Cytochrome c peroxidase compound ES is identical with horseradish peroxide compound I in iron-ligand distances

X-ray absorption studies of compound ES of cytochrome c peroxidase show a short iron-oxygen distance of 1.67 +/- 0.04 A, an iron-histamine distance of 1.91 +/- 0.03 A, and an iron-pyrrole nitrogen average distance of 2.02 +/- 0.02 A. This is identical within the error with the reported structure of horseradish peroxidase compound I [Chance, B., Powers, L., Ching, Y., Poulos, T., Yamazaki, I., & Paul, K. G. (1984) Arch. Biochem. Biophys. 235, 596-611]. Comparisons of the structures of myoglobin peroxide [Chance, M., Powers, L., Kumar, C., & Chance, B. (1986) Biochemistry (preceding paper in this issue)], compound ES, and the intermediates of horseradish peroxidase reveal the possible mechanisms for the stabilization of the free radical species generated during catalysis. The proximal histidine regulates the structure and function of the pyrrole nitrogens and the heme, allowing for the formation and maintenance of the characteristic intermediates.     

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.     

Crystallographic and spectroscopic studies of peroxide-derived myoglobin compound II and occurrence of protonated FeIV O

High resolution crystal structures of myoglobin in the pH range 5.2-8.7 have been used as models for the peroxide-derived compound II intermediates in heme peroxidases and oxygenases. The observed Fe-O bond length (1.86-1.90 A) is consistent with that of a single bond. The compound II state of myoglobin in crystals was controlled by single-crystal microspectrophotometry before and after synchrotron data collection. We observe some radiation-induced changes in both compound II (resulting in intermediate H) and in the resting ferric state of myoglobin. These radiation-induced states are quite unstable, and compound II and ferric myoglobin are immediately regenerated through a short heating above the glass transition temperature (<1 s) of the crystals. It is unclear how this influences our compound II structures compared with the unaffected compound II, but some crystallographic data suggest that the influence on the Fe-O bond distance is minimal. Based on our crystallographic and spectroscopic data we suggest that for myoglobin the compound II intermediate consists of an Fe(IV)-O species with a single bond. The presence of Fe(IV) is indicated by a small isomer shift of delta = 0.07 mm/s from M?ssbauer spectroscopy. Earlier quantum refinements (crystallographic refinement where the molecular-mechanics potential is replaced by a quantum chemical calculation) and density functional theory calculations suggest that this intermediate H species is protonated.     

An iron hydroxide moiety in the 1.35 Å resolution structure of hydrogen peroxide derived myoglobin compound II at pH 5.2

The biological conversions of O(2) and peroxides to water as well as certain incorporations of oxygen atoms into small organic molecules can be catalyzed by metal ions in different clusters or cofactors. The catalytic cycle of these reactions passes through similar metal-based complexes in which one oxygen- or peroxide-derived oxygen atom is coordinated to an oxidized form of the catalytic metal center. In haem-based peroxidases or oxygenases the ferryl (Fe(IV)O) form is important in compound I and compound II, which are two and one oxidation equivalents higher than the ferric (Fe(III)) form, respectively. In this study we report the 1.35 A structure of a compound II model protein, obtained by reacting hydrogen peroxide with ferric myoglobin at pH 5.2. The molecular geometry is virtually unchanged compared to the ferric form, indicating that these reactive intermediates do not undergo large structural changes. It is further suggested that at low pH the dominating compound II resonance form is a hydroxyl radical ferric iron rather than an oxo-ferryl form, based on the short hydrogen bonding to the distal histidine (2.70 A) and the Fe...O distance. The 1.92 A Fe...O distance is in agreement with an EXAFS study of compound II in horseradish peroxidase.     

A mechanism for NADPH inhibition of catalase compound II formation.

Catalase-bound NADPH both prevents and reverses the accumulation of inactive bovine liver catalase peroxide compound II generated by 'endogenous' donors under conditions of steady H2O2 formation without reacting rapidly with either compound I or compound II. It thus differs both from classical 2-electron donors of the ethanol type, and from 1-electron donors of the ferrocyanide/phenol type. NADPH also inhibits compound II formation induced by the exogenous one-electron donor ferrocyanide. A catalase reaction scheme is proposed in which the initial formation of compound II from compound I involves production of a neighbouring radical species. NADPH blocks the final formation of stable compound II by reacting as a 2-electron donor to compound II and to this free radical. The proposed behaviour resembles that of labile free radicals formed in cytochrome c peroxidase and myoglobin. Such radical migration patterns within haem enzymes are increasingly common motifs.     

