Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by 15N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN-/H2O2/HRP system and compared with the corresponding data on the SCN-/H2O2/LPO system. The NMR studies show that (SCN)2 is the oxidation product of thiocyanate in the SCN-/H2O2/HRP system, and its formation is maximum at pH less than or equal to 4 and that the oxidation does not take place at pH greater than or equal to 6. Since thiocyanate does not bind to HRP at pH greater than or equal to 6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H2O2]/[SCN-] = 4, (SCN)2 decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN-/H2O2/LPO system has been shown to be HOSCN/OSCN- which shows maximum inhibition of uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to LPO at the distal histidine while to HRP near 1- and 8-CH3 heme groups, the role of distal histidine in the activity of SCN-/H2O2/(LPO, HRP) systems is indicated.     

Reactions and reactivity of myeloperoxidase-derived oxidants: differential biological effects of hypochlorous and hypothiocyanous acids

Myeloperoxidase (MPO) is recognised to play important roles both in the immune system and during the development of numerous human pathologies. MPO is released by activated neutrophils, monocytes and some tissue macrophages, where it catalyses the conversion of hydrogen peroxide to hypohalous acids (HOX; X = Cl, Br, SCN) in the presence of halide and pseudo-halide ions. The major reactive species produced by MPO under physiological conditions are hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), with the ratio of these oxidants critically dependent on the concentration of thiocyanate ions (SCN?). The reactivity and selectivity of HOCl and HOSCN for biological targets are markedly different, indicating that SCN? ions have the potential to modulate both the extent and nature of oxidative damage in vivo. This article reviews recent developments in our understanding of the role of SCN? in modulating the formation of MPO-derived oxidants, particularly in respect to the differences in reaction kinetics and targets of HOCl compared to HOSCN and the ability of these two oxidants to induce damage in biological systems.     

A kinetic analysis of the catalase activity of myeloperoxidase

The predominant physiological activity of myeloperoxidase is to convert hydrogen peroxide and chloride to hypochlorous acid. However, this neutrophil enzyme also degrades hydrogen peroxide to oxygen and water. We have undertaken a kinetic analysis of this reaction to clarify its mechanism. When myeloperoxidase was added to hydrogen peroxide in the absence of reducing substrates, there was an initial burst phase of hydrogen peroxide consumption followed by a slow steady state loss. The kinetics of hydrogen peroxide loss were precisely mirrored by the kinetics of oxygen production. Two mols of hydrogen peroxide gave rise to 1 mol of oxygen. With 100 microM hydrogen peroxide and 6 mM chloride, half of the hydrogen peroxide was converted to hypochlorous acid and the remainder to oxygen. Superoxide and tyrosine enhanced the steady-state loss of hydrogen peroxide in the absence of chloride. We propose that hydrogen peroxide reacts with the ferric enzyme to form compound I, which in turn reacts with another molecule of hydrogen peroxide to regenerate the native enzyme and liberate oxygen. The rate constant for the two-electron reduction of compound I by hydrogen peroxide was determined to be 2 x 10(6) M(-1) s(-1). The burst phase occurs because hydrogen peroxide and endogenous donors are able to slowly reduce compound I to compound II, which accumulates and retards the loss of hydrogen peroxide. Superoxide and tyrosine drive the catalase activity because they reduce compound II back to the native enzyme. The two-electron oxidation of hydrogen peroxide by compound I should be considered when interpreting mechanistic studies of myeloperoxidase and may influence the physiological activity of the enzyme.     

Heme degradation as catalyzed by a recombinant bacterial heme oxygenase (Hmu O) from Corynebacterium diphtheriae.

Hmu O, a heme degradation enzyme in the pathogen Corynebacterium diphtheriae, catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. A bacterial expression system using a synthetic gene coding for the 215-amino acid, full-length Hmu O has been constructed. Expressed at very high levels in Escherichia coli BL21, the enzyme binds hemin stoichiometrically to form a hexacoordinate high spin hemin-Hmu O complex. When ascorbic acid is used as the electron donor, Hmu O converts hemin to biliverdin with alpha-hydroxyhemin and verdoheme as intermediates. The overall conversion rate to biliverdin is approximately 4-fold slower than that by rat heme oxygenase (HO) isoform 1. Reaction of the hemin-Hmu O complex with hydrogen peroxide yields a verdoheme species, the recovery of which is much less compared with rat HO-1. Reaction of the hemin complex with meta-chloroperbenzoic acid generates a ferryl oxo species. Thus, the catalytic intermediate species and the nature of the active form in the first oxygenation step of Hmu O appear to be similar to those of the mammalian HO. However, the considerably slow catalytic rate and low level of verdoheme recovery in the hydrogen peroxide reaction suggest that the active-site structure of Hmu O is different from that of its mammalian counterpart.     

