New insights into the heme cavity structure of catalase-peroxidase: a spectroscopic approach to the recombinant synechocystis enzyme and selected distal cavity mutants

Catalase-peroxidases (KatGs) are heme peroxidases with homology to yeast cytochrome cperoxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs exhibit a peroxidase activity of broad specificity and a high catalase activity, which strongly depends on the presence of a distal Trp as part of the conserved amino acid triad Arg-Trp-His. By contrast, both CCP and APX do not have a substantial catalase activity despite the presence of the same triad. Thus, to elucidate structure-function relationships of catalase-peroxidases (for which no crystal structure is available at the moment), we performed UV-Vis and resonance Raman studies of recombinant wild-type KatG from the cyanobacterium SynechocystisPCC 6803 and the distal side variants (His123-->Gln, Glu; Arg119-->Ala, Asn; Trp122-->Phe, Ala). The distal cavity of KatG is very similar to that of the other class I peroxidases. A H-bond network involving water molecules and the distal Trp, Arg, and His is present, which connects the distal and proximal sides of the heme pocket. However, distal mutation not only affects the heme Fe coordination state and perturbs the proximal Fe-Im bond, as previously observed for other peroxidases, but also alters the stability of the heme architecture. The charge of the distal residues appears particularly important for maintaining the heme architecture. Moreover, the Trp plays a significant role in the distal H-bonding, much more pronounced than in CCP. The relevance of these findings for the catalase activity of KatG is discussed in light of the complete loss of catalase activity in the distal Trp mutants.     

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 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.     

Probing the two-domain structure of homodimeric prokaryotic and eukaryotic catalase–peroxidases

Catalase–peroxidases (KatGs) are ancestral bifunctional heme peroxidases found in archaeons, bacteria and lower eukaryotes. In contrast to homologous cytochrome c peroxidase (CcP) and ascorbate peroxidase (APx) homodimeric KatGs have a two-domain monomeric structure with a catalytic N-terminal heme domain and a C-terminal domain of high sequence and structural similarity but without obvious function. Nevertheless, without its C-terminal counterpart the N-terminal domain exhibits neither catalase nor peroxidase activity. Except some hybrid-type proteins all other members of the peroxidase–catalase superfamily lack this C-terminal domain. In order to probe the role of the two-domain monomeric structure for conformational and thermal stability urea and temperature-dependent unfolding experiments were performed by using UV–Vis-, electronic circular dichroism- and fluorescence spectroscopy, as well as differential scanning calorimetry. Recombinant prokaryotic (cyanobacterial KatG from Synechocystis sp. PCC6803) and eukaryotic (fungal KatG from Magnaporthe grisea) were investigated. The obtained data demonstrate that the conformational and thermal stability of bifunctional KatGs is significantly lower compared to homologous monofunctional peroxidases. The N- and C-terminal domains do not unfold independently. Differences between the cyanobacterial and the fungal enzyme are relatively small. Data will be discussed with respect to known structure and function of KatG, CcP and APx.     

Hydrogen peroxide oxidation by catalase-peroxidase follows a non-scrambling mechanism

Despite catalyzing the same reaction (2 H2O2-->2 H2O+O2) heme-containing monofunctional catalases and bifunctional catalase-peroxidases (KatGs) do not share sequence or structural similarities raising the question of whether or not the reaction pathways are similar or different. The production of dioxygen from hydrogen peroxide by monofunctional catalases has been shown to be a two-step process involving the redox intermediate compound I which oxidizes H2O2 directly to O2. In order to investigate the origin of O2 released in KatG mediated H2O2 degradation we performed a gas chromatography-mass spectrometry investigation of the evolved O2 from a 50:50 mixture of H2(18)O2/H2(16)O2 solution containing KatGs from Mycobacterium tuberculosis and Synechocystis PCC 6803. The GC-MS analysis clearly demonstrated the formation of (18)O2 (m/e = 36) and (16)O2 (m/e = 32) but not (16)O(18)O (m/e = 34) in the pH range 5.6-8.5 implying that O2 is formed by two-electron oxidation without breaking the O-O bond. Also active site variants of Synechocystis KatG with very low catalase but normal or even enhanced peroxidase activity (D152S, H123E, W122F, Y249F and R439A) are shown to oxidize H2O2 by a non-scrambling mechanism. The results are discussed with respect to the catalatic mechanism of KatG.     