Observation of the FeIV=O stretching vibration of ferryl myoglobin by resonance Raman spectroscopy

We have directly observed the oxyferryl group of ferryl myoglobin by resonance Raman spectroscopy. The FeIV = O stretching vibration is observed at 797 cm-1 and confirmed by an 18O-induced isotopic shift to 771 cm-1. The porphyrin center-to-nitrogen distance of ferryl myoglobin is significantly less than that previously observed for horseradish peroxidase compound II, which also contains an FeIV = O heme. The FeIII-CN- stretch of myoglobin (FeIII) cyanide is observed at 454 cm-1, which shifts to 449 cm-1 upon substitution with [13C]cyanide.     

Heme-linked ionization of horseradish peroxidase compound II monitored by the resonance Raman Fe(IV)=O stretching vibration

Fe(IV)=O resonance Raman stretching vibrations were recently identified by this laboratory for horseradish peroxidase compound II and ferryl myoglobin. In the present report it is shown that Fe(IV)=O stretching frequency for horseradish peroxidase compound II will switch between two values depending on pH, with pKa values corresponding to the previously reported compound II heme-linked ionizations of pKa = 6.9 for isoenzyme A-2 and pKa = 8.5 for isoenzyme C. Similar pH-dependent shifts of the Fe(IV)=O frequency of ferryl myoglobin were not detected above pH 6. The Fe(IV)=O stretching frequencies of compound II of the horseradish peroxidase isoenzymes at pH values above the transition points were at a high value approaching the Fe(IV)=O stretching frequency of ferryl myoglobin. Below the transition points the horseradish peroxidase frequencies were found to be 10 cm-1 lower. Frequencies of the Fe(IV)=O stretching vibrations of horseradish peroxidase compound II for one set of isoenzymes were found to be sensitive to deuterium exchange below the transition point but not above. These results were interpreted to be indicative of an alkaline deprotonation of a distal amino acid group, probably histidine, which is hydrogen bonded to the oxyferryl group below the transition point. Deprotonation of this group at pH values above the pKa disrupts hydrogen bonding, raising the Fe(IV)=O stretching frequency, and is proposed to account for the lowering of compound II reactivity at alkaline pH. The high value of the Fe(IV)=O vibration of compound II above the transition point appears to be identical in frequency to what is believed to be the Fe(IV)=O vibration of compound X.     

Lignin peroxidase: resonance Raman spectral evidence for compound II and for a temperature-dependent coordination-state equilibrium in the ferric enzyme

Resonance Raman (RR) spectroscopy of lignin peroxidase (ligninase, dairylpropane oxygenase) from the basidiomycete Phanerochaete chrysosporium suggests two different coordination states for the native ferric enzyme. Evidence for a high-spin, hexacoordinate ferric protoporphyrin IX was presented by Andersson et al. [Andersson, L. A., Renganathan, V., Chiu, A.A., Loehr, T. M., & Gold, M. H. (1985) J. Biol. Chem. 260, 6080-6087], whereas Kuila et al. [Kuila, D., Tien, M., Fee, J. A., & Ondrias, M. R. (1985) Biochemistry 24, 3394-3397] proposed a high-spin, pentacoordinate ferric system. Because the two RR spectral studies were performed at different temperatures, we explored the possibility that lignin peroxidase might exhibit temperature-dependent coordination-state equilibria. Resonance Raman results presented herein indicate that this hypothesis is indeed correct. At or near 25 degrees C, the ferric iron of lignin peroxidase is predominantly high spin, pentacoordinate; however, at less than or equal to 2 degrees C, the high-spin, hexacoordinate state dominates, as indicated by the frequencies of well-documented spin- and coordination-state marker bands for iron protoporphyrin IX. The temperature-dependent behavior of lignin peroxidase is thus similar to that of cytochrome c peroxidase (CCP). Furthermore, lignin peroxidase, like horseradish peroxidase (HRP) and CCP, clearly has a vacant coordination site trans to the native fifth ligand at ambient temperature. High-frequency RR spectra of compound II of lignin peroxidase are also presented. The observed shifts to higher frequency for both the oxidation-state marker band v4 and the spin- and coordination-state marker band v10 are similar to those reported for the compound II forms of HRP and lactoperoxidase and for ferryl myoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the status of ferryl protonation