Hypochlorous acid-induced heme degradation from lactoperoxidase as a novel mechanism of free iron release and tissue injury in inflammatory diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H(2)O(2)) in the airways through its ability to oxidize thiocyanate (SCN(-)) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 μM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O(2), Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN(-). On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function.     

The reactivity of myeloperoxidase compound I formed with hypochlorous acid

The reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1 x 10(7) M(-1)s(-1) (pH 5), 2.0 x 10(8) M(-1)s(-1) (pH 7) and 2.0 x 10(6) M(-1)s(-1) (pH 9) at 15 degrees C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H2O2, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller.     

The inhibition of streptococci by lactoperoxidase, thiocyanate and hydrogen peroxide. The oxidation of thiocyanate and the nature of the inhibitory compound

1. The growth of the lactoperoxidase-sensitive Streptococcus cremoris 972 in a synthetic medium was inhibited by lactoperoxidase and thiocyanate. The glycolysis and oxygen uptake of suspensions of Strep. cremoris 972 in glucose or lactose were also inhibited. The lactoperoxidase-resistant Strep. cremoris 803 was not inhibited under these conditions but was inhibited in the absence of a source of energy. 2. Lactoperoxidase (EC 1.11.1.7), thiocyanate and hydrogen peroxide completely inhibited the hexokinases of non-metabolizing suspensions of both strains. The inhibition was reversible, hexokinase and glycolytic activities of Strep. cremoris 972 being restored by washing the cells free from inhibitor. The aldolase and 6-phosphogluconate-dehydrogenase activities of Strep. cremoris 972 were partially inhibited but several other enzymes were unaffected. 3. The resistance of Strep. cremoris 803 to inhibition was not due to the lack of hydrogen peroxide formation, to the destruction of peroxide, to the inactivation of lactoperoxidase or to the operation of alternative pathways of carbohydrate metabolism. 4. A ;reversal factor', which was partially purified from extracts of Strep. cremoris 803, reversed the inhibition of glycolysis of Strep. cremoris 972. The ;reversal factor' also catalysed the oxidation of NADH(2) in the presence of an intermediate oxidation product of thiocyanate and was therefore termed the NADH(2)-oxidizing enzyme. 5. The NADH(2)-oxidizing enzyme was present in lactoperoxidase-resistant streptococci but was absent from lactoperoxidase-sensitive streptococci.     

Spectral and kinetic studies on the formation of eosinophil peroxidase compound I and its reaction with halides and thiocyanate

Compound I of peroxidases takes part in both the peroxidation and the halogenation reaction. This study for the first time presents transient kinetic measurements of the formation of compound I of human eosinophil peroxidase (EPO) and its reaction with halides and thiocyanate, using the sequential-mixing stopped-flow technique. Addition of 1 equiv of hydrogen peroxide to native EPO leads to complete formation of compound I. At pH 7 and 15 degrees C, the apparent second-order rate constant is (4.3 +/- 0.4) x 10(7) M(-1) s(-1). The rate for compound I formation by hypochlorous acid is (5.6 +/- 0.7) x 10(7) M(-1) s(-1). EPO compound I is unstable and decays to a stable intermediate with a compound II-like spectrum. At pH 7, the two-electron reduction of compound I to the native enzyme by thiocyanate has a second-order rate constant of (1.0 +/- 0. 5) x 10(8) M(-1) s(-1). Iodide [(9.3 +/- 0.7) x 10(7) M(-1) s(-1)] is shown to be a better electron donor than bromide [(1.9 +/- 0.1) x 10(7) M(-1) s(-1)], whereas chloride oxidation by EPO compound I is extremely slow [(3.1 +/- 0.3) x 10(3) M(-1) s(-1)]. The pH dependence studies suggest that a protonated form of compound I is more competent in oxidizing the anions. The results are discussed in comparison with those of the homologous peroxidases myeloperoxidase and lactoperoxidase and with respect to the role of EPO in host defense and tissue injury.     

