Nucleotide sequence analysis, overexpression in Escherichia coli and kinetic characterization of Anacystis nidulans catalase-peroxidase

Bifunctional catalase-peroxidases are the least understood type of peroxidases. A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301) is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the examination of both the spectral characteristics and the reactivity of its redox intermediates by using the multi-mixing stopped-flow technique. The homodimeric acidic protein (pI = 4.6) contained high catalase activity (apparent K(m) = 4.8 mM and apparent k(cat) = 8850 s(-1)). Cyanide is shown to be an effective inhibitor of the catalase reaction. The second-order rate constant for cyanide binding to the ferric protein is (6.9 +/- 0.2) x 10(5) M(-1 )s(-1) at pH 7.0 and 15 degrees C and the dissociation constant of the cyanide complex is 17 microM. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. The apparent second-order rate constant for formation of compound I from the ferric enzyme and peroxoacetic acid is (1.3 +/- 0.3) x 10(4 )M(-1 )s(-1) at pH 7.0 and 15 degrees C. The spectrum of compound I is characterized by about 40% hypochromicity, a Soret region at 406 nm, and isosbestic points between the native enzyme and compound I at 355 and 428 nm. Rate constants for reduction of KatG compound I by o-dianisidine, pyrogallol, aniline and isoniazid are shown to be (7.3 +/- 0.4) x 10(6) M(-1 )s(-1), (5.4 +/- 0.3) x 10(5) M(-1 )s(-1), (1.6 +/- 0.3) x 10(5) M(-1 )s(-1) and (4.3 +/- 0.2) x 10(4) M(-1 )s(-1), respectively. The redox intermediate formed upon reduction of compound I did not exhibit the classical red-shifted peroxidase compound II spectrum which characterizes the presence of a ferryl oxygen species. Its spectral features indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.     

Catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803: cloning, overexpression in Escherichia coli, and kinetic characterization.

The Synechocystis PCC 6803 katG gene encodes a dual-functional catalase-peroxidase (EC 1.11.1.7). We have established a system for the high level expression of a fully active recombinant form of this enzyme. Its entire coding DNA was extended using a synthetic oligonucleotide encoding a hexa-histidine tag at the C-terminus and expressed in Escherichia coli [BL21-(DE3)pLysS] using the pET-3a vector. Hemin was added to the culture medium to ensure its proper association with KatG upon induction. The expressed protein was purified to homogeneity by two chromatography steps including a metal chelate affinity and hydrophobic interaction chromatography. The homodimeric acidic protein (pl = 5.4) had a molecular mass of 170 kDa and a Reinheitszahl (A406/A280) of 0.64. The recombinant protein contained high catalase activity (apparent Km = 4.9 +/- 0.25 mM and apparent kcat = 3500 s(-1)) and an appreciable peroxidase activity with o-dianisidine, guaiacol and pyrogallol, but not with NAD(P)H, ferrocytochrome c, ascorbate or glutathione as electron donors. By using both conventional and sequential stopped-flow spectroscopy, formation of compound I with peroxoacetic acid was calculated to be (8.74 +/- 0.26) x 10(3) M(-1) s(-1), whereas compound I reduction by o-dianisidine, pyrogallol and ascorbate was determined to be (2.71 +/- 0.03) x 10(6) M(-1) S(-1), (8.62 +/- 0.21) x 10(4) M(-1) S(-1), and (5.43 +/- 0.19) x 10(3) M(-1) S(-1), respectively. Cyanide binding studies on native and recombinant enzyme indicated that both have the same heme environment. An apparent second-order rate constant for cyanide binding of (4.8 +/- 0.1) x 10(5) M(-1) S(-1) was obtained.     

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.     

