Ethylene enhances reactivity of superoxide with peroxidase to form the oxy-ferrous complex

Ethylene and its analogues acetylene, carbon monoxide, and propylene inhibited the rate of oxidation of indole-3-acetic acid by peroxidase. Annulment of this effect by addition of superoxide dismutase showed that inhibition occurred only in the presence of the superoxide anion radical (O2-.). Kinetic and spectral data established that ethylene and its analogues enhanced markedly the rate of reaction of O2-. with peroxidase. This reaction resulted in the formation of compound III, an oxy-ferrous complex of peroxidase. In the presence of indole-3-acetic acid, the interaction between ethylene, peroxidase, and O2-. activated the reduced peroxidase in equilibrium compound III shuttle. O2-. is a major product of this shuttle, and compound III constitutes the dominant steady-state form of peroxidase. These interactions may help to explain the mechanism of action of ethylene as a plant growth regulator.     

A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin

We report the first characterization and classification of Orf13 (S. refuineus) as a heme-dependent peroxidase catalyzing the ortho-hydroxylation of L-tyrosine to L-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis, and maximal L-tyrosine conversion to L-DOPA is observed in the presence of hydrogen peroxide. Preincubation of L-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for L-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady-state kinetic analysis of L-tyrosine hydroxylation revealed similar catalytic efficiency for both L-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO(g) UV-vis spectrum of Orf13 and electron paramagnetic resonance of ferric heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for L-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of L-tyrosine to L-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases.     

A study of the supposed hydroxylation of tyrosine catalysed by peroxidase.

The claim that peroxidase (rather than tyrosinase) is the enzyme responsible for the conversion of tyrosine into dopa (3,4-dihydroxyphenylalanine) in melanogenesis was investigated. The spectral changes that occurred during the action of horseradish peroxidase in the presence of H2O2 on dopa, tyrosine and mixtures of dopa with tyrosine or other phenolic compounds were studied. The effect of ascorbic acid or dihydroxyfumaric acid on some of these changes was also investigated. No evidence was found that tyrosine was hydroxylated by peroxidase in the presence of H2O2 and dopa as cofactor, although tyrosine or other phenolic compounds increased the rate of oxidation of dopa to dopachrome (indoline-5,6-quinone-2-carboxylic acid). Peroxidase was, however, effective in oxidizing tyrosine to dopa in the presence of dihydroxyfumaric acid and oxygen.     

Similarities in the optical spectra of prostaglandin H synthase during its cyclooxygenase and peroxidase reactions

The spectral behavior of the enzyme prostaglandin H synthase was studied in the Soret region under conditions that permitted comparison of enzyme intermediates involved in peroxidase and cyclooxygenase activities. First, the peroxidase activity was examined. The enzyme's spectral behavior upon reacting with 5-phenyl-pent-4-enyl-1-hydroperoxide was different depending on the presence or absence of the reducing substrate, phenol. In the reaction of prostaglandin H synthase with the peroxide in the absence of phenol, formation of the enzyme intermediate compound I is observed followed by partial conversion to compound II and then by enzyme bleaching. In the reaction with both peroxide and phenol the absorbance decreases and a steady-state spectrum is observed which is a mixture of native enzyme and compound II. The steady state is followed by an increase in absorbance back to that of the native enzyme with no bleaching. The difference can be explained by the reactivity of phenol as a reducing substrate with the prostaglandin H synthase intermediate compounds. Cyclooxygenase activity with arachidonic acid could not be examined in the absence of diethyldithiocarbamate because extensive bleaching occurred. In the presence of diethyldithiocarbamate, enzyme spectral behavior similar to that seen in the reaction of the peroxide and phenol was observed. The similarity of the spectra strongly suggests that the enzyme intermediates involved in both the peroxidase and cyclooxygenase reactions are the same.     

Myeloperoxidase-oxidase oxidation of cysteamine.

Cysteamine oxidation was shown to be catalysed by nanomolar concentrations of myeloperoxidase in a peroxidase-oxidase reaction, i.e. an O2-consuming oxidation of a compound catalysed by peroxidase without H2O2 addition. When auto-oxidation of the thiol was prevented by the metal-ion chelator diethylenetriaminepenta-acetic acid, native, but not heat-inactivated, myeloperoxidase induced changes in the u.v.-light-absorption spectrum of cysteamine. These changes were consistent with disulphide (cystamine) formation. Concomitantly, O2 was consumed and superoxide radical anion formation could be detected by Nitro Blue Tetrazolium reduction. Both superoxide dismutase and catalase inhibited the reaction, whereas the hydroxyl-radical scavengers mannitol and ethanol did not. O2 consumption increased with increasing pH (between pH 6.0 and 8.0), and 50% inhibition was exhibited by about 3 mM-NaCl at pH 7.0 and by about 100 mM-NaCl at pH 8.0. Cysteamine was about 5 times as active (in terms of increased O2 consumption at pH 7.5) as the previously reported peroxidase-oxidase substrates NADPH, dihydroxyfumaric acid and indol-3-ylacetic acid. A possible reaction pathway for the myeloperoxidase-oxidase oxidation of cysteamine is discussed. These results indicate that cysteamine is a very useful substrate for studies on myeloperoxidase-oxidase activity.     