The Pseudomonas toxin pyocyanin inhibits the dual oxidase-based antimicrobial system as it imposes oxidative stress on airway epithelial cells

The dual oxidase-thiocyanate-lactoperoxidase (Duox/SCN(-)/LPO) system generates the microbicidal oxidant hypothiocyanite in the airway surface liquid by using LPO, thiocyanate, and Duox-derived hydrogen peroxide released from the apical surface of the airway epithelium. This system is effective against several microorganisms that infect airways of cystic fibrosis and other immunocompromised patients. We show herein that exposure of airway epithelial cells to Pseudomonas aeruginosa obtained from long-term cultures inhibits Duox1-dependent hydrogen peroxide release, suggesting that some microbial factor suppresses Duox activity. These inhibitory effects are not seen with the pyocyanin-deficient P. aeruginosa strain PA14 Phz1/2. We show that purified pyocyanin, a redox-active virulence factor produced by P. aeruginosa, inhibits human airway cell Duox activity by depleting intracellular stores of NADPH, as it generates intracellular superoxide. Long-term exposure of human airway (primary normal human bronchial and NCI-H292) cells to pyocyanin also blocks induction of Duox1 by Th2 cytokines (IL-4, IL-13), which was prevented by the antioxidants glutathione and N-acetylcysteine. Furthermore, we showed that low concentrations of pyocyanin blocked killing of wild-type P. aeruginosa by the Duox/SCN(-)/LPO system on primary normal human bronchial epithelial cells. Thus, pyocyanin can subvert Pseudomonas killing by the Duox-based system as it imposes oxidative stress on the host. We also show that lactoperoxidase can oxidize pyocyanin, thereby diminishing its cytotoxicity. These data establish a novel role for pyocyanin in the survival of P. aeruginosa in human airways through competitive redox-based reactions between the pathogen and host.     

Two-electron reduction and one-electron oxidation of organic hydroperoxides by human myeloperoxidase

The reaction of native myeloperoxidase (MPO) and its redox intermediate compound I with hydrogen peroxide, ethyl hydroperoxide, peroxyacetic acid, t-butyl hydroperoxide, 3-chloroperoxybenzoic acid and cumene hydroperoxide was studied by multi-mixing stopped-flow techniques. Hydroperoxides are decomposed by MPO by two mechanisms. Firstly, the hydroperoxide undergoes a two-electron reduction to its corresponding alcohol and heme iron is oxidized to compound I. At pH 7 and 15 degrees C, the rate constant of the reaction between 3-chloroperoxybenzoic acid and ferric MPO was similar to that with hydrogen peroxide (1.8x10(7) M(-1) s(-1) and 1.4x10(7) M(-1) s(-1), respectively). With the exception of t-butyl hydroperoxide, the rates of compound I formation varied between 5.2x10(5) M(-1) s(-1) and 2.7x10(6) M(-1) s(-1). Secondly, compound I can abstract hydrogen from these peroxides, producing peroxyl radicals and compound II. Compound I reduction is shown to be more than two orders of magnitude slower than compound I formation. Again, with 3-chloroperoxybenzoic acid this reaction is most effective (6. 6x10(4) M(-1) s(-1) at pH 7 and 15 degrees C). Both reactions are controlled by the same ionizable group (average pK(a) of about 4.0) which has to be in its conjugated base form for reaction.     

Redox properties of the couple compound I/native enzyme of myeloperoxidase and eosinophil peroxidase.

The standard reduction potential of the redox couple compound I/native enzyme has been determined for human myeloperoxidase (MPO) and eosinophil peroxidase (EPO) at pH 7.0 and 25 degrees C. This was achieved by rapid mixing of peroxidases with either hydrogen peroxide or hypochlorous acid and measuring spectrophotometrically concentrations of the reacting species and products at equilibrium. By using hydrogen peroxide, the standard reduction potential at pH 7.0 and 25 degrees C was 1.16 +/- 0.01 V for MPO and 1.10 +/- 0.01 V for EPO, independently of the concentration of hydrogen peroxide and peroxidases. In the case of hypochlorous acid, standard reduction potentials were dependent on the hypochlorous acid concentration used. They ranged from 1.16 V at low hypochlorous acid to 1.09 V at higher hypochlorous acid for MPO and from 1.10 V to 1.03 V for EPO. Thus, consistent results for the standard reduction potentials of redox couple compound I/native enzyme of both peroxidases were obtained with all hydrogen peroxide and at low hypochlorous acid concentrations: possible reasons for the deviation at higher concentrations of hypochlorous acid are discussed. They include instability of hypochlorous acid, reactions of hypochlorous acid with different amino-acid side chains in peroxidases as well as the appearance of a compound I-chloride complex.     

