Chemical and transient state kinetic studies on the formation and decomposition of horseradish peroxidase compounds XI and XII

Previous studies on the chlorination reaction catalyzed by horseradish peroxidase using chlorite as the source of chlorine detected the formation of a chlorinating intermediate that was termed Compound X (Shahangian, S., and Hager, L.P. (1982) J. Biol. Chem. 257, 11529-11533). These studies indicated that at pH 10.7, the optical absorption spectrum of Compound X was similar to the spectrum of horseradish peroxidase Compound II. Compound X was shown to be quite stable at alkaline pH values. This study was undertaken to examine the relationship between the oxidation state of the iron protoporphyrin IX heme prosthetic group in Compound X and the chemistry of the halogenating intermediate. The experimental results show that the optical absorption properties and the oxidation state of the heme prosthetic group in horseradish peroxidase are not directly related to the presence of the activated chlorine atom in the intermediate. The oxyferryl porphyrin heme group in alkaline Compound X can be reduced to a ferric heme species that still retains the activated chlorine atom. Furthermore, the reaction of chlorite with horseradish peroxidase at acidic pH leads to the secondary formation of a green intermediate that has the spectral properties of horseradish peroxidase Compound I (Theorell, H. (1941) Enzymologia 10, 250-252). The green intermediate also retains the activated chlorine atom. By analogy to peroxidase Compound I chemistry, the heme prosthetic group in the green chlorinating intermediate must be an oxyferryl porphyrin pi-cation radical species (Roberts, J. E., Hoffman, B. M., Rutter, R. J., and Hager, L. P. (1981) J. Am. Chem. Soc. 103, 7654-7656). To be consistent with traditional peroxidase nomenclature, the red alkaline form of Compound X has been renamed Compound XII, and the green acidic form has been named Compound XI. The transfer of chlorine from the chlorinating intermediate to an acceptor molecule follows an electrophilic (rather than a free radical) path. A mechanism for the reaction is proposed in which the activated chlorine atom is bonded to a heteroatom on an active-site amino acid side chain. Transient state kinetic studies show that the initial intermediate, Compound XII, is formed in a very fast reaction. The second-order rate constant for the formation of Compound XII is approximately 1.1 x 10(7) M-1 s-1. The rate of formation of Compound XII is strongly pH-dependent. At pH 9, the second-order rate constant for the formation of Compound XII drops to 1.5 M-1 s-1. At acidic pH values, Compound XII undergoes a spontaneous first-order decay to yield Compound XI.(ABSTRACT TRUNCATED AT 400 WORDS)     

Compound X. An intermediate in enzymatic halogenation.

Previous studies have shown that chlorite serves as a halogenation substrate for horseradish peroxidase. In its substrate role, chlorite serves both as a halogen donor and as a source of oxidizing equivalents in the chlorination reaction. We now show that a new spectral intermediate, which we have termed Compound X, can be detected as the initial product of the reaction of chlorite with horseradish peroxidase. The reaction of chlorite with horseradish peroxidase to form Compound X is a relatively fast reaction especially at acidic pH values. The second order rate constant (Kf) for the formation of Compound X at pH 4.5 (optimum pH) is 0.9 X 10(6) M-1 S-1. Compound X, in the absence of a halogen acceptor, decomposes to Compound I and chloride ion. The first order rate constant (Kd) for the decay of Compound X to Compound I is 0.2 s-1 at pH 4.5. The pH optimum for enzymatic chlorination with chlorite compares favorably with the pH profile for the lifetime of Compound X (Kf/Kd). These observations indicate that Compound X is the halogenating intermediate in the chlorite reaction and that the rate of enzymatic chlorination is directly related to the stability of Compound X. We propose an -OCl ligand on a ferric heme as the most likely structure for Compound X.     

Proton stoichiometry of the cytochrome c peroxidase mechanism as a function of pH.

The proton stoichiometry for the oxidation of cytochrome c peroxidase (ferrocytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) to cytochrome c peroxidase Compound I by H2O2, for the reduction of cytochrome c peroxidase Compound I to cytochrome c peroxidase Compound II by ferrocyanide, and for the reduction of cytochrome c peroxidase Compound II to the native enzyme by ferrocyanide has been determined as a function of pH between pH 4 and 8. The basic stoichiometry for the reaction is that no protons are required for the oxidation of the native enzyme to Compound I, while one proton is required for the reduction of Compound I to Compound II, and one proton is required for the reduction of Compound II to the native enzyme. Superimposed upon the basic stoichiometry is a contribution due to the perturbation of two ionizable groups in the enzyme by the redox reactions. The pKa values for the two groups are 4.9 +/- 0.3 and 5.7 +/- 0.2 in the native enzyme, 4.1 +/- 0.4 and 7.8 +/- 0.2 in Compound I, and 4.3 +/- 0.4 and 6.7 +/- 0.2 in Compound II.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

The oxidation-reduction potentials of compound I/compound II and compound II/ferric couples of horseradish peroxidases A2 and C.