Engineering the proximal heme cavity of catalase-peroxidase

Catalase-peroxidases (KatGs) are prokaryotic heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs, CCP and APXs contain identical amino acid triads in the heme pocket (distal Arg/Trp/His and proximal His/Trp/Asp), but differ dramatically in their reactivities towards hydrogen peroxide and various one-electron donors. Only KatGs have high catalase activity in addition to a peroxidase activity of broad specificity. Here, we investigated the effect of mutating the conserved proximal triad on KatG catalysis. With the exception of W341F, all variants (H290Q, W341A, D402N, D402E) exhibited a catalase activity <1% of wild-type KatG and spectral properties indicating alterations in heme coordination and spin states. Generally, the peroxidase activity was much less effected by these mutations. Compared with wild-type KatG the W341F variant had a catalase and halogenation activity of about 40% and an even increased overall peroxidase activity. This variant, for the first time, allowed to monitor the hydrogen peroxide mediated transitions of ferric KatG to compound I and back to the resting enzyme. Compound I reduction by aromatic one-electron donors (o-dianisidine, pyrogallol, aniline) was not influenced by exchanging Trp by Phe. The findings are discussed in comparison with the data known from CCP and APX and a reaction mechanism for the multifunctional activity of the W341F variant is suggested.     

Redox thermodynamics of the ferric-ferrous couple of wild-type synechocystis KatG and KatG(Y249F)

Crystal structures and mass spectrometric analyses of catalase-peroxidases (KatGs) from different organisms revealed the existence of a peculiar distal Met-Tyr-Trp cross-link. The adduct appears to be important for the catalase but not the peroxidase activity of bifunctional KatG. To examine the effect of the adduct on enzyme redox properties and functions, we have determined the thermodynamics of ferric reduction for wild-type KatG and KatG(Y249F), whose tyrosine-to-phenylalanine mutation prevents cross-link formation. At 25 degrees C and pH 7.0, the reduction potential of wild-type KatG is found to be -226 +/- 10 mV, remarkably lower than the published literature values. The reduction potential of KatG(Y249F) is very similar (-222 +/- 10 mV), but variable temperature experiments revealed compensatory differences in reduction enthalpies and entropies. In both proteins, the oxidized state is enthalpically stabilized over the reduced state, but entropy is lost on reduction, which is in strong contrast to horseradish peroxidase, which also features a much more pronounced enthalpic stabilization of the ferriheme. With both proteins, the midpoint potential increased linearly with decreasing pH. We discuss whether the observed redox thermodynamics reflects the differences in structure and function between bifunctional KatG and monofunctional peroxidases.     

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.     

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.     

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.     

Distal side tryptophan, tyrosine and methionine in catalase-peroxidases are covalently linked in solution

Distal side tryptophan and tyrosine have been shown to be essential in the catalase but not the peroxidase activity of bifunctional catalase-peroxidases (KatGs). Recently published crystal structures suggest that both residues could be part of a novel adduct including in addition a conserved methionine. A mass spectrometric analysis of the tryptic peptides from recombinant wild-type Synechocystis KatG and the variants Trp122Phe, Tyr249Phe and Met275Ile confirms that this novel adduct really exists in solution and thus may be common to all KatGs. Exchange of either Trp122 or Tyr249 prevents cross-linking, whereas exchange of Met275 still allowed bond formation between Trp122 and Tyr249. It is proposed that the covalent bond between Trp and Tyr may form before that between Tyr and Met. The findings are discussed with respect to the mechanism of cross-linking and its role in KatG catalysis.     