Total conversion of bifunctional catalase-peroxidase (KatG) to monofunctional peroxidase by exchange of a conserved distal side tyrosine

Catalase-peroxidases (KatGs) are unique peroxidases exhibiting a high catalase activity and a peroxidase activity with a wide range of artificial electron donors. Exchange of tyrosine 249 in Synechocystis KatG, a distal side residue found in all as yet sequenced KatGs, had dramatic consequences on the bifunctional activity and the spectral features of the redox intermediate compound II. The Y249F variant lost catalase activity but retained a peroxidase activity (substrates o-dianisidine, pyrogallol, guaiacol, tyrosine, and ascorbate) similar to the wild-type protein. In contrast to wild-type KatG and similar to monofunctional peroxidases, the formation of the redox intermediate compound I could be followed spectroscopically even by addition of equimolar hydrogen peroxide to ferric Y249F. The corresponding bimolecular rate constant was determined to be (1.1 +/- 0.1) x 107 m-1 s-1 (pH 7 and 15 degrees C), which is typical for most peroxidases. Additionally, for the first time a clear transition of compound I to an oxoferryl-like compound II with peaks at 418, 530, and 558 nm was monitored when one-electron donors were added to compound I. Rate constants of reaction of compound I and compound II with tyrosine ((5.0 +/- 0.3) x 104 m-1 s-1 and (1.7 +/- 0.4) x 102 m-1 s-1) and ascorbate ((1.3 +/- 0.2) x 104 m-1 s-1 and (8.8 +/- 0.1) x 101 m-1 s-1 at pH 7 and 15 degrees C) were determined by using the sequential stopped-flow technique. The relevance of these findings is discussed with respect to the bifunctional activity of KatGs and the recently published first crystal structure.     

Role of the Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG) as revealed by KatG(M255I)

Catalase-peroxidases (KatGs) are bifunctional enzymes possessing both catalase and peroxidase activities. Four crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link which has been suggested to impart catalatic activity to the KatGs. To decipher the individual roles of the two cross-links in the Met-Tyr-Trp adduct, we have focused on recombinant Mycobacterium tuberculosis KatG(M255I). UV-visible spectroscopic and mass spectrometric studies of the peptide fragments resulting from tryptic digestion of KatG(M255I) confirmed the presence of the single Tyr-Trp cross-link, as well as a 2e- oxidized form which is postulated to be an intermediate generated during Met-Tyr-Trp cross-link formation. KatG(M255I) lacking the Tyr-Trp cross-link was also prepared, and incubation with peroxyacetic acid, but not 2-methyl-1-phenyl-2-propyl hydroperoxide, resulted in complete formation of the Tyr-Trp cross-link. A mechanism for Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies with KatG(M255I) were performed, allowing characterization of compounds I, II, and III. Interestingly, two compound II intermediates were identified: (KatG*)(Por)Fe(III)-OH, where KatG* represents a protein-based radical, and oxoferryl (KatG)(Por)Fe(IV)=O. Insight into the contributions of the individual Tyr-Trp and Met-Tyr cross-links to catalase activity is presented, as is the overall contribution of the Met-Tyr-Trp cross-link to the structure-function-spectroscopy relationship and catalase-peroxidase mechanism in KatG.     

The Met-Tyr-Trp cross-link in Mycobacterium tuberculosis catalase-peroxidase (KatG): autocatalytic formation and effect on enzyme catalysis and spectroscopic properties

Catalase-peroxidases (KatG) are bifunctional enzymes possessing both catalase and peroxidase activities. Three crystal structures of different KatGs revealed the presence of a novel Met-Tyr-Trp cross-link that has been suggested to impart catalatic activity to the KatGs. High-performance liquid chromatographic separation of the peptide fragments resulting from tryptic digestion of recombinant Mycobacterium tuberculosis WT KatG identified a peptide with unusual UV-visible spectroscopic features attributable to the Met(255)-Tyr(229)-Trp(107) cross-link, whose structure was confirmed by mass spectrometry. WT KatG lacking the Met-Tyr-Trp cross-link was prepared, making possible studies of its formation under oxidizing conditions that generate either compound I (peroxyacetic acid, PAA) or compound II (2-methyl-1-phenyl-2-propyl hydroperoxide, MPPH). Incubation of this "cross-link-free" WT KatG with PAA revealed complete formation of the Met-Tyr-Trp structure after six equivalents of peracid were added, whereas MPPH was unable to promote cross-link formation. A mechanism for Met-Tyr-Trp autocatalytic formation by KatG compound I is proposed from these studies. Optical stopped-flow studies of WT KatG and KatG(Y229F), a mutant in which the cross-link cannot be formed, were performed with MPPH and revealed an unusual compound II spectrum for WT KatG, best described as (P.)Fe(III), where P. represents a protein-based radical. This contrasts with the oxoferryl compound II spectrum observed for KatG(Y229F) under identical conditions. The structure-function-spectroscopy relationship in KatG is discussed with relevance to the role that the Met-Tyr-Trp cross-link plays in the catalase-peroxidase mechanism.     