Thiocyanate catalyzes myeloperoxidase-initiated lipid oxidation in LDL

There is evidence that LDL oxidation may render the lipoprotein atherogenic. The myeloperoxidase-hydrogen peroxide (MPO/H2O2) system of activated phagocytes may be involved in this process. Chloride is supposed to be the major substrate for MPO, generating reactive hypochlorous acid (HOCl), modifying LDL. The pseudo-halide thiocyanate (SCN-) has been shown to be a suitable substrate for MPO, forming reactive HOSCN/SCN*. As relatively abundant levels of SCN- are found in plasma of smokers--a well-known risk group for cardiovascular disease--the ability of SCN- to act as a catalyst of LDL atherogenic modification by MPO/H2O2 was tested. Measurement of conjugated diene and lipid hydroperoxide formation in LDL preparations exposed to MPO/H2O2 revealed that SCN- catalyzed lipid oxidation in LDL. Chloride did not diminish the effect of SCN- on lipid oxidation. Surprisingly, SCN inhibited the HOCl-mediated apoprotein modification in LDL. Nitrite--recently found to be a substrate for MPO--showed some competing properties. MPO-mediated lipid oxidation was inhibited by heme poisons (azide, cyanide) and catalase. Ascorbic acid was the most effective compound in inhibiting the SCN- -catalyzed reaction. Bilirubin showed some action, whereas tocopherol was ineffective. When LDL oxidation was performed with activated human neutrophils, which employ the MPO pathway, SCN- catalyzed the cell-mediated LDL oxidation. The MPO/H2O2/SCN- system may have the potential to play a significant role in the oxidative modification of LDL--an observation further pointing to the link between the long-recognized risk factors of atherosclerosis: elevated levels of LDL and smoking.     

The reactivity of myeloperoxidase compound I formed with hypochlorous acid

Abstract

The reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl^-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1×10⁷ M^-1s^-1 (pH 5), 2.0×10⁸ M^-1s^-1 (pH 7) and 2.0×10⁶ M^-1s^-1 (pH 9) at 15°C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H₂O₂, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller.     

Polarographic Studies on Sarkomycin

Nicotinic acid hydrazid (NH) gave three waves at the dropping mercury electrode and can be polarographically determined. Sarkomycin nicotinic acid hydrazid derivative (SK-NH), consisting of sarkomycin (SK) and NH, gave three polarographic waves, one of which disappeared in alkaline pH region. The half-wave potential of the second wave of SK-NH was the same as that of the first wave of SK and the half-wave potential of the third wave in the acid pH region was the same as that of the second wave of NH. The relation between limiting current and concentration of SK-NH was linear, but the regression obtained by one lot of sample was different from that by another lot of sample. The regression of limiting current of the third wave at pH 8.5 upon NH content was coincident within error throughout the samples used herein.     

Polarographic Assay of Hydrogen Peroxide Accumulation in Microbial Cultures

A method is described for determining low concentrations of hydrogen peroxide by using a polarographic oxygen electrode to measure the oxygen released into solution on addition of catalase. A sample can be assayed directly without prior manipulation in 3 min. The method is capable of assaying hydrogen peroxide concentrations as low as 7 μM. The method has proved extremely useful for the assay of hydrogen peroxide secreted into milk by lactic acid bacteria.     

A combined experimental and quantum chemical study on the putative protonophoric activity of thiocyanate

Inhibition of gastric acid secretion by thiocyanate is explained by a protonophoric mechanism assuming that thiocyanate induces a H(+) back flux from the acidic gastric lumen into the parietal cells of gastric mucosa. Protonophoric activity of thiocyanate was examined by swelling measurements using rat liver mitochondria and theoretically by quantum chemical methods. Mitochondria suspended in K-thiocyanate medium plus nigericin (an H/K-exchanger) swelled when the medium pH was acidic, indicating that SCN(-) initiates a transfer of H(+) across the inner membrane. To rationalize the protonophoric activity of thiocyanate, we considered the dehydration of SCN(-) to be critical for transmembranal H(+) transfer. For modeling this process, various hydrate clusters of SCN(-) and Cl(-) were generated and optimized by density functional theory (DFT) at the B3-LYP/6-311++G(d,p) level. The cluster hydration energy was lower for SCN(-) than for Cl(-). The total Gibbs free energies of hydration of the ions were estimated by a hybrid supermolecule-continuum approach based on DFT. The calculated hydration energies also led to the conclusion that SCN(-) is less efficiently solvated than Cl(-). Due to the easier removal of the hydration shell of SCN(-) relative to Cl(-), SCN(-) is favored in going across the membrane, giving rise to the protonophoric activity.     

Leukocyte-mediated inactivation of alpha 1-proteinase inhibitor is inhibited by amino analogues of alpha-tocopherol.

