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Catalase-peroxidases are bifunctional peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity. Here we present a kinetic study of the formation and reduction of the key intermediate compound I by probing the role of the conserved tryptophan at the distal haem cavity site. Two wild-type proteins and three mutants of Synechocystis catalase-peroxidase (W122A and W122F) and Escherichia coli catalase-peroxidase (W105F) have been investigated by steady-state and stopped-flow spectroscopy. W122F and W122A completely lost their catalase activity whereas in W105F the catalase activity was reduced by a factor of about 5000. However, the mutations did not influence both formation of compound I and its reduction by peroxidase substrates. It was demonstrated unequivocally that the rate of compound I reduction by pyrogallol or o-dianisidine sometimes even exceeded that of the wild-type enzyme. This study demonstrates that the indole ring of distal Trp in catalase-peroxidases is essential for the two-electron reduction of compound I by hydrogen peroxide but not for compound I formation or for peroxidase reactivity (i.e. the one-electron reduction of compound I).

Myeloperoxidase is the most abundant protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central role in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidized by myeloperoxidase, and we have determined the features that enable them to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxidase. The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s-1 (15 degrees C, pH 7.0) and were relatively insensitive to the substituents on the aromatic ring. Rate constants for reduction of compound II varied between 6.5 x 10(5) M-1 s-1 for ABAH and 1.3 x 10(3) M-1 s-1 for 4-nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compound I and compound II by BAHs adhered to the Hammett rule, and there were significant correlations with Brown-Okamoto substituent constants. This indicates that the rates of these reactions were simply determined by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydrogen peroxide because it underwent auto-oxidation. Although BAHs generally reacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxidase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constants. BAHs containing more hydrophilic substituents were more effective at converting the enzyme to compound III. This implies that BAH radicals must hydrogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate constants for reduction of compounds I or II. We propose that BAHs destroy the heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex.

In the course of oxidation of thiols by peroxidases thiyl radicals are formed which are known to undergo several free‐radical conjugative reactions, among others leading to hydrogen peroxide formation. The present paper for the first time presents a comparative transient‐state and steady‐state investigation of the reaction of 15 aliphatic and aromatic mono‐ and dithiols with horseradish peroxidase (HRP). Both sequential‐stopped‐flow spectrophotometric investigations of the reaction of HRP intermediates Compound I (k2) and Compound II (k3) with thiols and measurements of the overall thiol oxidation and the simultaneous oxygen consumption in the presence and absence of exogenously added hydrogen peroxide (10 μM) have been performed. With HRP as thiyl radical generator it was shown that three groups of thiols have to be distinguished: (i) Aromatic thiols (e.g. thiophenol, 2‐mercaptopurine) were excellent electron donors of both Compounds (k2: 104–107M−1s−1and k3: 103–106M−1s−1); however, the overall reaction was shown to depend on addition of hydrogen peroxide, indicating insufficient peroxide regeneration by arylthiyl radicals. (ii) Aliphatic thiols which were extremely bad substrates (k3<10 M−1s−1) for HRP (e.g. homocysteine, glutathione) and/or have a pKa,SH>9.5 (e.g. N‐acetylcysteine, α‐lipoic acid) were also shown to depend on exogenously added H2O2to maintain the peroxidasic reaction, whereas (iii) with those thiols with rates of k3between 11 and 1600 M−1s−1(e.g. cysteine, cysteamine, cysteine methyl ester, cysteine ethyl ester) and/or with a pKa,SH<8 (penicillamine) thiol oxidation was independent of exogenously added hydrogen peroxide, indicating sufficient hydrogen peroxide regeneration.