The reversibility of the stepwise reduction of Compound I to the ferric state via Compound II was confirmed in horseradish peroxidases A2 and C. The values of E'o (compound I/Compound II) and E'O (Compound II/ferric) were measured from equilibrium data coupled with the K2IrCl6-K3IrCl6 system in a narrow region of pH near 6.3. The ferric enzymes were also oxidized by ferricyanide to Compound II at alkaline pH and the values of E'O (Compound II/ferric) were measured from the equilibrium data. The pH dependence of E'O (Compound II/ferric) was in accord with the equation: E'O = EO + 0.058 log (Kr[H+] + [H+]2)/(KO + [H+]), where Kr and KO are proton dissociation constants in the ferric enzyme and Compound II, respectively. The pH-E'O (Compound I/Compound II) curves were likewise obtained from the equation, E'O = EO + 0.058 log (Kr + [H+]), where Kr is the proton dissociation constant in Compound II. The forward and backward rate constants were measured in each of one-electron transfer reactions of the peroxidases with the K2IrCl6-K3IrCl6 system at various pH values. The E'O values calculated on the assumption that the ratio of the rate constants equals the equilibrium constant were compared with those obtained from the equilibrium data.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Stoichiometry of the reaction between horseradish peroxidase and p-cresol.

Over a wide range of pH horseradish peroxidase compound I can be reduced quantitatively via compound II to the native enzyme by only 1 molar equivalent of p-cresol. Since 2 molar equivalents of electrons are required for the single turnover of the enzymatic cycle, p-cresol behaves as a 2-electron reductant. With p-cresol and compound I in a 1:1 ratio compound II and p-methylphenoxy radicals are obtained in the transient state. Compound II is then reduced to the native enzyme. A possible explanation for the facile reduction of compound II involves reaction with the dimerization product of these radicals, 1/2 molar equivalent of 2,2'-dihydroxy-5,5'-dimethylbiphenyl. If only 1/2 molar equivalent of p-cresol is present, than at high pH the reduction stops at compound II. The major steady state peroxidase oxidation product of p-cresol (with p-cresol in large excess compared to the enzyme concentration) is Pummerer's ketone. Pummerer's ketone is only reactive at pH values greater than about 9 where significant amounts of the enol can be formed via the enolate anion. Therefore, in alkaline solution it is reactive with compound I, but not with compound II, which is converted into an unreactive basic form. These results indicate that Pummerer's ketone cannot be the intermediate free radical product responsible for reducing compound II in the single turnover experiments. It is postulated that Pummerer's ketone is formed only in the steady state by the reaction of the p-methylphenoxy radical with excess p-cresol.     

Oxidation of p-cresol by horseradish peroxidase compound I.

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

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.     

Pre-steady-state kinetic study on the formation of Compound I and II of ligninase

The reaction between ligninase and hydrogen peroxide yielding Compound I has been investigated using a stopped-flow rapid-scan spectrophotometer. The optical absorption spectrum of Compound I appears different to that reported by Andrawis, A. et al. (1987) and Renganathan, V. and Gold, M.H. (1986), in that the Soret-maximum is at 401 nm rather than 408 nm. The second-order rate constant (4.2·10⁵ M⁻¹·s⁻¹) for the formation of Compound I was independent of pH (pH 3.0–6.0). In the absence of external electron donors, Compound I decayed to Compound II with a half-life of 5–10 s at pH 3.1. The rate of this reaction was not affected by the H₂O₂ concentration used. In the presence of either veratryl alcohol or ferrocyanide, Compound II was rapidly generated. With ferrocyanide, the second-order rate constant increased from 1.9·10⁴ M⁻¹·s⁻¹ to 6.8·10⁶ M⁻¹·s⁻¹ when the pH was lowered from 6.0 to 3.1. With veratryl alcohol as an electron donor, the second-order rate constant for the formation of Compound II increased from 7.0·10³ M⁻¹·s⁻¹ at pH 6.0 to 1.0·10⁵ M⁻¹·s⁻¹ at pH 4.5. At lower pH values the rate of Compound II formation no longer followed an exponential relationship and the steady-state spectral properties differed to those recorded in the presence of ferrocyanide. Our data support a model of enzyme catalysis in which veratryl alcohol is oxidized in one-electron steps and strengthen the view that veratryl alcohol oxidation involves a substrate-modified Compound II intermediate which is rapidly reduced to the native enzyme.     