We examine the issue of ferryl protonation in heme proteins. An analysis of the results obtained from X-ray crystallography, resonance Raman spectroscopy, and extended X-ray absorption spectroscopy (EXAFS) is presented. Fe-O bond distances obtained from all three techniques are compared using Badger's rule. The long Fe-O bond lengths found in the ferryl crystal structures of myoglobin, cytochrome c peroxidase, horseradish peroxidase, and catalase deviate substantially from the values predict by Badger's rule, while the oxo-like distances obtained from EXAFS measurements are in good agreement with the empirical formula. Density functional calculations, which suggest that M?ssbauer spectroscopy can be used to determine ferryl protonation states, are presented. Our calculations indicate that the quadrupole splitting (DeltaE(Q)) changes significantly upon ferryl protonation. New resonance Raman data for horse-heart myoglobin compound II (Mb-II, pH 4.5) are also presented. An Fe-O stretching frequency of 790cm(-1) (shifting to 754cm(-1) with (18)O substitution) was obtained. This frequency provides a Badger distance of r(Fe-O)=1.66A. This distance is in agreement with the 1.69A Fe-O bond distance obtained from EXAFS measurements but is significantly shorter than the 1.93A bond found in the crystal structure of Mb-II (pH 5.2). In light of the available evidence, we conclude that the ferryl forms of myoglobin (pKa4), horseradish peroxidase (pKa4), cytochrome c peroxidase (pKa4), and catalase (pKa7) are not basic. They are authentic Fe(IV)oxos with Fe-O bonds on the order of 1.65A.     

Formation of compound I and compound II ferryl species in the reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide

The formation of ferryl heme (Fe(IV) = O) species, i.e., compound I and compound II, has been identified as the main intermediates in heme protein peroxidative reactions. We report stopped-flow kinetic measurements which illustrate that the reaction of hemoglobin I (HbI) from Lucina pectinata with hydrogen peroxide produce ferryl intermediates compound I and compound II. Compound I appears relatively stable displaying an absorption at 648 nm. The rate constant value (k'(2)) for the conversion of compound I to compound II is 3.0 x 10(-2) s(-1), more than 100 times smaller than that reported for myoglobin. The rate constant value for the oxidation of the ferric heme (k'(12) + k'(13)) is 2.0 x 10(2) M(-1) s(-1). These values suggest an alternate route for the formation of compound II (by k'(13)) avoiding the step from compound I to compound II (k'(2)). In HbI from L. pectinata the stabilization of compound I is attribute to the unusual collection of amino acids residues (Q64, F29, F43, F68) in the heme pocket active site of the protein.     

Peroxidase-catalyzed formation of triplet acetone and chemiluminescence from isobutyraldehyde and molecular oxygen

It has been established that the horseradish peroxidase/O2/isobutyraldehyde (IBAL) system leads to triplet acetone and formic acid formation followed by phosphorescence of the triplet acetone (see, for example, Bechara, E.J.H., Faria Oliveira, O.M.M., Durán, N., Casadei de Baptista, R., and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110). In this paper many of the mechanistic details are established. The reaction is initiated by the autoxidation of IBAL to form the peracid (CH3)2CHC = O(OOH). The peracid converts horseradish peroxidase into compound I which in turn is converted into compound II by abstracting the alcoholic hydrogen atom from the enol form of IBAL. This creates a free radical with two resonance forms. (Formula: see text) Addition of molecular oxygen to the latter resonance form creates a peroxy radical which abstracts a hydrogen atom near the active site of the enzyme. The newly formed alpha-peroxide in turn forms a dioxetane-type of intermediate which rapidly decomposes into triplet acetone and formic acid. Compound II reacts with the enol by the same pathway as compound I. Thus native horseradish peroxidase is regenerated. The hydrogen atom abstraction near the enzyme active site may occur directly from ethanol, present to solubilize IBAL or from a group on the enzyme, in which case ethanol participates in a repair mechanism. Phosphate buffer is necessary because it catalyzes the keto-enol conversion of IBAL. Thus horseradish peroxidase participates in a normal peroxidatic cycle. The only chain reaction is the uncatalyzed autoxidation of IBAL, most of which occurs prior to the mixing of IBAL with the oxygenated horseradish peroxidase solution.     