In the course of oxidation of thiols by peroxidases thiyl radicals are formed which are known to undergo several free-radical conjugative reactions, among others leading to hydrogen peroxide formation. The present paper for the first time presents a comparative transient-state and steady-state investigation of the reaction of 15 aliphatic and aromatic mono- and dithiols with horseradish peroxidase (HRP). Both sequential-stopped-flow spectrophotometric investigations of the reaction of HRP intermediates Compound I ( k2) and Compound II ( k3) with thiols and measurements of the overall thiol oxidation and the simultaneous oxygen consumption in the presence and absence of exogenously added hydrogen peroxide (10 μM) have been performed. With HRP as thiyl radical generator it was shown that three groups of thiols have to be distinguished: (i) Aromatic thiols (e.g. thiophenol, 2-mercaptopurine) were excellent electron donors of both Compounds ( k2: 10 4–10 7M −1s −1and k3: 10 3–10 6M −1s −1); however, the overall reaction was shown to depend on addition of hydrogen peroxide, indicating insufficient peroxide regeneration by arylthiyl radicals. (ii) Aliphatic thiols which were extremely bad substrates ( k3<10 M −1s −1) for HRP (e.g. homocysteine, glutathione) and/or have a p Ka,SH>9.5 (e.g. N-acetylcysteine, α-lipoic acid) were also shown to depend on exogenously added H 2O 2to maintain the peroxidasic reaction, whereas (iii) with those thiols with rates of k3between 11 and 1600 M −1s −1(e.g. cysteine, cysteamine, cysteine methyl ester, cysteine ethyl ester) and/or with a p Ka,SH<8 (penicillamine) thiol oxidation was independent of exogenously added hydrogen peroxide, indicating sufficient hydrogen peroxide regeneration.

The described staining techniques for glucose oxidase activity on polyacrylamide gels are based on the fact that glucose oxidase reacts with one-electron scavengers in a reaction dependent on pH and pO2. Glucose oxidase separated in PAGE shows reduction of ferricyanide, cuprisulfate, ferricytochrome c and nitrotetrazolium blue reduction leading to sharp coloured bands under aerobic conditions. In contrast to the conventional peroxidase/o-dianisidine-assay, the staining procedures only need about 30 min and are specific for glucose oxidase using glucose as electron donor. Enzyme activity can be detected down to 0.2 units.

Wild-typeEscherichia coliHPII catalase (heme d containing) has 15% the activity of beef liver enzyme per heme. The rate constant for compound I formation with H2O2is 1.3 × 106m−1s−1. HPII compound I reacts with H2O2to form O2with a rate constant of 1.8 × 106m−1s−1. Forty percent of HPII hemes are in the compound I state during turnover. Compound I is reduced by ethanol and formate at rates of 5 and 13m−1s−1(pH 7.0), respectively. Incubation of HPII compound I with ferrocyanide and ascorbate does not form a compound II species. Mutation of His128 to alanine or asparagine gives inactive protoheme proteins. Mutation of Asn-201 gives partially active heme d forms. Asn201Ala has 24%, Asn201Asp 10%, and Asn201Gln 0.4% of wild-type activity. Asn201His contains protoheme when isolated and converts this via protoheme compound I to a heme d species. Both distal heme cavity residues His128 and Asn201 are implicated in catalytic activity, compound I formation, andin situheme d biosynthesis. HPII Asn201, like the corresponding residue in protoheme catalases, may promote H+transfer to His128 imidazole, facilitating (i) peroxide anion binding to heme and (ii) stabilization of a transition state for heterolytic cleavage of the O–O bond.

Cytosolic extracts of the cyanobacteriumAnacystis nidulansexhibit both catalase and o-dianisidine peroxidase activity. Native polyacrylamide gel electrophoresis demonstrates one distinct enzyme, which has been purified to essential homogeneity and found to be composed of two identical subunits of equal size (80.5 kDa). The isoelectric point is at pH 4.7. It is a very efficient catalase with a broad pH optimum between 6.5 and 7.5 and a Kmfor H2O2of 4.3 mM, a calculated turnover number of 7200 s−1, and an overall-rate constant of 3.5 × 106M−1s−1. The behaviour of this protoheme-enzyme is typical of the class of prokaryotic catalase-peroxidases, which is sensitive to cyanide (Ki= 27.2 μM) and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole. The enzyme accepts electrons from o-dianisidine, but not from ascorbate, glutathione, and NADH. With hydrogen peroxide in steady-state conditions the enzyme is mainly in the ferric state indicating that Compound I is much faster reduced by H2O2than it is formed. The native enzyme is in the high-spin state, which is transformed to low-spin upon addition of cyanide. With peroxoacetic acid Compound I is formed at a rate of 5.9 × 104M−1s−1at pH 7.0 and 25°C with about 50% hypochromicity, a Soret-maximum at 405 nm and isosbestic points at 354 and 427 nm.