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Douglas C. Goodwin - One of the best experts on this subject based on the ideXlab platform.
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The kinetic properties producing the perfunctory pH profiles of Catalase-Peroxidases.
Biochimica et biophysica acta, 2008Co-Authors: Robert L. Moore, Luke J. Powell, Douglas C. GoodwinAbstract:Many structure-function relationship studies performed on the Catalase-Peroxidase enzymes are based on limited kinetic data. To provide a more substantive understanding of Catalase-Peroxidase function, we undertook a more exhaustive evaluation of Catalase-Peroxidase catalysis as a function of pH. Kinetic parameters across a broad pH range for the catalase and peroxidase activities of E. coli catalase peroxidase (KatG) were obtained, including the separate analysis of the oxidizing and reducing substrates of the peroxidase catalytic cycle. This investigation identified ABTS-dependent inhibition of peroxidase activity, particularly at low pH, unveiling that previously reported pH optima are clearly skewed. We show that turnover and efficiency of peroxidase activity increases with decreasing pH until the protein unfolds. The data also suggest that the catalase pH optimum is more complex than it is often assumed to be. The apparent optimum is in fact the intersection of the optimum for binding (7.00) and the optimum for activity (5.75). We also report the apparent pK(a)s for binding and catalysis of catalase activity as well as approximate values for certain peroxidatic and catalatic steps.
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Catalase−Peroxidase Active Site Restructuring by a Distant and “Inactive” Domain†
Biochemistry, 2006Co-Authors: Ruletha D. Baker, Carma O. Cook, Douglas C. GoodwinAbstract:Catalase−peroxidases are composed of two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. The C-terminal domain does not bind heme, has no catal...
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Properties of catalase–peroxidase lacking its C-terminal domain
Biochemical and biophysical research communications, 2004Co-Authors: Ruletha D. Baker, Carma O. Cook, Douglas C. GoodwinAbstract:Catalase-Peroxidases have a two-domain structure. The N-terminal domain contains the bifunctional active site, but the function of the C-terminal domain is unknown. We produced Catalase-Peroxidase containing only its N-terminal domain (KatG(Nterm)). Removal of the C-terminal domain did not result in unexpected changes in secondary structure as evaluated by CD, but KatG(Nterm) had neither catalase nor peroxidase activity. Partial recovery of both activities was achieved by incubating KatG(Nterm) with the separately expressed and isolated KatG C-terminal domain. Spectroscopic measurements revealed a shift in heme environment from a mixture of high-spin species (wtKatG) to exclusively hexacoordinate, low-spin (KatG(Nterm)). Moreover, a > 1000-fold lower kon for CN- binding was observed for KatG(Nterm). EPR spectra for KatG(Nterm) and the results of site-specific substitution of active site histidines suggested that the distal histidine was the sixth ligand. Thus, one important role for the C-terminal domain may be to support the architecture of the active site, preventing heme ligation by this catalytically essential residue.
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Vital roles of an interhelical insertion in Catalase-Peroxidase bifunctionality.
Biochemical and biophysical research communications, 2004Co-Authors: Douglas C. GoodwinAbstract:Abstract The loop connecting the F and G helices of catalase–peroxidases contains a ∼35 amino acid structure (the FG insertion) that is absent from monofunctional peroxidases. These two groups of enzymes share highly similar active sites, yet the monofunctional peroxidases lack appreciable catalase activity. Thus, the FG insertion may serve a role in catalase–peroxidase bifunctionality, despite its peripheral location relative to the active site. We produced a variant of Escherichia coli catalase–peroxidase (KatG) lacking its FG insertion (KatG ΔFG ). Absorption spectra indicated the heme environment of KatG ΔFG was highly similar to wild-type KatG, but the variant retained only 0.2% catalase activity. In contrast, the deletion reduced peroxidase activity by only 50%. Kinetic parameters for the peroxidase and residual catalase activities of KatG ΔFG as well as pH dependence studies suggested that the FG insertion supports hydrogen-bonded networks critical for reactions involving H 2 O 2 . The structure also appears to regulate access of electron donors to the active site.
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System for the expression of recombinant hemoproteins in Escherichia coli.