Characterization of the W321F mutant of Mycobacterium tuberculosis catalase–peroxidase KatG

A single amino acid mutation (W321F) in Mycobacterium tuberculosis catalase-peroxidase (KatG) was constructed by site-directed mutagenesis. The purified mutant enzyme was characterized using optical and electron paramagnetic resonance spectroscopy, and optical stopped-flow spectrophotometry. Reaction of KatG(W321F) with 3-chloroperoxybenzoic acid, peroxyacetic acid, or t-butylhydroperoxide showed formation of an unstable intermediate assigned as Compound I (oxyferryl iron:porphyrin pi-cation radical) by similarity to wild-type KatG, although second-order rate constants were significantly lower in the mutant for each peroxide tested. No evidence for Compound II was detected during the spontaneous or substrate-accelerated decay of Compound I. The binding of isoniazid, a first-line anti-tuberculosis pro-drug activated by catalase-peroxidase, was noncooperative and threefold weaker in KatG(W321F) compared with wild-type enzyme. An EPR signal assigned to a protein-based radical tentatively assigned as tyrosyl radical in wild-type KatG, was also observed in the mutant upon reaction of the resting enzyme with alkyl peroxide. These results show that mutation of residue W321 in KatG does not lead to a major alteration in the identity of intermediates formed in the catalytic cycle of the enzyme in the time regimes examined here, and show that this residue is not the site of stabilization of a radical as might be expected based on homology to yeast cytochrome c peroxidase. Furthermore, W321 is indicated to be important in KatG for substrate binding and subunit interactions within the dimer, providing insights into the origin of isoniazid resistance in clinically isolated KatG mutants.     

Kinetics of interconversion of ferrous enzymes, compound II and compound III, of wild-type synechocystis catalase-peroxidase and Y249F: proposal for the catalatic mechanism

With the exception of catalase-peroxidases, heme peroxidases show no significant ability to oxidize hydrogen peroxide and are trapped and inactivated in the compound III form by H2O2 in the absence of one-electron donors. Interestingly, some KatG variants, which lost the catalatic activity, form compound III easily. Here, we compared the kinetics of interconversion of ferrous enzymes, compound II and compound III of wild-type Synechocystis KatG, the variant Y249F, and horseradish peroxidase (HRP). It is shown that dioxygen binding to ferrous KatG and Y249F is reversible and monophasic with apparent bimolecular rate constants of (1.2 +/- 0.3) x 10(5) M(-1) s(-1) and (1.6 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C), similar to HRP. The dissociation constants (KD) of the ferrous-dioxygen were calculated to be 84 microm (wild-type KatG) and 129 microm (Y249F), higher than that in HRP (1.9 microm). Ferrous Y249F and HRP can also heterolytically cleave hydrogen peroxide, forming water and an oxoferryl-type compound II at similar rates ((2.4 +/- 0.3) x 10(5) M(-1) s(-1) and (1.1 +/- 0.2) x 10(5) M(-1) s(-1) (pH 7, 25 degrees C)). Significant differences were observed in the H2O2-mediated conversion of compound II to compound III as well as in the spectral features of compound II. When compared with HRP and other heme peroxidases, in Y249F, this reaction is significantly faster ((1.2 +/- 0.2) x 10(4) M(-1) s(-1))). Ferrous wild-type KatG was also rapidly converted by hydrogen peroxide in a two-phasic reaction via compound II to compound III (approximately 2.0 x 10(5) M(-1) s(-1)), the latter being also efficiently transformed to ferric KatG. These findings are discussed with respect to a proposed mechanism for the catalatic activity.     