Comparison between catalase-peroxidase and cytochrome c peroxidase. The role of the hydrogen-bond networks for protein stability and catalysis

A detailed resonance Raman and electronic absorption investigation has been carried out on a series of novel distal and proximal variants of recombinant catalase-peroxidase from the cyanobacterium Synechocystis PCC 6803. In particular, variants of the distal triad Pro-Asp-Asn and the proximal triad His-Asp-Trp have been studied in their ferric and ferrous states at various pH. The data suggest marked differences in the structural role of the conserved residues and hydrogen-bond networks in KatG and CCP, which might be connected to the different catalytic activity. In particular, in KatG the proximal residues have a major role in the stability of the protein architecture because the disruption of the proximal Trp-Asp hydrogen bond by mutation weakens heme binding to the protein. On the distal side, replacing the hydrogen-acceptor carboxamide group of Asn153 by an aspartate carboxylate group or an aliphatic residue alters or disrupts the hydrogen bond with the distal His. As a consequence, the basicity of His123 is altered. The effect of mutation on Asp152 is noteworthy. Replacement of the Asp152 with Ser makes the architecture of the protein very similar to that of CCP. The Asp152 residue, which has been shown to be important in the hydrogen peroxide oxidation reaction, is expected to be hydrogen bonded to the nitrogen atom of Ile248 which is part of the KatG-specific insertion LL1, as in other KatGs. This insertion is at one edge of the heme, and connects the distal side with the proximal helices E and F, the latter carrying the proximal His ligand. We found that the distal Asp-Ile hydrogen bond is important for the stability of the heme architecture and its alteration changes markedly the proximal His-Asp hydrogen-bond interaction.     

Intracellular targeting of ascomycetous catalase-peroxidases (KatG1s)

Bifunctional catalase-peroxidases (KatGs) are heme oxidoreductases widely spread among bacteria, archaea and among lower eukaryotes. In fungi, two KatG groups with different localization have evolved, intracellular (KatG1) and extracellular (KatG2) proteins. Here, the cloning, expression analysis and subcellular localization of two novel katG1 genes from the soil fungi Chaetomium globosum and Chaetomium cochliodes are reported. Whereas, the metalloenzyme from Ch. globosum is expressed constitutively, Ch. cochliodes KatG1 reveals a slight increase in expression after induction of oxidative stress by cadmium ions and hydrogen peroxide. The intronless open reading frames of both Sordariomycetes katG1 genes as well as of almost all fungal katG1s possess two peroxisomal targeting signals (PTS1 and PTS2). Peroxisomal localization of intracellular eukaryotic catalase-peroxidases was verified by organelle separation and immunofluorescence microscopy. Co-localization with the peroxisomal enzyme 3-ketoacyl-CoA-thiolase was demonstrated for KatGs from Magnaporthe grisea, Chaetomium globosum and Chaetomium cochliodes. The physiological role of fungal catalase-peroxidases is discussed.     

Eukaryotic extracellular catalase-peroxidase from Magnaporthe grisea - Biophysical/chemical characterization of the first representative from a novel phytopathogenic KatG group

All phytopathogenic fungi have two catalase–peroxidase paralogues located either intracellularly (KatG1) or extracellularly (KatG2). Here, for the first time a secreted bifunctional, homodimeric catalase–peroxidase (KatG2 from the rice blast fungus Magnaporthe grisea) has been produced heterologously with almost 100% heme occupancy and comprehensively investigated by using a broad set of methods including UV–Vis, ECD and resonance Raman spectroscopy (RR), thin-layer spectroelectrochemistry, mass spectrometry, steady-state &; presteady-state spectroscopy. RR spectroscopy reveals that MagKatG2 shows a unique mixed-spin state, non-planar heme b, and a proximal histidine with pronounced imidazolate character. At pH 7.0 and 25 °C, the standard reduction potential E°′ of the Fe(III)/Fe(II) couple for the high-spin native protein was found to fall in the range typical for the KatG family. Binding of cyanide was relatively slow at pH 7.0 and 25 °C and with a K_d value significantly higher than for the intracellular counterpart. Demonstrated by mass spectrometry MagKatG2 has the typical Trp118-Tyr251-Met277 adduct that is essential for its predominantly catalase activity at the unique acidic pH optimum. In addition, MagKatG2 acts as a versatile peroxidase using both one- and two-electron donors. Based on these data, structure–function relationships of extracellular eukaryotic KatGs are discussed with respect to intracellular KatGs and possible role(s) in host–pathogen interaction.     