Horseradish peroxidase-catalyzed aerobic oxidation and peroxidation of indole-3-acetic acid. I. Optical spectra.

A study of the indole-3-acetate reaction with horse-radish peroxidase, in the absence or presence of hydrogen peroxide, has been performed, employing rapid scan and conventional spectrophotometry. We present here the first clear spectral evidence, obtained on the millisecond time scale, indicating that at pH 5.0 and for high [enzyme/substrate] ratios peroxidase compound III is formed. Most, if not all, of the compound III is formed by oxygenation of the ferrous peroxidase. There is an inhibitory effect of superoxide dismutase and histidine on compound III formation which indicates the involvement of the active oxygen species superoxide and singlet oxygen. It is concluded that the oxidation of indole-3-acetate by horseradish peroxidase at pH 5.0 proceeds through compound III formation to the catalytically inactive forms P-670 and P-630. A reaction path in which the enzyme is directly reduced by indole-3-acetate might be involved as an initiation step. Rapid scan spectral data, which indicate differences in the formation and decay of enzyme intermediate compounds at pH 7.0, in comparison with those observed at pH 5.0, are also presented. At pH 7.0 compound II is a key intermediate in oxidation--peroxidation of substrate. Mechanisms of reactions consistent with the experimental data are proposed and discussed.     

Horseradish peroxidase—catalyzed hydroxylation of phenol: I. Thermodynamic analysis

We analyzed the horseradish peroxidase (HRP)—catalyzed hydroxylation of phenol in the presence of dihydroxy-fumaric acid and oxygen. All of the intermediate forms of the enzyme are reviewed. The last step of hydroxylation, consisting of the production of OH^• radicals that further react on phenol, is emphasized. Possible OH^• radicals production reactions were compiled and analyzed with respect to the available thermodynamic data. Some results of electrochemical experiments were also used to choose the correct set of reactions. At the end of analysis only two reactions for producing OH^• seemed to be consistent with the thermodynamic and experimental data. Neither of these reactions involved compound III or any other intermediate form of HRP. The last step of hydroxylation was thus totally independent of the pure catalytic cycle of the enzyme. As a consequence, HRP cannot be used as an hydroxylation enzyme in place of the P₄₅₀ cytochrome, as is sometimes suggested.     

An enzymatic method for removal of phenol from industrial effluent

Phenols in an aqueous solution were removed after treatment with peroxidase in the presence of hydrogen peroxide. Phenols occur in wastewater of a number of industries, such as high temperature coal conversion, petroleum refining, resin and plastic, wood and dye industries, etc. It can be toxic when present at elevated levels and is known to be carcinogeneous. Thus, removal of such compound from these industrial effluents is of great importance. An enzymatic method for removal of phenols from industrial wastewater, using turnip peroxidase, has been developed. Phenol-containing industrial wastewater was treated with immobilized turnip peroxidase in the presence of hydrogen peroxide. In the reaction, a number of phenols are oxidized to form the corresponding free radicals in the presence of hydrogen peroxide as an oxidant. Free radicals polymerize to form substances that are less soluble in water than the original substances. The precipitates were removed by conventional methods and residual phenol was estimated. The present report describes the immobilization of turnip peroxidase on silica via covalent coupling, and its utility in phenol removal. A comparative study was also carried out with other immobilization techniques, viz., calcium alginate entrapment, polyacrylamide gel entrapment, etc. Peroxidase, covalently bound to silica, showed 95% removal of phenol, whereas naphthol was removed up to 99%.     

A model of peroxidase activity with inhibition by hydrogen peroxide

The rate of color formation in an activity assay consisting of phenol and hydrogen peroxide as substrates and 4-aminoantipyrine as chromogen is significantly influenced by hydrogen peroxide concentration due to its inhibitory effect on catalytic activity. A steady-state kinetic model describing the dependence of peroxidase activity on hydrogen peroxide concentration is presented. The model was tested for its application to soybean peroxidase (SBP) and horseradish peroxidase (HRP) reactions based on experimental data which were measured using simple spectrophotometric techniques. The model successfully describes the dependence of enzyme activity for SBP and HRP over a wide range of hydrogen peroxide concentrations. Model parameters may be used to compare the rate of substrate utilization for different peroxidases as well as their susceptibility to compound III formation. The model indicates that SBP tends to form more compound III and is catalytically slower than HRP during the oxidation of phenol.     