Characterization of the oxidized states of bromoperoxidase

Bromoperoxidase Compound I has been formed in reactions between bromoperoxidase and organic peroxide substrates. The absorbance spectrum of bromoperoxidase Compound I closely resembles the Compound I spectra of other peroxidases. The pH dependence of the second order rate constant for the formation of Compound I with hydrogen peroxide demonstrates the presence of an ionizable group at the enzyme active site having a pKa of 5.3. Protonation of this acidic group inhibits the rate of Compound I formation. This pKa value is higher than that determined for other peroxidases but the overall pH rate profiles for Compound I formation are similar. The one-electron reduction of bromoperoxidase Compound I yields Compound II and a second reduction yields native enzyme. Bromoperoxidase Compound II readily forms Compound III in the presence of an excess of hydrogen peroxide. Compound III passes through an as yet uncharacterized intermediate (III) in its decay to native enzyme. Compound III is produced and accumulates in enzymatic bromination reactions to become the predominate steady state form of the enzyme. Since Compound III is inactive as catalyst for enzymatic bromination, its accumulation leads to an idling reaction pathway which displays an unusual kinetic pattern for the bromination of monochlorodimedone.     

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.     

A steady-state study on the formation of Compounds II and III of myeloperoxidase

The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a pKa of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5.10(4) M-1.s-1. During the reaction of myeloperoxidase with H2O2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a pKa of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6.10(7) M-1.s-1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III (k = 50 M-1.s-1) proceeded via two different pathways, one of which was inhibitable by tetranitromethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3-11.     

Mechanisms of electron transfer from sulfite to horseradish peroxidase-hydroperoxide compounds.

Using a rapid-scan spectrophotometer equipped with a stopped-flow apparatus, reactions of sulfite with compounds I and II of two horseradish peroxidase isoenzymes A and C were investigated. The direct two-electron reduction of peroxidase compound I by sulfite occurred at acidic pH but the mechanism gradually changed to the two-step reduction with the intermediate formation of compound II as the pH increased. The pH at which the one- and two-electron changes occurred at the same speed was 4.5 for peroxidase A and 7.7 for peroxidase C. A new peroxidase intermediate was found in the reaction between peroxidase compound II and sulfite. The sulfite compound showed a characteristic absorption band at 850 nm and the optical spectrum was similar to that of isoporphyrins but was quite different from that of sulfhemoproteins. The rate (k) of conversion from the sulfite-compound II complex to the sulfite compound was proportional to the concentration of H+ and the log k vs. pH plot for peroxidase A moved to the acidic side by 1.1 pH unit from that for peroxidase C.     

Kinetics of the oxidation of p-aminobenzoic acid catalyzed by horseradish peroxidase compounds I and II.

The kinetics of p-aminobenzoic acid oxidation catalyzed by horseradish peroxidase Compounds I and II was investigated intensively as a function of pH at 25 degrees in aqueous solutions of ionic strength 0.11. All of the rate data were collected from single turnover experiments involving reactions of a single enzyme compound. In reactions of both compounds, deviations from first order behavior with respect to the enzyme were observed at high pH values which were explained in terms of a free radical interaction of product with the enzyme. The effect could be eliminated with sufficient excess of substrate. Kinetic behavior which deviated from first order in substrate, observed at low pH, was explained by a mechanism involving an enzyme-substrate complex which reacted with an additional molecule of substrate but at a slower rate. The pH dependence of the second order rate constants for the reaction of p-aminobenzoic acid with free Compounds I and II is similar to results obtained for the comparable reactions of ferrocyanide, suggesting similar proton-transfer mechanisms for both reducing substrates. The reduction of Compound II by p-aminobenzoic acid appeared to be influenced by two ionizable groups on the enzyme which affect the electronic environment of the heme. The lack of influence of substrate ionizable groups on the rate of the Compound II reaction indicated that potential differences in reactivities of NH2C6H4COO- and NH2C6H4COOH were levelled by the diffusion-controlled limit in the acid region of pH. The reduction of Compound I by p-aminobenzoic acid was not diffusion-controlled and the rate-pH profile could be explained in terms of three acid ionizations, two on the substrate and one on Compound I.     