Protein expression and purification, 2004Co-Authors: Cornelius L. Varnado, Douglas C. GoodwinAbstract:Expression of recombinant hemoproteins in Escherichia coli is often limited because a vast majority of the protein produced lacks the heme necessary for function. This is compounded by the fact that standard laboratory strains of E. coli have a limited capacity to withdraw heme from the extracellular environment. We are developing a new tool designed to increase the heme content of our proteins of interest by simply supplementing the expression medium with low concentrations of hemin. This hemoprotein expression (HPEX) system is based on plasmids (pHPEX1-pHPEX3) that encode an outermembrane-bound heme receptor (ChuA) from E. coli O157:H7. This heme receptor, and others like it, confers on the host the ability to more effectively internalize exogenous heme. Transformation of a standard laboratory E. coli protein expression strain (BL-21 [DE3]) with the pHPEX plasmid led to the expression of a new protein with the appropriate molecular weight for ChuA. The receptor was functional as demonstrated by the ability of the transformant to grow on iron-deficient media supplemented with hemin, an ability that the unmodified expression strain lacked. Expression of our proteins of interest, Catalase-Peroxidases, using this system led to a dramatic and parallel increase in heme content and activity. On a per-heme basis, the spectral and kinetic properties of HPEX-derived Catalase-Peroxidase were the same as those observed for Catalase-Peroxidases expressed in standard E. coli-based systems. We suggest that the pHPEX plasmids may be a useful addition to other E. coli expression systems and may help address a broad range of problems in hemoprotein structure and function.
Christian Obinger - One of the best experts on this subject based on the ideXlab platform.
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Turning points in the evolution of peroxidase–catalase superfamily: molecular phylogeny of hybrid heme peroxidases
Cellular and Molecular Life Sciences, 2014Co-Authors: Marcel Zámocký, Bernhard Gasselhuber, Paul G. Furtmüller, Christian ObingerAbstract:Heme peroxidases and catalases are key enzymes of hydrogen peroxide metabolism and signaling. Here, the reconstruction of the molecular evolution of the peroxidase–catalase superfamily (annotated in pfam as PF00141) based on experimentally verified as well as numerous newly available genomic sequences is presented. The robust phylogenetic tree of this large enzyme superfamily was obtained from 490 full-length protein sequences. Besides already well-known families of heme b peroxidases arranged in three main structural classes, completely new (hybrid type) peroxidase families are described being located at the border of these classes as well as forming (so far missing) links between them. Hybrid-type A peroxidases represent a minor eukaryotic subfamily from Excavates, Stramenopiles and Rhizaria sharing enzymatic and structural features of ascorbate and cytochrome c peroxidases. Hybrid-type B peroxidases are shown to be spread exclusively among various fungi and evolved in parallel with peroxidases in land plants. In some ascomycetous hybrid-type B peroxidases, the peroxidase domain is fused to a carbohydrate binding (WSC) domain. Both here described hybrid-type peroxidase families represent important turning points in the complex evolution of the whole peroxidase–catalase superfamily. We present and discuss their phylogeny, sequence signatures and putative biological function.
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The reaction of synechocystis Catalase-Peroxidase (KatG) with Isoniazid Investigated by multifrequency (9-285 GHz) EPR Spectroscopy
Applied Magnetic Resonance, 2009Co-Authors: Julie Colin, Christa Jakopitsch, Christian Obinger, Anabella IvancichAbstract:Three distinct electron paramagnetic resonance (EPR) spectra of radical intermediates formed as reactive intermediates in the catalytic cycle of Synechocystis catalase–peroxidase were identified. Multifrequency EPR spectroscopy, combined with site-directed mutagenesis and selective deuterium labeling of Trp and Tyr residues, allowed us to unequivocally assign such intermediates to an [Fe(IV) = O Por ·+] species, the first committed intermediate in monofunctional peroxidases and two protein-based radicals, identified as Trp106 · and a Tyr · , formed subsequently to the [Fe(IV) = O Por ·+] species by intramolecular electron transfer. Our recent characterization of the Mycobacterium tuberculosis catalase–peroxidase showed that the Trp · sites differ among these enzymes, and that the [Fe(IV) = O Trp · ] species was the reactive intermediate with the prodrug isoniazid. Accordingly, the question to address was whether the dissimilarity in the sites for the formation of the Trp · intermediates and in the geometry of the distal side was reflected by differences in the peroxidase-like reaction of Synechocystis and Mycobacterium tuberculosis catalase–peroxidases with the prodrug isoniazid. Our findings show that in the Synechocystis enzyme, the isoniazid substrate can get closer to the heme distal side and can react readily with the [Fe(IV) = O Por ·+] species, at variance to the situation in the M. tuberculosis catalase–peroxidase. These results indicate that, as in the case of monofunctional peroxidases, the difference in the sites for the formation of the Trp · as alternative reactive intermediates to the [Fe(IV) = O Por ·+] species is correlated to differences in substrate binding sites.