The reactions of horseradish peroxidase, lactoperoxidase, and myeloperoxidase with enzymatically generated superoxide

The formation and decay of intermediate compounds of horseradish peroxidase, lactoperoxidase, and myeloperoxidase formed in the presence of the superoxide/hydrogen peroxide-generating xanthine/xanthine oxidase system has been studied by observation of spectral changes in both the Soret and visible spectral regions and both on millisecond and second time scales. It is tentatively concluded that in all cases compound III is formed in a two-step reaction of native enzyme with superoxide. The presence of superoxide dismutase completely inhibited compound III formation; the presence of catalase had no effect on the process. Spectral data which indicate differences in the decay of horseradish peroxidase compound III back to the native state in comparison with compounds III of lactoperoxidase and myeloperoxidase are also presented.     

Purification of the agent inducing lipid peroxidation with dihydroxyfumaric acid in rat liver mitochondria

Dihydroxyfumaric acid induces lipid peroxidation in rat liver mitochondria as reported previously. When the mitochondria were solubilized with 0.35% ($\text{W}\text{V}$) sodium cholate, the supernatant itself could not catalyze lipid peroxidation with dihydroxyfumaric acid, but the precipitate slightly induced the reaction. The supernatant produced lipid peroxide in the presence of the precipitate and dihydroxyfumaric acid. The supernatant was heat sensitive contrary to the stability of the precipitate. An attempt was made to isolate active entity through a sephadex G-200 column and a DEAE-cellulose column, resulting in about 10-fold purification. At 408–410 nm the partially purified agent showed a maximum absorption, which disappeared rapidly after reduction with sodium dithionite and was slowly diminished with dihydroxyfumaric acid. The molecular weight was much larger than that of oxidized cytochrome c.     

Catalytic pathways of Euphorbia characias peroxidase reacting with hydrogen peroxide

The reaction of Euphorbia characias latex peroxidase (ELP) with hydrogen peroxide as the sole substrate was studied by conventional and stopped-flow spectrophotometry. The reaction mechanism occurs via three distinct pathways. In the first (pathway I), ELP shows catalase-like activity: H2O2 oxidizes the native enzyme to compound I and subsequently acts as a reducing substrate, again converting compound I to the resting ferric enzyme. In the presence of an excess of hydrogen peroxide, compound I is still formed and further reacts in two other pathways. In pathway II, compound I initiates a series of cyclic reactions leading to the formation of compound II and compound III, and then returns to the native resting state. In pathway III, the enzyme is inactivated and compound I is converted into a bleached inactive species; this reaction proceeds faster in samples illuminated with bright white light, demonstrating that at least one of the intermediates is photosensitive. Calcium ions decrease the rate of pathway I and accelerate the rate of pathways II and III. Moreover, in the presence of calcium the inactive stable verdohemochrome P670 species accumulates. Thus, Ca2+ ions seem to be the key for all catalytic pathways of Euphorbia peroxidase.     

A simple colorimetric method for determination of hydrogen peroxide in plant tissues

A simple colorimetric method for determination of hydrogen peroxide in plant materials is described. The method is based on hydrogen peroxide producing a stable red product in reaction with 4-aminoantipyrine and phenol in the presence of peroxidase. Plant tissues was ground with trichloroacetic acid (5% w/v) and extracts were adjusted to pH 8.4 with ammonia solution. Activated charcoal was added to the homogenate to remove pigments, antioxidants and other interfering substances. The colorimetric reagent (pH 5.6) consisted of 4-aminoantipyrine, phenol, and peroxidase. With this method, we have determined the hydrogen peroxide concentration in leaves of eight species which ranged from 0.2 to 0.8 μmol g⁻¹ FW. Changes in hydrogen peroxide concentration of Stylosanthes guianensis in response to heat stress are also analyzed using this method.     

Reduction of prostaglandin H synthase compound II by phenol and hydroquinone, and the effect of indomethacin.