High Conformational Stability of Secreted Eukaryotic Catalase-peroxidases: ANSWERS FROM FIRST CRYSTAL STRUCTURE AND UNFOLDING STUDIES

Catalase-peroxidases (KatGs) are bifunctional heme enzymes widely spread in archaea, bacteria, and lower eukaryotes. Here we present the first crystal structure (1.55 Å resolution) of an eukaryotic KatG, the extracellular or secreted enzyme from the phytopathogenic fungus Magnaporthe grisea. The heme cavity of the homodimeric enzyme is similar to prokaryotic KatGs including the unique distal ⁺Met-Tyr-Trp adduct (where the Trp is further modified by peroxidation) and its associated mobile arginine. The structure also revealed several conspicuous peculiarities that are fully conserved in all secreted eukaryotic KatGs. Peculiarities include the wrapping at the dimer interface of the N-terminal elongations from the two subunits and cysteine residues that cross-link the two subunits. Differential scanning calorimetry and temperature- and urea-mediated unfolding followed by UV-visible, circular dichroism, and fluorescence spectroscopy combined with site-directed mutagenesis demonstrated that secreted eukaryotic KatGs have a significantly higher conformational stability as well as a different unfolding pattern when compared with intracellular eukaryotic and prokaryotic catalase-peroxidases. We discuss these properties with respect to the structure as well as the postulated roles of this metalloenzyme in host-pathogen interactions.     

Integral role of the I′-helix in the function of the “inactive” C-terminal domain of catalase–peroxidase (KatG)

Catalase–peroxidases (KatGs) have two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. Though the C-terminal domain lacks the ability to bind heme or directly catalyze any reaction, it has been proposed to serve as a platform to direct the folding of the N-terminal domain. Toward such a purpose, its I′-helix is highly conserved and appears at the interface between the two domains. Single and multiple substitution variants targeting highly conserved residues of the I′-helix were generated for intact KatG as well as the stand-alone C-terminal domain (KatG^C). Single variants of intact KatG produced only subtle variations in spectroscopic and catalytic properties of the enzyme. However, the double and quadruple variants showed substantial increases in hexa-coordinate low-spin heme and diminished enzyme activity, similar to that observed for the N-terminal domain on its own (KatG^N). The analogous variants of KatG^C showed a much more profound loss of function as evaluated by their ability to return KatG^N to its active conformation. All of the single variants showed a substantial decrease in the rate and extent of KatG^N reactivation, but with two substitutions, KatG^C completely lost its capacity for the reactivation of KatG^N. These results suggest that the I′-helix is central to direct structural adjustments in the adjacent N-terminal domain and supports the hypothesis that the C-terminal domain serves as a platform to direct N-terminal domain conformation and bifunctionality.     

Disruption of the H-bond network in the main access channel of catalase-peroxidase modulates enthalpy and entropy of Fe(III) reduction

Catalase-peroxidases are the only heme peroxidases with substantial hydrogen peroxide dismutation activity. In order to understand the role of the redox chemistry in their bifunctional activity, catalatically-active and inactive mutant proteins have been probed in spectroelectrochemical experiments. In detail, wild-type KatG from Synechocystis has been compared with variants with (i) disrupted KatG-typical adduct (Trp122-Tyr249-Met275), (ii) mutation of the catalytic distal His123-Arg119 pair, and (iii) altered accessibility to the heme cavity (Asp152, Ser335) and modified charge at the substrate channel entrance (Glu253). A valuable insight into the mechanism of reduction potential (E°′) modulation in KatG has been obtained from the parameterization of the corresponding enthalpic and entropic components, determined from the analysis of the temperature dependence of E°′. Moreover, model structures of ferric and ferrous Synechocystis KatG have been computed and used as reference to analyze and discuss the experimental data. The results, discussed by reference to published resonance Raman data on the strength of the proximal iron-imidazole bond and catalytic properties, demonstrate that E°′ of the Fe(III)/Fe(II) couple is not strongly correlated with the bifunctional activity. Besides the importance of an intact Trp-Tyr-Met adduct, it is the architecture of the long and constricted main channel that distinguishes KatGs from monofunctional peroxidases. An ordered matrix of oriented water dipoles is important for H₂O₂ oxidation. Its disruption results in modification of enthalpic and entropic contributions to E°′ that reflect reduction-induced changes in polarity, electrostatics, continuity and accessibility of solvent to the metal center as well as alterations in solvent reorganization.     