The crystal structure of peroxymyoglobin generated through cryoradiolytic reduction of myoglobin compound III during data collection

Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallographic data collection. We have generated and solved the 1.30 A (1 A=0.1 nm) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe-O distance of 1.8 A and O-O bond of 1.3 A in accordance with a Fe(II)-O-O- (or Fe(III)-O-O2-) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metalloproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O-O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.     

Magnetic circular dichroism studies on horseradish peroxidase.

Magnetic circular dichroism (MCD) spectra were observed for native (Fe(III)) horseradish peroxidase (peroxidase, EC 1.11.1.7), its alkaline form and fluoro- and cyano-derivatives, and also for reduced (Fe(II)) horseradish peroxidase and its carbonmonoxy-- and cyano- derivatives. MCD spectra were obtained for the cyano derivative of Fe(III) horseradish peroxidase, and reduced horseradish peroxidase and its carbonmonoxy- derivative nearly identical with those for the respective myoglobin derivatives. The alkaline form of horseradish peroxidase exhibits a completely different MCD spectrum from that of myoglobin hydroxide. Thus it shows an MCD spectrum which falls into the ferric low-spin heme grouping. Native horseradish peroxidase and its fluoro derivatives show almost identical MCD spectra with those for the respective myoglobin derivatives in the visible region, though some changes were detected in the Soret region. Therefore it is concluded that the MCD spectra on the whole are sensitive to the spin state of the heme iron rather than to the porphyrin structures. The cyanide derivative of reduced horseradish peroxidase exhibited a characteristic MCD spectrum of the low-spin ferrous derivative like oxy-myoglobin.     

Kinetic studies on the formation and decomposition of compounds II and III. Reactions of lignin peroxidase with H2O2.

The present study characterizes the serial reactions of H2O2 with compounds I and II of lignin peroxidase isozyme H1. These two reactions constitute part of the pathway leading to formation of the oxy complex (compound III) from the ferric enzyme. Compounds II and III are the only complexes observed; no compound III* is observed. Compound III* is proposed to be an adduct of compound III with H2O2, formed from the complexation of compound III with H2O2 (Wariishi, H., and Gold, M. H. (1990) J. Biol. Chem. 265, 2070-2077). We provide evidence that demonstrates that the spectral data, on which the formation of compound III* is based, are merely an artifact caused by enzyme instability and, therefore, rule out the existence of compound III*. The reactions of compounds II and III with H2O2 are pH-dependent, similar to that observed for reactions of compounds I and II with the reducing substrate veratryl alcohol. The spontaneous decay of the compound III of lignin peroxidase results in the reduction of ferric cytochrome c. The reduction is inhibited by superoxide dismutase, indicating that superoxide is released during the decay. Therefore, the lignin peroxidase compound III decays to the ferric enzyme through the dissociation of superoxide. This mechanism is identical with that observed with oxymyoglobin and oxyhemoglobin but different from that for horseradish peroxidase. Compound III is capable of reacting with small molecules, such as tetranitromethane (a superoxide scavenger) and fluoride (a ligand for the ferric enzyme), resulting in ferric enzyme and fluoride complex formation, respectively.     