The reduction of prostaglandin H synthase compound II to native enzyme by phenol and by hydroquinone, in the presence of diethyldithiocarbamate as a stabilizing agent, was studied by rapid scan spectrometry and transient state kinetics at 4.0 +/- 0.5 degrees C in 0.1 M phosphate buffer, pH 8.0. The plot of pseudo-first-order rate constants for the conversion of prostaglandin H synthase compound II to native enzyme versus phenol concentration was linear with a non-zero intercept. The second-order rate constant was determined from the slope to be (5.3 +/- 0.3) x 10(5) M-1 s-1. For the reduction by hydroquinone, the second-order rate constant was determined from pointwise measurements of the pseudo-first-order rate constant to be (2.1 +/- 0.4) x 10(6) M-1 s-1. Rapid scan spectrum results also showed the reduction of compound I to compound II by both phenol and hydroquinone. Thus reduction of both compound I and compound II is one electron process. Our results suggest that the tyrosyl radical, detected in the presence of oxidizing agents, is formed by intramolecular electron transfer from the tyrosyl residue to the porphyrin pi-cation radical, and this reaction tends to disappear in the presence of sufficient reducing substrate. These in vitro results support speculation that there is a role of the peroxidase component of prostaglandin H synthase in benzene-induced toxicity. In the present work, the effect of indomethacin on the reduction of prostaglandin H synthase compound II by diethyldithiocarbamate, phenol, and hydroquinone was also investigated. Results revealed, for the first time, that indomethacin is an inhibitor of the peroxidase activity of prostaglandin H synthase, although not as effectively as in its well-known inhibition of cyclooxygenase activity.     

Chloroperoxidase-catalyzed enantioselective oxidation of methyl phenyl sulfide with dihydroxyfumaric acid/oxygen or ascorbic acid/oxygen as oxidants

The chloroperoxidase catalyzed oxidation of methyl phenyl sulfide to (R)-methyl phenyl sulfoxide was investigated, both in batch and membrane reactors, using as oxidant H2O2, or O2 in the presence of either dihydroxyfumaric acid or ascorbic acid. The effects of pH and nature and concentration of the oxidants on the selectivity, stability, and productivity of the enzyme were evaluated. The highest selectivity was displayed by ascorbic acid/O2, even though the activity of chloroperoxidase with this system was lower than that obtained with the others. When the reaction was carried out in a membrane reactor, it was possible to reuse the enzyme for several conversion cycles. The results obtained with ascorbic acid/O2 and dihydroxyfumaric acid/O2 as oxidants do not seem to be compatible with either a mechanism involving hydroxyl radicals as the active species or with the hypothesis that oxidation occurs through the initial formation of H2O2. Copyright 1999 John Wiley & Sons, Inc.     

Mechanism of hydrogen peroxide formation catalyzed by NADPH oxidase in thyroid plasma membrane

The thyroid plasma membrane contains a Ca2(+)-regulated NADPH-dependent H2O2 generating system which provides H2O2 for the thyroid peroxidase-catalyzed biosynthesis of thyroid hormones. The plasma membrane fraction contains a Ca2(+)-independent cytochrome c reductase activity which is not inhibited by superoxide dismutase. But it is not known whether H2O2 is produced directly from molecular oxygen (O2) or formed via dismutation of super-oxide anion (O2-). Indirect evidence from electron scavenger studies indicate that the H2O2 generating system does not liberate O2-, but studies using the modified peroxidase, diacetyldeuteroheme horseradish peroxidase, to detect O2- indicate that H2O2 is provided via the dismutation of O2-. The present results provide indirect evidence that the cytochrome c reductase activity is not a component of the NADPH-dependent H2O2 generator, since it was removed by washing the plasma membranes with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid without affecting H2O2 generation. Spectral studies with diacetyldeuteroheme-substituted horseradish peroxidase showed that the thyroid NADPH-dependent H2O2 generator does not catalyze superoxide anion formation. The O2- adduct compound (compound III) was formed but was completely inhibited by catalase, indicating that the initial product was H2O2. The rate of NADPH oxidation also increased in the presence of diacetylheme peroxidase. This increase was blocked by catalase and was greatly enhanced by superoxide dismutase. The O2- adduct compound (compound III) was produced in the presence of NADPH when glucose-glucose oxidase (which does not produce O2-) was used as the H2O2 generator. NADPH oxidation occurred simultaneously and was enhanced by superoxide dismutase. We conclude that O2- formation occurs in the presence of an H2O2 generator, diacetylheme peroxidase and NADPH, but that it is not the primary product of the H2O2 generator. We suggest that O2- formation results from oxidation of NADPH, catalyzed by the diacetylheme peroxidase compound I, producing NADP degree, which in turn reacts with O2 to give O2-.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Generation of hydrogen peroxide, superoxide and hydroxyl radicals during the oxidation of dihydroxyfumaric acid by peroxidase.