Catalase-peroxidase from synechocystis is capable of chlorination and bromination reactions

Catalase-peroxidases (KatGs) are multifunctional heme peroxidases exhibiting an overwhelming catalase activity and a substantial peroxidase activity of broad specificity. Here, we show that catalase-peroxidases are also haloperoxidases capable of oxidizing chloride, bromide, and iodide in a peroxide- and enzyme-dependent manner. Recombinant KatG and the variants R119A, W122F, and W122A from the cyanobacterium Synechocystis PCC 6803 have been tested for their halogenation activity. Halogenation of monochlorodimedon (MCD), formation of triiodide and tribromide, and bromide- and chloride-mediated oxidation of glutathione have been tested. Halogenation of MCD by chloride, bromide, and iodide was shown to be catalyzed by wild-type KatG and the variant R119A. Generally, rates of halogenation increased in the order Cl(-) < Br(-) < I(-) and/or by decreasing pH. The halogenation activity of R119A was about 7-9% that of the wild-type enzyme. Upon exchange of the distal Trp122 by Phe and Ala, both the catalase and halogenation activities were lost but the overall peroxidase activity was increased. The findings suggest that the same redox intermediate is involved in H(2)O(2) and halide oxidation and that distal Trp122 is involved in both two-electron reactions. That halides compete with H(2)O(2) for the same redox intermediate is also emphasized by the fact that the polarographically measured catalase activity is influenced by halides, with bromide being more effective than chloride.     

Reduced affinity for Isoniazid in the S315T mutant of Mycobacterium tuberculosis KatG is a key factor in antibiotic resistance

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis is responsible for the activation of the antitubercular drug isonicotinic acid hydrazide (INH) and is important for survival of M. tuberculosis in macrophages. Characterization of the structure and catalytic mechanism of KatG is being pursued to provide insights into drug (INH) resistance in M. tuberculosis. Site-directed mutagenesis was used to prepare the INH-resistant mutant KatG[S315T], and the overexpressed enzyme was characterized and compared with wild-type KatG. KatG[S315T] exhibits a reduced tendency to form six-coordinate heme, because of coordination of water to iron during purification and storage, and also forms a highly unstable Compound III (oxyferrous enzyme). Catalase activity and peroxidase activity measured using t-butylhydroperoxide and o-dianisidine were moderately reduced in the mutant compared with wild-type KatG. Stopped-flow spectrophotometric experiments revealed a rate of Compound I formation similar to wild-type KatG using peroxyacetic acid to initiate the catalytic cycle, but no Compound I was detected when bulkier peroxides (chloroperoxybenzoic acid, t-butylhydroperoxide) were used. The affinity of resting (ferric) KatG[S315T] for INH, measured using isothermal titration calorimetry, was greatly reduced compared with wild-type KatG, as were rates of reaction of Compound I with the drug. These observations reveal that although KatG[S315T] maintains reasonably good steady state catalytic rates, poor binding of the drug to the enzyme limits drug activation and brings about INH resistance.     

Modification of the active site of Mycobacterium tuberculosis KatG after disruption of the Met-Tyr-Trp cross-linked adduct

Mycobacterium tuberculosis catalase-peroxidase (Mtb KatG) is a bifunctional enzyme that possesses both catalase and peroxidase activities and is responsible for the activation of the antituberculosis drug isoniazid. Mtb KatG contains an unusual adduct in its distal heme-pocket that consists of the covalently linked Trp107, Tyr229, and Met255. The KatG(Y229F) mutant lacks this adduct and has decreased steady-state catalase activity and enhanced peroxidase activity. In order to test a potential structural role of the adduct that supports catalase activity, we have used resonance Raman spectroscopy to probe the local heme environment of KatG(Y229F). In comparison to wild-type KatG, resting KatG(Y229F) contains a significant amount of 6-coordinate, low-spin heme and a more planar heme. Resonance Raman spectroscopy of the ferrous-CO complex of KatG(Y229F) suggest a non-linear Fe-CO binding geometry that is less tilted than in wild-type KatG. These data provide evidence that the Met-Tyr-Trp adduct imparts structural stability to the active site of KatG that seems to be important for sustaining catalase activity.