Distal site aspartate is essential in the catalase activity of catalase-peroxidases

Structural and biochemical characterization of aspartate 152 at the distal heme side of catalase-peroxidase (KatG) from Synechocystis PCC 6803 reveals an important functional role for this residue. In the wild-type protein, the side chain carboxyl group of Asp152 is 7.8 A apart from the heme iron and is hydrogen-bonded to two water molecules and a KatG-specific large loop. We have prepared the site-specific variants Asp152Asn, Asp152Ser, Asp152Trp, and Pro151Ala. Exchange of Asp152 exhibited dramatic consequences on the bifunctional activity of this unique peroxidase. The turnover number of catalase activity of Asp152Asn is 2.7%, Asp152Ser 5.7%, and Asp152Trp is 0.6% of wild-type activity. By contrast, the peroxidase activity of the Asp152 variants was 2-7 times higher than that of wild-type KatG or Pro151Ala. The KatG-specific pH profile of the catalase activity was completely different in these variants and exchange of Asp152 made it possible to follow the transition of the ferric enzyme to the redox intermediate compound I by hydrogen peroxide spectroscopically and to determine the corresponding bimolecular rate constant to be 7.5 x 10(6) M(-1) s(-1) (pH 7 and 15 degrees C). The reactivity of compound I toward aromatic one-electron donors was enhanced in the Asp152 variants compared with the wild-type protein, whereas the reactivity toward hydrogen peroxide was dramatically decreased. A mechanism for the hydrogen peroxide oxidation, which is different from monofunctional catalases and involves the distal residues Trp122 and Asp152, is proposed.     

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.     

Influence of the unusual covalent adduct on the kinetics and formation of radical intermediates in synechocystis catalase peroxidase: a stopped-flow and EPR characterization of the MET275, TYR249, and ARG439 variants

Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp(122), Tyr(249), and Met(275) (Synechocysis KatG numbering). The known crystal structures suggest that Tyr(249) and Met(275) could be within hydrogen-bonding distance to Arg(439). To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg(439) variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr(249) and Trp(122). In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferryl-porphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por(.-)(+) --> Trp(.-) (or Trp(.-)(+)) --> Tyr(.-). The M275I variant did not form the Trp(.-) species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp(122)-Tyr(249) adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.     

Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism

Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme.     

Differential substrate behaviour of phenol and aniline derivatives during oxidation by horseradish peroxidase: kinetic evidence for a two-step mechanism

The catalytic constant (k(cat)) and the second-order association constant of compound II with reducing substrate (k(5)) of horseradish peroxidase C (HRPC) acting on phenols and anilines have been determined from studies of the steady-state reaction velocities (V(0) vs. [S(0)]). Since k(cat)=k(2)k(6)/k(2)+k(6), and k(2) (the first-order rate constant for heterolytic cleavage of the oxygen-oxygen bond of hydrogen peroxide during compound I formation) is known, it has been possible to calculate the first-order rate constant for the transformation of each phenol or aniline by HRPC compound II (k(6)). The values of k(6) are quantitatively correlated to the sigma values (Hammett equation) and can be rationalized by an aromatic substrate oxidation mechanism in which the substrate donates an electron to the oxyferryl group in HRPC compound II, accompanied by two proton additions to the ferryl oxygen atom, one from the substrate and the other the protein or solvent. k(6) is also quantitatively correlated to the experimentally determined (13)C-NMR chemical shifts (delta(1)) and the calculated ionization potentials, E (HOMO), of the substrates. Similar dependencies were observed for k(cat) and k(5). From the kinetic analysis, the absolute values of the Michaelis constants for hydrogen peroxide and the reducing substrates (K(M)(H(2)O(2)) and K(M)(S)), respectively, were obtained.     

Characterization of the binding of isoniazid and analogues to Mycobacterium tuberculosis catalase-peroxidase