1. Dihydroxyfumarate slowly autoxidizes at pH6. This reaction is inhibited by superoxide dismutase but not by EDTA. Mn2+ catalyses dihydroxyfumarate oxidation by reacting with O2 leads to to form Mn3+, which seems to oxidize dihydrofumarate rapidly. Cu2+ also catalyses dihydroxyfumarate oxidation, but by a mechanism that does not involve O2 leads to. 2. Peroxidase catalyses oxidation of dihydroxyfumarate at pH6; addition of H2O2 does not increase the rate. Experiments with superoxide dismutase and catalase suggest that there are two types of oxidation taking place: an enzymic, H2O2-dependent oxidation of dihydroxyfumarate by peroxidase, and a non-enzymic reaction involving oxidation of dihydroxyfumarate by O2 leads to. The latter accounts for most of the observed oxidation of dihydroxyfumarate. 3. During dihydroxyfumarate oxidation, most peroxidase is present as compound III, and the enzymic oxidation may be limited by the low rate of breakdown of this compound. 4. Addition of p-coumaric acid to the peroxidase/dihydroxyfumarate system increases the rate of dihydroxyfumarate oxidation, which is now stimulated by addition of H2O2, and is more sensitive to inhibition by catalase but less sensitive to superoxide dismutase. Compound III is decomposed in the presence of p-coumaric acid. p-Hydroxybenzoate has similar, but much smaller, effects on dihydroxyfumarate oxidation. However, salicylate affects neither the rate nor the mechanism of dihydroxyfumarate oxidation. 5. p-Hydroxybenzoate, salicylate and p-coumarate are hydroxylated by the peroxidase/dihydroxyfumarate system. Experiments using scavengers of hydroxyl radicals shown that OH is required. Ability to increase dihydroxyfumarate oxidation is not necessary for hydroxylation to occur.     

Oxidase Reaction of the Hybrid Mn-Peroxidase of the Fungus <Emphasis Type="Italic">Panus tigrinus</Emphasis> 8/18

The hybrid Mn-peroxidase of the fungus Panus tigrinus 8/18 oxidized NADH in the absence of hydrogen peroxide, this being accompanied by the consumption of oxygen. The reaction of NADH oxidation started after a period of induction and completely depended on the presence of Mn(II). The reaction was inhibited in the presence of catalase and super-oxide dismutase. Oxidation of NADH by the enzyme or by manganese(III)acetate was accompanied by the production of hydrogen peroxide and superoxide radicals. In the presence of NADH, the enzyme was transformed into a catalytically inactive oxidized form (compound III), and the latter was inactivated with bleaching of the heme. The substrate of the hybrid Mn-peroxidase (Mn(II)) reduced compound III to yield the native form of the enzyme and prevented its inactivation. It is assumed that the hybrid Mn-peroxidase used the formed hydrogen peroxide in the usual peroxidase reaction to produce Mn(III), which was involved in the formation of hydrogen peroxide and thus accelerated the peroxidase reaction. The reaction of NADH oxidation is a peroxidase reaction and the consumption of oxygen is due to its interaction with the products of NADH oxidation. The role of Mn(II) in the oxidation of NADH consisted in the production of hydrogen peroxide and the protection of the enzyme from inactivation.__________Translated from Biokhimiya, Vol. 70, No. 4, 2005, pp. 568–574.Original Russian Text Copyright © 2005 by Lisov, Leontievsky, Golovleva.     

The Mechanism of the Scopoletin-induced Inhibition of the Peroxidase Catalyzed Degradation of Indole-3-acetate

The naturally occurring coumarin, scopoletin, has been found to modify horseradish peroxidase rapidly to give a stable, spectroscopically distinguishable form of the enzyme. Peroxidase treated with scopoletin is less active in reactions with molecular oxygen and indole-3-acetic acid. Kinetic data for the degradation of this growth regulator were obtained with a continuously monitored fluorometric procedure. Lineweaver-Burk plots of the reciprocal rate of degradation against the reciprocal substrate concentration were markedly curved in the presence of the inhibitor, scopoletin. Excess indole-3-acetate restored the scopoletin-treated enzyme to a reactive state. In the presence of molecular oxygen, concentrations of indole-3-acetic acid which were at least 10-fold greater than the inhibitor concentration led to the rapid oxidation of the coumarin and converted peroxidase to compound III as expected from previous studies. This form of the enzyme is the catalytically active species in the oxidative degradation of the growth regulator. The kinetically preferential reaction of scopoletin or related coumarins with peroxidase and the suppression of indole-3-acetic acid degradation may provide a possible control mechanism over the oxidative degradation of indole-3-acetate by this plant enzyme.