Effect ofpH on the formation of Compounds II and III of horseradish peroxidase

The formation of Compounds II and III of horseradish peroxidase from Compound I and potassium ferrocyanide and from Compound II and excess hydrogen peroxide, respectively, was studied as a function ofpH at 25°C and a constant ionic strength of 0.11. The yield of Compound II obtained increases progressively with increase inpH; a mixture of Compounds I and II is produced at acidicpH. Pure Compound III is obtained at allpH values, but the highest yield is obtained atpH values between 6.0 and 7.0. The yield of p-670, formed when Compound III is allowed to stand for 60 min, decreases with increase inpH, while the decay of Compound III also decreases with increase inpH. Therefore p-670 is the decay product of Compound III.     

Effect ofpH on the formation of Compounds II and III of horseradish peroxidase

The formation of Compounds II and III of horseradish peroxidase from Compound I and potassium ferrocyanide and from Compound II and excess hydrogen peroxide, respectively, was studied as a function ofpH at 25°C and a constant ionic strength of 0.11. The yield of Compound II obtained increases progressively with increase inpH; a mixture of Compounds I and II is produced at acidicpH. Pure Compound III is obtained at allpH values, but the highest yield is obtained atpH values between 6.0 and 7.0. The yield of p-670, formed when Compound III is allowed to stand for 60 min, decreases with increase inpH, while the decay of Compound III also decreases with increase inpH. Therefore p-670 is the decay product of Compound III.     

Resonance Raman spectra of bovine liver catalase compound II. Similarity of the heme environment to horseradish peroxidase compound II

Resonance Raman spectroscopy has been used to investigate the structure and environment of the heme group in bovine liver catalase compound II. Both Soret- and Q-band excitation have been employed to observe and assign the skeletal stretching frequencies of the porphyrin ring. The oxidation state marker band v4 increases in frequency from 1373 cm-1 in ferricatalase to 1375 cm-1 in compound II, consistent with oxidation of the iron atom to the Fe(IV) state. Oxidation of five-coordinate, high-spin ferricatalase to compound II is accompanied by a marked increase of the porphyrin core marker frequencies that is consistent with a six-coordinate low-spin state with a contracted core. An Fe(IV) = O stretching band is observed at 775 cm-1 for compound II at neutral pH, indicating that there is an oxo ligand at the sixth site. At alkaline pH, the Fe(IV) = O stretching band shifts to 786 cm-1 in response to a heme-linked ionization that is attributed to the distal His-74 residue. Experiments carried out in H218O show that the oxo ligand of compound II exchanges with bulk water at neutral pH, but not at alkaline pH. This is essentially the same behavior exhibited by horseradish peroxidase compound II and the exchange reaction at neutral pH for both enzymes is attributed to acid/base catalysis by a distal His residue that is believed to be hydrogen-bonded to the oxo ligand. Thus, the structure and environment of the heme group of the compound II species of catalase and horseradish peroxidase are very similar. This indicates that the marked differences in their reactivities as oxidants are probably due to the manner in which the protein controls access of substrates to the heme group.     

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.     

Kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II

The kinetics of the oxidation of ferrocyanide by lactoperoxidase compound II has been studied over the pH range 5.2-9.9 at 25 degrees C and an ionic strength of 0.11 M. For all pH values, exponential decay curves are obtained for the reaction of compound II in the presence of ferrocyanide which yielded pseudo-first-order rate constants kobs. The spontaneous decay of compound II in the absence of ferrocyanide occurs at an appreciable rate which was measured independently and used in the data analysis. At all pH values two striking effects were observed when the rate of the decay reaction in the presence of ferrocyanide, kobs, was plotted against ferrocyanide concentration: a saturation effect and positive intercepts which are attributable to the spontaneous decay. The plots of kobs versus ferrocyanide concentration were analyzed in terms of the following parameters: a first-order rate constant k3,obs, a Michaelis constant Km,obs and a spontaneous-decay rate constant k4. The parameters k3,obs and Km,obs describe the reaction of compound II with ferrocyanide, independently of the spontaneous decay. The parameter k4 has only a small pH dependence, whereas plots of the logs of k3,obs and Km,obs versus pH have slopes of -1 at high pH. The major part of the pH dependence can be explained by the influence of a single heme-linked acid group in the LPO-compound-II-ferrocyanide complex.