Human leukocytes stimulated by opsonized zymosan increase their NADPH oxidase-catalysed reduction of molecular oxygen. This leads to enhanced formation of superoxyl radicals and subsequently hydrogen peroxide. The leukocyte enzyme myeloperoxidase generates the strong microbicidal oxidant hypochlorite from hydrogen peroxide and chloride anions. Hypochlorite inactivates serum alpha 1-proteinase inhibitor, a protein which protects host tissue from digestion by proteinases, that are also secreted by stimulated leukocytes. Micromolar concentrations of a water-soluble, quaternary ammonium analogue of alpha-tocopherol (vitamin E) (3,4-dihydro-6-hydroxy-N,N,N-2,5,7,8-heptamethyl-2H-1-benzopyran-2 -ethanaminium 4-methylbenzenesulfonate) and its tertiary amine derivative (3,4-dihydro-2- (2-dimethylaminoethyl)-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol hydrochloride) were able to protect alpha 1-proteinase inhibitor from inactivation by stimulated human leukocytes. The mechanism of action of the quaternary ammonium analogue was further investigated. Selective inhibition of hydrogen peroxide formation is assumed to be the reason for its protective effect. This compound rapidly reacts with superoxyl radicals, but not with hydrogen peroxide, and is only a weak hypochlorite scavenger. It neither impedes exocytosis of elastase, nor effectively inhibits NADPH oxidase or myeloperoxidase. In contrast, superoxide dismutase, which enhances hydrogen peroxide formation, cannot protect alpha 1-proteinase inhibitor from inactivation.     

Electron paramagnetic resonance and spectrophotometric studies of the peroxide compounds of manganese-substituted horseradish peroxidase, cytochrome-c peroxidase and manganese-porphyrin model complexes

Peroxide compounds of manganese protoporphyrin IX and its complexes with apo-horseradish peroxidase and apocytochrome-c peroxidase were characterized by electronic absorption and electron paramagnetic resonance spectroscopies. An intermediate formed upon titration of Mn(III)-horseradish peroxidase with hydrogen peroxide exhibited a new electron paramagnetic resonance absorption at g = 5.23 with a definite six-lined 55Mn hyperfine (AMn = 8.2 mT). Neither a porphyrin pi-cation radical nor any other radical in the apoprotein moiety could be observed. The reduced form of Mn-horseradish peroxidase, Mn(II)-horseradish peroxidase, reacted with a stoichiometric amount of hydrogen peroxide to form a peroxide compound whose electronic absorption spectrum was identical with that formed from Mn(III)-horseradish peroxidase. The electronic state of the peroxide compound of manganese horseradish peroxidase was thus concluded to be Mn(IV), S = 3/2. Mn(III)-cytochrome-c peroxidase reacted with stoichiometry quantities of hydrogen peroxide to form a catalytically active intermediate. The electronic absorption spectrum was very similar to that of a higher oxidation state of manganese porphyrin, Mn(V). Since the peroxide compound of manganese cytochrome-c peroxidase retained two oxidizing equivalents per mol of the enzyme (Yonetani, T. and Asakura, T. (1969) J. Biol. Chem. 244, 4580-4588), this peroxide compound might contain an Mn(V) center.     

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 oxidation of reduced nicotinamide nucleotides by hydrogen peroxide in the presence of lactoperoxidase and thiocyanate, iodide or bromide

Lactoperoxidase (EC 1.11.1.7) catalysed the oxidation of NADH by hydrogen peroxide in the presence of either thiocyanate, iodide or bromide. In the presence of thiocyanate, net oxidation of thiocyanate occurred simultaneously with the oxidation of NADH, but in the presence of iodide or bromide, only the oxidation of NADH occurred to a significant extent. In the presence of thiocyanate or bromide, NADH was oxidized to NAD(+) but in the presence of iodide, an oxidation product with spectral and chemical properties distinct from NAD(+) was formed. Thiocyanate, iodide and bromide appeared to function in the oxidation of NADH by themselves being oxidized to products which in turn oxidized NADH, rather than by activating the enzyme. Iodine, which oxidized NADH non-enzymically, appeared to be an intermediate in the oxidation of NADH in the presence of iodide. NADPH was oxidized similarly under the same conditions. An assessment was made of the rates of these oxidation reactions, together with the rates of other lactoperoxidase-catalysed reactions, at physiological concentrations of thiocyanate, iodide and bromide. The results indicated that in milk and saliva the oxidation of thiocyanate to a bacterial inhibitor was likely to predominate over the oxidation of NADH.