The first-line antituberculosis drug isonicotinic hydrazide (INH) is a prodrug whose bactericidal function requires activation by Mycobacterium tuberculosis catalase-peroxidase (KatG) to produce an acyl-NAD adduct. Peroxidation of INH is considered a required catalytic process for drug action. The binding of INH and a series of hydrazide analogues to resting KatG was examined using optical and calorimetric techniques to provide thermodynamic parameters, binding stoichiometries, and kinetic constants (on and off rates). This work revealed high-affinity binding of these substrates to a small fraction of ferric enzyme in a six-coordinate heme iron form, a species most likely containing a weakly bound water molecule, which accumulates during storage of the enzyme. The binding of hydrazides is associated with a large enthalpy loss (>100 kcal/mol); dissociation constants are in the range of 0.05-1.6 microM, and optical stopped-flow measurements demonstrated kon values in the range of 0.5-27 x 10(3) M-1 s-1 with very small koff rates. Binding parameters did not depend on pH in the range 5-8. High-affinity binding of INH is disrupted in two mutant enzymes bearing replacements of key distal side residues, KatG[W107F] and KatG[Y229F]. The rates of reduction of KatG Compound I by hydrazides parallel the on rates for association with the resting enzyme. In a KatG-mediated biomimetic activation assay, only isoniazid generated in good yield the acyl-NAD adduct which is considered a key molecule in INH action, providing a better understanding of the action mechanism of INH.     

Reaction of lactoperoxidase compound I with halides and thiocyanate

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions, including milk, tears, and saliva, and has physiological significance in antimicrobial defense which involves (pseudo-) halide oxidation. This study for the first time presents transient kinetic measurements of the reactivity of its competent redox intermediate compound I with halides and thiocyanate, using the sequential stopped-flow technique. Compound I was produced with either H(2)O(2) [(1.1 +/- 0.1) x 10(7) M(-1) s(-1)] or hypochlorous acid [(3.2 +/- 0.1) x 10(7) M(-1) (s-1)]. At pH 7 and 15 degrees C, the two-electron reduction of compound I to native LPO by bromide and iodide has a second-order rate constant of (4.1 +/- 0.1) x 10(4) M(-1) s(-1) and (1.2 +/- 0.04) x 10(8) M(-1) s(-1), respectively. With thiocyanate the reaction is extremely fast (2.0 x 10(8) M(-1) s(-1)), whereas chloride cannot function as electron donor. The results are discussed with respect to known kinetic data of homologous mammalian peroxidases and to the physiological role of LPO in antimicrobial defense.     

The catalytic role of the distal site asparagine-histidine couple in catalase-peroxidases.

Catalase-peroxidases (KatGs) are unique in exhibiting an overwhelming catalase activity and a peroxidase activity of broad specificity. Similar to other peroxidases the distal histidine in KatGs forms a hydrogen bond with an adjacent conserved asparagine. To investigate the catalytic role(s) of this potential hydrogen bond in the bifunctional activity of KatGs, Asn153 in Synechocystis KatG was replaced with either Ala (Asn153-->Ala) or Asp (Asn153-->Asp). Both variants exhibit an overall peroxidase activity similar with wild-type KatG. Cyanide binding is monophasic, however, the second-order binding rates are reduced to 5.4% (Asn153-->Ala) and 9.5% (Asn153-->Asp) of the value of native KatG [(4.8 +/- 0.4) x 105 m-1.s-1 at pH 7 and 15 degrees C]. The turnover number of catalase activity of Asn153-->Ala is 6% and that of Asn153-->Asp is 16.5% of wild-type activity. Stopped-flow analysis of the reaction of the ferric forms with H2O2 suggest that exchange of Asn did not shift significantly the ratio of rates of H2O2-mediated compound I formation and reduction. Both rates seem to be reduced most probably because (a) the lower basicity of His123 hampers its function as acid-base catalyst and (b) Asn153 is part of an extended KatG-typical H-bond network, the integrity of which seems to be essential to provide optimal conditions for binding and oxidation of the second H2O2 molecule necessary in the catalase reaction.     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Redox intermediates in the catalase cycle of catalase-peroxidases from Synechocystis PCC 6803, Burkholderia pseudomallei, and Mycobacterium tuberculosis

