adequately describes the solvent effects found for lipase-catalyzed kinetic resolutions in binary mixtures of solvents and possibly in series of homologous organic solvents.     

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions

Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.     

Higher oxidation states of prostaglandin H synthase. Rapid electronic spectroscopy detected two spectral intermediates during the peroxidase reaction with prostaglandin G2

The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 degree C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1 = 1.4 x 10(7) M-1 s-1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+.FeIVO]. Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2 = 65 s-1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome-c peroxidase, was assigned to a two-electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+. which was formed by an intramolecular electron transfer from tyrosine to the porphyrin-pi-cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.     

Activity,stability and conformational flexibility of seed coat soybean peroxidase

Seed coat soybean peroxidase (SBP) belongs to class III of the plant peroxidase superfamily that includes the classical peroxidase, namely horseradish peroxidase (HRP). We have measured the catalytic activity (k(cat)) and catalytic efficiency (k(cat)/K(M)) of SBP and that of HRP-C for the oxidation of ABTS [2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonate)] by hydrogen peroxide at 25 degrees C. We observed that the k(cat) and k(cat)/K(M) values for SBP are much higher than those for HRP-C at all pH values, rendering SBP a more potent peroxidase. This is attributed to the relatively more solvent exposed delta-meso heme edge in SBP. We observed that the maximum catalytic activity and conformational stability of SBP is at pH approximately 5.5. A pH maximum of 5.0 for the catalytic activity of SBP has recently been reported. Estimation of secondary structural elements at various pH values indicated that there is a maximal reduction of beta-strands and beta-turns at pH 5.5 causing the heme to be further exposed to the solvent and increasing the overall conformational flexibility of the protein.     

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

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

Structural Change and Catalytic Activity of Horseradish Peroxidase in Oxidative Polymerization of Phenol

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

Comparative study of the interaction of polyuridylic acid with 30S subunits and 70S ribosomes of Escherichia coli.

Fractionated polyuridylic acid with an average chain length of 55 nucleotides forms binary complexes with 30S subunits with a stoichiometry of I:I. These complexes are heterogeneous in stability. The more stable one is characterized by an association constant K2 - 5.5xI09 M-I, and the less stable-by KI = I06xM-I, at 20 mM Mg2+, 200 mM NH4(+) and 0 degrees C. The main reason for this heterogeneity is the presence or absence of the ribosomal protein SI in the presence or absence of the ribosomal protein SI in the subunits. Decrease of Mg2+ concentration down to 5 mM hardly changes the K2 values but reduction of the NH4(+) concentration to 50 mM results in a 25-fold increase of K2. Association constants K2 for the stable complex, i.e. in the presence of SI protein, were measured at different temperatures (0 - 30 degrees C) and the thermodynamic parameters of binding (delta H degrees, delta S degrees, delta G degrees) were determined. Analogous experiments were made with 70S ribosomes. K2 values as well as delta H degrees, delta S degrees, delta G degrees appeared the same both for 30S and 70S ribosomes in all conditions examined. This is strong evidence that the 50S subunits do not contribute to the interaction of poly(U) with the complete 70S ribosomes.     

New trends in cryoenzymology: probing the functional role of protein dynamics by single-step kinetics

Cryoenzymology was initially used to slow down enzyme-catalyzed reactions so as to stabilize intermediates for further study. During the course of this early work, it became clear that cryoenzymology could be extended to other ends and some of these are described. First, the use of a cryosolvent on its own (or together with temperature) as a perturbant has allowed a resolution of the substrate binding steps of certain enzymes (myosin, D-amino acid oxidase, peroxidase and cytochrome P450). Second, by the use of cryosolvent and temperature, coupled with the classical physico-chemical perturbants, one can selectively modulate the various steps of an enzyme pathway. This approach can lead to an understanding of the mechanism of enzyme regulation. Finally, by carrying out experiments over a wide range of temperatures (-30 degrees C- +30 degrees C) and pressure (up to several kbars) in specially constructed fast reaction equipment, one can study the thermodynamic properties of the individual rate constants describing the interconversions of reaction intermediates. Experiments with creatine kinase, cytochrome P450 and peroxidase are described. The thermodynamic parameters delta H, delta G, delta S and delta V are thus measured and when this is done under different solvent conditions one can, at least within the theories available, attempt an approach to the problem of protein dynamics.     