Comparison of the heme structures of horseradish peroxidase compounds X and II by resonance Raman spectroscopy

Horseradish peroxidase will catalyze the chlorination of certain substrates by sodium chlorite through an intermediate known as compound X. A chlorite-derived chlorine atom is known to be retained by compound X and has been proposed to be located at the heme active site. Although several heme structures have been proposed for compound X, including an Fe(IV)-OCl group, preliminary data previously reported by our laboratory suggested that compound X contained a heme Fe(IV) = O group, based on the similarity of a compound X resonance Raman band at 788 cm-1 to resonance Raman Fe(IV) = O stretching vibrations recently identified for horseradish peroxidase compound II and ferryl myoglobin. Isotopic studies now confirm that the 788 cm-1 resonance Raman band of compound X is, in fact, due to a heme Fe(IV) = O group, with the oxygen atom derived from chlorite. The Fe(IV) = O frequency of compound X, of horseradish peroxidase isoenzymes B and C, undergoes a pH-induced frequency shift, with behavior which appears to be the same as that previously reported for compound II, formed from the same isoenzymes. These observations strongly suggest that compounds II and X have very similar, if not identical, heme structures. The chlorine atom thus appears not to be heme-bound and may rather be located on an amino acid residue. The studies on compound X reported here were done in a pH region above pH 8, where compound X is moderately stable. The present results do not necessarily apply to compound X below pH 8.     

Titration study of guaiacol oxidation by horseradish peroxidase.

Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 0.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound II. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound II. The direct two-electron transfer from guaiacol to compound I without a dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer.     

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.     

Redox properties of the couples compound I/compound II and compound II/native enzyme of human myeloperoxidase

Myeloperoxidase (MPO) is an important component of the neutrophil's antimicrobial armory and has been implicated in promoting tissue damage in numerous inflammatory diseases. For the first time the standard reduction potential of the redox couple compound II/native enzyme has been determined to be (0.97+/-0.01)V at pH 7.0 and 25 degrees C. This was achieved by rapid mixing of preformed compound II with either tyrosine or nitrite by using the sequential-mixing stopped-flow technique and measuring spectrophotometrically the concentrations of the reacting species and products at equilibrium. Using the recently determined standard reduction potential for the couple compound I/native enzyme (1.16 V), the reduction potential of the couple compound I/compound II was calculated to be 1.35 V at pH 7 and 25 degrees C. These data reveal substantial differences between the two known heme peroxidase superfamilies and reflect the dramatic differences observed in the oxidisability of substrates by the MPO redox intermediates compound I and compound II.     

Effects of cyanogen bromide modification of the distal histidine on the spectroscopic and ligand binding properties of myoglobin: magnetic circular dichroism spectroscopy as a probe of distal water ligation in ferric high-spin histidine-bound heme proteins

Magnetic circular dichroism (MCD) spectroscopy has been utilized to characterize the change in coordination structure in native ferric sperm whale myoglobin upon cyanogen bromide-modification. Comparison of the MCD properties of the ferric high-spin state of cyanogen bromide-modified myoglobin (BrCN-Mb) with those of native ferric horseradish peroxidase and Aplysia myoglobin suggests that ferric BrCN-Mb is a potential MCD model for the pentacoordinate state of ferric high-spin histidine-ligated heme proteins. These five-coordinate heme proteins afford a relatively weak and unsymmetric signal in the Soret region of the MCD spectrum. In contrast, native ferric myoglobin and the benzohydroxamic acid adduct of ferric horseradish peroxidase show a strong and symmetric derivative-shaped Soret MCD signal which is indicative of hexacoordination with water and histidine axial ligands. Therefore it seems that MCD spectroscopy could be used to probe the presence of water ligated to the distal side of ferric high-spin heme proteins. The MCD spectra of the ferric-azide, ferrous-deoxy and ferrous-CO forms of BrCN-Mb have also been measured and compared to those of analogous native myoglobin complexes. The present MCD study has been extended to include new ligands, NO, thiocyanate and cyanate, which bind to ferric BrCN-Mb. With exogenous ligands such as CO, NO and thiocyanate, the coordination structures of the BrCN-Mb complexes are similar to those of the respective native myoglobin adducts. In the case of ferrous-deoxy and ferric-cyanate BrCN-Mb, however, the altered MCD spectra (and EPR for the latter) reveal changes in electronic structure which likely correlate with alterations of the coordination environment of these BrCN-Mb derivatives. Data are also presented which support the proposed tetrazole-bound structure for azide-treated BrCN-Mb (Hori, H., Fujii, M., Shiro, Y., Iizuka, T., Adachi, S. and Morishima, I. (1989) J. Biol. Chem. 264, 5715-5719) and the inability of the distal histidine of BrCN-Mb to stabilize the ferric ligand-bound state.     

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.