Monofunctional catalases (EC 1.11.1.6) and catalase-peroxidases (KatGs, EC 1.11.1.7) have neither sequence nor structural homology, but both catalyze the dismutation of hydrogen peroxide (2H2O2 --> 2H2O + O2). In monofunctional catalases, the catalatic mechanism is well-characterized with conventional compound I [oxoiron(IV) porphyrin pi-cation radical intermediate] being responsible for hydrogen peroxide oxidation. The reaction pathway in KatGs is not as clearly defined, and a comprehensive rapid kinetic and spectral analysis of the reactions of KatGs from three different sources (Synechocystis PCC 6803, Burkholderia pseudomallei, and Mycobacterium tuberculosis) with peroxoacetic acid and hydrogen peroxide has focused on the pathway. Independent of KatG, but dependent on pH, two low-spin forms dominated in the catalase cycle with absorbance maxima at 415, 545, and 580 nm at low pH and 418 and 520 nm at high pH. By contrast, oxidation of KatGs with peroxoacetic acid resulted in intermediates with different spectral features that also differed among the three KatGs. Following the rate of H2O2 degradation by stopped-flow allowed the linking of reaction intermediate species with substrate availability to confirm which species were actually present during the catalase cycle. Possible reaction intermediates involved in H2O2 dismutation by KatG are discussed.     

Kinetic and molecular orbital studies on the rate of oxidation of monosubstituted phenols and anilines by horseradish peroxidase compound II

The second-order rate constant (k4) for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by horseradish peroxidase (HRP) compound II was examined with a stopped-flow apparatus. The electronic states of these substrates were calculated by an ab initio molecular orbital method. It was found that in both phenols and anilines log k4 values correlate well with the highest occupied molecular orbital (HOMO) energy level and the lowest unoccupied molecular orbital (LUMO) energy level, but not with the net charge or frontier electron density on atoms of these molecules. The HOMO and LUMO energy levels of phenols and anilines further showed linear relationships with Hammett's sigma values with negative slopes. Similar results were obtained in the oxidation of substrates by HRP compound I, except that the rate of reaction was much higher than in the case of HRP compound II. In addition, the rates of oxidation of phenols by compound I or II were found to be about 1000 times higher than those of anilines with similar HOMO energy levels. On the basis of these results, the mechanism of electron transfer from the substrate to the heme iron of HRP compound II is discussed.     

Kinetics of oxidation of aliphatic and aromatic thiols by myeloperoxidase compounds I and II

Myeloperoxidase (MPO) is the most abundant protein in neutrophils and plays a central role in microbial killing and inflammatory tissue damage. Because most of the non-steroidal anti-inflammatory drugs and other drugs contain a thiol group, it is necessary to understand how these substrates are oxidized by MPO. We have performed transient kinetic measurements to study the oxidation of 14 aliphatic and aromatic mono- and dithiols by the MPO intermediates, Compound I (k3) and Compound II (k4), using sequential mixing stopped-flow techniques. The one-electron reduction of Compound I by aromatic thiols (e.g. methimidazole, 2-mercaptopurine and 6-mercaptopurine) varied by less than a factor of seven (between 1.39 +/- 0.12 x 10(5) M(-1) s(-1) and 9.16 +/- 1.63 x 10(5) M(-1) s(-1)), whereas reduction by aliphatic thiols was demonstrated to depend on their overall net charge and hydrophobic character and not on the percentage of thiol deprotonation or redox potential. Cysteamine, cysteine methyl ester, cysteine ethyl ester and alpha-lipoic acid showed k3 values comparable to aromatic thiols, whereas a free carboxy group (e.g. cysteine, N-acetylcysteine, glutathione) diminished k3 dramatically. The one-electron reduction of Compound II was far more constrained by the nature of the substrate. Reduction by methimidazole, 2-mercaptopurine and 6-mercaptopurine showed second-order rate constants (k4) of 1.33 +/- 0.08 x 10(5) M(-1) s(-1), 5.25 +/- 0.07 x 10(5) M(-1) s(-1) and 3.03 +/- 0.07 x 10(3) M(-1) s(-1). Even at high concentrations cysteine, penicillamine and glutathione could not reduce Compound II, whereas cysteamine (4.27 +/- 0.05 x 10(3) M(-1) s(-1)), cysteine methyl ester (8.14 +/- 0.08 x 10(3) M(-1) s(-1)), cysteine ethyl ester (3.76 +/- 0.17 x 10(3) M(-1) s(-1)) and alpha-lipoic acid (4.78 +/- 0.07 x 10(4) M(-1) s(-1)) were demonstrated to reduce Compound II and thus could be expected to be oxidized by MPO without co-substrates.