Reactions of the class II peroxidases, lignin peroxidase and Arthromyces ramosus peroxidase, with hydrogen peroxide. Catalase-like activity, compound III formation, and enzyme inactivation

The reactions of the fungal enzymes Arthromyces ramosus peroxidase (ARP) and Phanerochaete chrysosporium lignin peroxidase (LiP) with hydrogen peroxide (H(2)O(2)) have been studied. Both enzymes exhibited catalase activity with hyperbolic H(2)O(2) concentration dependence (K(m) approximately 8-10 mm, k(cat) approximately 1-3 s(-1)). The catalase and peroxidase activities of LiP were inhibited within 10 min and those of ARP in 1 h. The inactivation constants were calculated using two independent methods; LiP, k(i) approximately 19 x 10(-3) s(-1); ARP, k(i) approximately 1.6 x 10(-3) s(-1). Compound III (oxyperoxidase) was detected as the majority species after the addition of H(2)O(2) to LiP or ARP, and its formation was accompanied by loss of enzyme activity. A reaction scheme is presented which rationalizes the turnover and inactivation of LiP and ARP with H(2)O(2). A similar model is applicable to horseradish peroxidase. The scheme links catalase and compound III forming catalytic pathways and inactivation at the level of the [compound I.H(2)O(2)] complex. Inactivation does not occur from compound III. All peroxidases studied to date are sensitive to inactivation by H(2)O(2), and it is suggested that the model will be generally applicable to peroxidases of the plant, fungal, and prokaryotic superfamily.     

Reaction of bovine cytochrome c oxidase with hydrogen peroxide produces a tryptophan cation radical and a porphyrin cation radical

Oxidized bovine cytochrome c oxidase reacts with hydrogen peroxide to generate two electron paramagnetic resonance (EPR) free radical signals (Fabian, M., and Palmer, G. (1995) Biochemistry 34, 13802-13810). These radicals are associated with the binuclear center and give rise to two overlapped EPR signals, one signal being narrower in line width (DeltaHptp = 12 G) than the other (DeltaHptp = 45 G). We have used electron nuclear double resonance (ENDOR) spectrometry to identify the two different chemical species giving rise to these two EPR signals. Comparison of the ENDOR spectrum associated with the narrow signal with that of compound I of horseradish peroxidase (formed by reaction of that enzyme with hydrogen peroxide) demonstrates that the two species are virtually identical. The chemical species giving rise to the narrow signal is therefore identified as an exchange-coupled porphyrin cation radical similar to that formed in horseradish peroxidase compound I. Comparison of the ENDOR spectrum of compound ES (formed by the reaction of hydrogen peroxide with cytochrome c peroxidase) with that of the broad signal indicates that the chemical species giving rise to the broad EPR signal in cytochrome c oxidase is probably an exchange coupled tryptophan cation radical. This is substantiated using H(2)O/D(2)O solvent exchange experiments where the ENDOR difference spectrum of the broad EPR signal of cytochrome c oxidase shows a feature consistent with hyperfine coupling to the exchangeable N(1) proton of a tryptophan cation radical.     

Preparation and initial characterization of the compound I, II, and III states of iron methylchlorin-reconstituted horseradish peroxidase and myoglobin: models for key intermediates in iron chlorin enzymes

To better understand the spectral properties of high valent and oxyferrous states in naturally occurring iron chlorin-containing proteins, we have prepared the oxoferryl compound I derivative of iron methylchlorin-reconstituted horseradish peroxidase (MeChl-HRP) and the compound II and oxyferrous compound III states of iron MeChl-reconstituted myoglobin. Initial spectral characterization has been carried out with UV-visible absorption and magnetic circular dichroism. In addition, the peroxidase activity of iron MeChl-HRP in pyrogallol oxidation has been found to be 40% of the rate for native HRP. Previous studies of oxoferryl chlorins have employed tetraphenylchlorins in organic solvents at low temperatures; stable oxyferrous chlorins have not been previously examined. The present study describes the compound I, II, and III states of histidine-ligated iron chlorins in a protein environment for the first time.     

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

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

Mechanism of reaction of horseradish peroxidase with chlorite and chlorine dioxide

It is demonstrated that horseradish peroxidase (HRP) mixed with chlorite follows the whole peroxidase cycle. Chlorite mediates the two-electron oxidation of ferric HRP to compound I (k(1)) thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of both compound I (k(2)) and compound II (k(3)) forming chlorine dioxide. The strong pH-dependence of all three reactions clearly suggests that chlorous acid is the reactive species. Typical apparent bimolecular rate constants at pH 5.6 are 1.4 x 10(5)M(-1)s(-1) (k(1)), 2.25 x 10(5)M(-1)s(-1) (k(2)), and 2.4 x 10(4)M(-1)s(-1) (k(3)), respectively. Moreover, the reaction products hypochlorous acid and chlorine dioxide, which are known to induce heme bleaching and amino acid modification upon longer incubation times, also mediate the oxidation of ferric HRP to compound I (2.4 x 10(7)M(-1)s(-1) and 2.7 x 10(4)M(-1)s(-1), respectively, pH 5.6) but do not react with compounds I and II. A reaction scheme is presented and discussed from both a mechanistic and thermodynamic point of view. It helps to explain the origin of contradictory data so far found in the literature on this topic.