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Two distinct groups of fungal catalase/peroxidases.
Biochemical Society Transactions, 2009Co-Authors: Marcel Zámocký, Paul G. Furtmüller, Christian ObingerAbstract:Catalase/peroxidases (KatGs) are bifunctional haem b -containing (Class I) peroxidases with overwhelming catalase activity and substantial peroxidase activity with various one-electron donors. These unique oxidoreductases evolved in ancestral bacteria revealing a complex gene-duplicated structure. Besides being found in numerous bacteria of all phyla, katG genes were also detected in genomes of lower eukaryotes, most prominently of sac and club fungi. Phylogenetic analysis demonstrates the occurrence of two distinct groups of fungal KatGs that differ in localization, structural and functional properties. Analysis of lateral gene transfer of bacterial katG s into fungal genomes reveals that the most probable progenitor was a katG from a bacteroidetes predecessor. The putative physiological role(s) of both fungal KatG groups is discussed with respect to known structure–function relationships in bacterial KatGs and is related with the acquisition of (phyto)pathogenicity in fungi.
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Identification of Trp106 as the tryptophanyl radical intermediate in Synechocystis PCC6803 Catalase-Peroxidase by multifrequency Electron Paramagnetic Resonance spectroscopy.
Journal of inorganic biochemistry, 2006Co-Authors: Christa Jakopitsch, Christian Obinger, Anabella IvancichAbstract:The reactive intermediates formed in the Catalase-Peroxidase from Synechocystis PCC6803 upon reaction with peroxyacetic acid, and in the absence of peroxidase substrates, are the oxoferryl-porphyrin radical and two subsequent protein-based radicals that we have previously assigned to a tyrosyl (Tyr()) and tryptophanyl (Trp()) radicals by using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with deuterium labeling and site-directed mutagenesis. In this work, we have further investigated the Trp() in order to identify the site for the tryptophanyl radical formation, among the 26 Trp residues of the enzyme and to possibly understand the protein constraints that determine the selective formation of this radical. Based on our previous findings about the absence of the Trp() intermediate in four of the Synechocystis Catalase-Peroxidase variants on the heme distal side (W122F, W106A, H123Q, and R119A) we constructed new variants on Trp122 and Trp106 positions. Trp122 is very close to the iron on the heme distal side while Trp106 belongs to a short stretch (11 amino acid residues on the enzyme surface) that is highly conserved in Catalase-Peroxidases. We have used EPR spectroscopy to characterize the changes on the heme microenvironment induced by these mutations as well as the chemical nature of the radicals formed in each variant. Our findings identify Trp106 as the tryptophanyl radical site in Synechocystis Catalase-Peroxidase. The W122H and W106Y variants were specially designed to mimic the hydrogen-bond interactions of the naturally occurring Trp residues. These variants clearly demonstrated the important role of the extensive hydrogen-bonding network of the heme distal side, in the formation of the tryptophanyl radical. Moreover, the fact that W106Y is the only Synechocystis Catalase-Peroxidase variant of the distal heme side that recovers a catalase activity comparable to the WT enzyme, strongly indicates that the integrity of the extensive hydrogen-bonding network is also essential for the catalatic activity of the enzyme.
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Protein-based radicals in the Catalase-Peroxidase of synechocystis PCC6803: a multifrequency EPR investigation of wild-type and variants on the environment of the heme active site.
Journal of the American Chemical Society, 2003Co-Authors: Anabella Ivancich, Christa Jakopitsch, Markus Auer, Christian ObingerAbstract:Catalase-Peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of Catalase-Peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 Catalase-Peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to th...
Peter C. Loewen - One of the best experts on this subject based on the ideXlab platform.
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Electron Transfer in Catalases and Catalase-Peroxidases
Encyclopedia of Biophysics, 2018Co-Authors: Anabella Ivancich, Peter C. LoewenAbstract:Catalases (EC 1.11.1.6) are enzymes that catalyze the disproportionation of hydrogen peroxide into water and molecular oxygen by means of a heme iron or a dimanganese active site. They are crucial metalloproteins regulating the cellular concentration of hydrogen peroxide, which has a concentration-dependent dual role in cell signaling and oxidative stress. Catalase-Peroxidases, also named KatGs from the encoding katG gene, are heme-containing oxidoreductases capable of both the disproportionation of hydrogen peroxide and the oxidation of substrates via high-valent heme intermediates. KatGs also generate superoxide via an oxidase reaction and activate the antitubercular prodrug isoniazid (INH) through its conversion to isonicotinyl-NAD.
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Encyclopedia of Inorganic and Bioinorganic Chemistry - Catalase‐Peroxidase: KatG
Encyclopedia of Inorganic and Bioinorganic Chemistry, 2015Co-Authors: Ignacio Fita, Peter C. Loewen, Xavi CarpenaAbstract:Even in the absence of the catalase fold, an efficient catalase activity is also found in the heme-containing Catalase-Peroxidase proteins. The structure of these broad substrate range enzymes, reported for the first time less than 10 years ago from the halophilic archaebacterium Haloarcula marismortui (HmKatG) and from the bacterium Burkholderia pseudomallei (BpKatG), and more recently, from the eukaryotic fungus Magnaporthe grisea (MgKatG), showed a heme pocket closely related to that of plant peroxidases, although with a number of unique modifications that enable the catalase reaction. 3D Structure Schematic representation of the structure of Burkholderia pseudomallei (BpKatG) dimer viewed down the molecular twofold axis (PDB code: 1 MWV). Heme groups are shown as ball-and-sticks for the two subunits (depicted in light and dark teal color). The iron atom is indicated in red. All structural figures were prepared with program PyMOL. Keywords: catalatic reaction; peroxidatic reaction; Catalase-Peroxidase enzymes; KatG; heme enzymes; isoniazid
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Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase-like reactivity of Bacillus pumilus catalase monitored by X-ray crystallography and EPR spectroscopy
Proteins - Structure Function and Bioinformatics, 2015Co-Authors: Peter C. Loewen, Jacylyn Villanueva, Jacek Switala, Lynda J. Donald, Anabella IvancichAbstract:Heme-containing catalases and Catalase-Peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual Catalase-Peroxidase led us to investigate the enzyme for comparison with other Catalase-Peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (kcat = 339,000 s−1). In addition, the enzyme supported a much slower (kcat = 20 s−1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2-chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low-spin conversion of the FeIII high-spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.
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Oxygen Binding to Catalase-Peroxidase
The Journal of Physical Chemistry Letters, 2011Co-Authors: Pietro Vidossich, Peter C. Loewen, Ignacio Fita, Xavi Carpena, Carme RoviraAbstract:By means of quantum mechanics/molecular mechanics calculations, we show that binding of dioxygen to the FeIII enzyme Catalase-Peroxidase (KatG), responsible for activating the antitubercular drug isoniazid, is possible in the absence of an external reducing agent, thanks to the unique electronic properties of the active site Met-Tyr-Trp adduct. The calculations give support to recent experimental observations suggesting that KatG activates molecular oxygen and suggest that dioxygen activation may be achieved in other enzymes by inserting a residue with low ionization potential near the active site.
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Versatility of the electronic structure of compound I in Catalase-Peroxidases.
Journal of the American Chemical Society, 2007Co-Authors: Pietro Vidossich, Peter C. Loewen, Xavi Carpena, Mercedes Alfonso-prieto, And Ignacio Fita, Carme RoviraAbstract:Catalase-Peroxidases (KatGs) are bifunctional heme proteins, belonging to the family of class I peroxidases, that are able to catalyze both catalatic and peroxidatic reactions within a peroxidase-like structure. We investigated the electronic structure of reaction intermediates of the catalytic cycle of KatGs by means of density functional theory (DFT) QM/MM calculations. The outcome was that the ionization state of the KatG-specific covalent adduct (Met264-Tyr238-Trp111) affects the radical character of compound I (Cpd I). Specifically, in the optimized structures, substantial radical character is observed on the proximal Trp330 when Tyr238 is protonated, whereas when Tyr238 is deprotonated the radical localizes on the Met+-Tyr(O-)-Trp adduct. These findings are not affected by protein thermal fluctuations, although details of the spin density distribution are affected by the geometry of the active site. Calculations provide structures in good agreement with the crystal structure of BpKatG Cpd I. They al...
Enrique Olmos - One of the best experts on this subject based on the ideXlab platform.
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graft union formation in tomato plants peroxidase and catalase involvement
Annals of Botany, 2004Co-Authors: Nieves Fernandezgarcia, Micaela Carvajal, Enrique OlmosAbstract:• Background and Aims The use of grafted plants in vegetable crop production is now being expanded greatly. However, few data are available on the formation of graft unions in vegetables. In this work, the structural development of the graft union formation in tomato plants is studied, together with the possible relationship with activities of peroxidases and catalases. • Methods Tomato (Lycopersicon esculentum Mill.) seedlings of cultivar Fanny were grafted on the rootstock of cultivar AR‐9704 using the ‘tongue approach grafting’ method, and were grown in a crop chamber. A study of the structural development of the graft union and the involvement of peroxidases and catalases in the process of graft formation was carried out during the first stages of the graft union (4, 8 and 15 d after grafting). • Key Results Observation of the structure of the graft union showed formation of xylem and phloem vessels through the graft union 8 d after grafting. In addition, root hydraulic conductance, L0, indicate that the graft union is fully functional 8 d after grafting, which coincided with an increase of peroxidase and catalase activities. • Conclusions These results suggest that increased peroxidase and catalase activities might be implicated in graft development in tomato plants.
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Graft Union Formation in Tomato Plants: Peroxidase and Catalase Involvement
Annals of botany, 2003Co-Authors: Nieves Fernández-garcía, Micaela Carvajal, Enrique OlmosAbstract:The use of grafted plants in vegetable crop production is now being expanded greatly. However, few data are available on the formation of graft unions in vegetables. In this work, the structural development of the graft union formation in tomato plants is studied, together with the possible relationship with activities of peroxidases and catalases. Tomato (Lycopersicon esculentum Mill.) seedlings of cultivar Fanny were grafted on the rootstock of cultivar AR-9704 using the 'tongue approach grafting' method, and were grown in a crop chamber. A study of the structural development of the graft union and the involvement of peroxidases and catalases in the process of graft formation was carried out during the first stages of the graft union (4, 8 and 15 d after grafting). Observation of the structure of the graft union showed formation of xylem and phloem vessels through the graft union 8 d after grafting. In addition, root hydraulic conductance, L0, indicate that the graft union is fully functional 8 d after grafting, which coincided with an increase of peroxidase and catalase activities. These results suggest that increased peroxidase and catalase activities might be implicated in graft development in tomato plants.
Christa Jakopitsch - One of the best experts on this subject based on the ideXlab platform.
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The reaction of synechocystis Catalase-Peroxidase (KatG) with Isoniazid Investigated by multifrequency (9-285 GHz) EPR Spectroscopy
Applied Magnetic Resonance, 2009Co-Authors: Julie Colin, Christa Jakopitsch, Christian Obinger, Anabella IvancichAbstract:Three distinct electron paramagnetic resonance (EPR) spectra of radical intermediates formed as reactive intermediates in the catalytic cycle of Synechocystis catalase–peroxidase were identified. Multifrequency EPR spectroscopy, combined with site-directed mutagenesis and selective deuterium labeling of Trp and Tyr residues, allowed us to unequivocally assign such intermediates to an [Fe(IV) = O Por ·+] species, the first committed intermediate in monofunctional peroxidases and two protein-based radicals, identified as Trp106 · and a Tyr · , formed subsequently to the [Fe(IV) = O Por ·+] species by intramolecular electron transfer. Our recent characterization of the Mycobacterium tuberculosis catalase–peroxidase showed that the Trp · sites differ among these enzymes, and that the [Fe(IV) = O Trp · ] species was the reactive intermediate with the prodrug isoniazid. Accordingly, the question to address was whether the dissimilarity in the sites for the formation of the Trp · intermediates and in the geometry of the distal side was reflected by differences in the peroxidase-like reaction of Synechocystis and Mycobacterium tuberculosis catalase–peroxidases with the prodrug isoniazid. Our findings show that in the Synechocystis enzyme, the isoniazid substrate can get closer to the heme distal side and can react readily with the [Fe(IV) = O Por ·+] species, at variance to the situation in the M. tuberculosis catalase–peroxidase. These results indicate that, as in the case of monofunctional peroxidases, the difference in the sites for the formation of the Trp · as alternative reactive intermediates to the [Fe(IV) = O Por ·+] species is correlated to differences in substrate binding sites.
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Identification of Trp106 as the tryptophanyl radical intermediate in Synechocystis PCC6803 Catalase-Peroxidase by multifrequency Electron Paramagnetic Resonance spectroscopy.
Journal of inorganic biochemistry, 2006Co-Authors: Christa Jakopitsch, Christian Obinger, Anabella IvancichAbstract:The reactive intermediates formed in the Catalase-Peroxidase from Synechocystis PCC6803 upon reaction with peroxyacetic acid, and in the absence of peroxidase substrates, are the oxoferryl-porphyrin radical and two subsequent protein-based radicals that we have previously assigned to a tyrosyl (Tyr()) and tryptophanyl (Trp()) radicals by using multifrequency Electron Paramagnetic Resonance (EPR) spectroscopy combined with deuterium labeling and site-directed mutagenesis. In this work, we have further investigated the Trp() in order to identify the site for the tryptophanyl radical formation, among the 26 Trp residues of the enzyme and to possibly understand the protein constraints that determine the selective formation of this radical. Based on our previous findings about the absence of the Trp() intermediate in four of the Synechocystis Catalase-Peroxidase variants on the heme distal side (W122F, W106A, H123Q, and R119A) we constructed new variants on Trp122 and Trp106 positions. Trp122 is very close to the iron on the heme distal side while Trp106 belongs to a short stretch (11 amino acid residues on the enzyme surface) that is highly conserved in Catalase-Peroxidases. We have used EPR spectroscopy to characterize the changes on the heme microenvironment induced by these mutations as well as the chemical nature of the radicals formed in each variant. Our findings identify Trp106 as the tryptophanyl radical site in Synechocystis Catalase-Peroxidase. The W122H and W106Y variants were specially designed to mimic the hydrogen-bond interactions of the naturally occurring Trp residues. These variants clearly demonstrated the important role of the extensive hydrogen-bonding network of the heme distal side, in the formation of the tryptophanyl radical. Moreover, the fact that W106Y is the only Synechocystis Catalase-Peroxidase variant of the distal heme side that recovers a catalase activity comparable to the WT enzyme, strongly indicates that the integrity of the extensive hydrogen-bonding network is also essential for the catalatic activity of the enzyme.
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Protein-based radicals in the Catalase-Peroxidase of synechocystis PCC6803: a multifrequency EPR investigation of wild-type and variants on the environment of the heme active site.
Journal of the American Chemical Society, 2003Co-Authors: Anabella Ivancich, Christa Jakopitsch, Markus Auer, Christian ObingerAbstract:Catalase-Peroxidases are bifunctional heme enzymes with a high structural homology to peroxidases from prokaryotic origin and a catalatic activity comparable to monofunctional catalases. These unique features of Catalase-Peroxidases make them good systems to study and understand the role of alternative electron pathways both in catalases and peroxidases. In particular, it is of interest to study the poorly understood role of tyrosyl and tryptophanyl radicals as alternative cofactors in the catalytic cycle of catalases and peroxidases. In this work, we have used a powerful combination of multifrequency EPR spectroscopy, isotopic labeling of tryptophan and tyrosine residues, and site-directed mutagenesis to unequivocally identify the reactive intermediates formed by the wild-type Synechocystis PCC6803 Catalase-Peroxidase. Selected variants of the heme distal and proximal sides of the Synechocystis enzyme were investigated. Variants on the aromatic residues of the short stretch located relatively close to th...
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New Insights into the Heme Cavity Structure of Catalase-Peroxidase: A Spectroscopic Approach to the Recombinant Synechocystis Enzyme and Selected Distal Cavity Mutants†
Biochemistry, 2002Co-Authors: Hendrik A. Heering, Christian Obinger, Christa Jakopitsch, Günther Regelsberger, Giulietta SmulevichAbstract:Catalase-Peroxidases (KatGs) are heme peroxidases with homology to yeast cytochrome c peroxidase (CCP) and plant ascorbate peroxidases (APXs). KatGs exhibit a peroxidase activity of broad specifici...
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Catalase-Peroxidase from synechocystis is capable of chlorination and bromination reactions.
Biochemical and biophysical research communications, 2001Co-Authors: Christa Jakopitsch, Paul G. Furtmüller, Günther Regelsberger, Florian Rüker, Günter A. Peschek, Christian ObingerAbstract: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.