Rapid formation of compound II and a tyrosyl radical in the Y229F mutant of Mycobacterium tuberculosis catalase-peroxidase disrupts catalase but not peroxidase function

Catalase-peroxidases (KatG), which belong to Class I heme peroxidase enzymes, have high catalase activity and substantial peroxidase activity. The Y229F mutant of Mycobacterium tuberculosis KatG was prepared and characterized to investigate the functional role of this conserved residue unique to KatG enzymes. Purified, overexpressed KatG[Y229F] exhibited severely reduced steady-state catalase activity while the peroxidase activity was enhanced. Optical stopped-flow experiments showed rapid formation of Compound (Cmpd) II (oxyferryl heme intermediate) in the reaction of resting KatG[Y229F] with peroxyacetic acid or chloroperoxybenzoic acid, without detectable accumulation of Cmpd I (oxyferryl heme pi-cation radical intermediate), the latter being readily observed in the wild-type enzyme under similar conditions. Facile formation of Cmpd III (oxyferrous enzyme) also occurred in the mutant in the presence of micromolar hydrogen peroxide. Thus, the lost catalase function may be explained in part because of formation of intermediates that do not participate in catalatic turnover. The source of the reducing equivalent required for generation of Cmpd II from Cmpd I was shown by rapid freeze-quench electron paramagnetic resonance spectroscopy to be a tyrosine residue, just as in wild-type KatG. The kinetic coupling of radical generation and Cmpd II formation was shown in KatG[Y229F]. Residue Y229, which is a component of a newly defined three amino acid adduct in catalase-peroxidases, is critically important for protecting the catalase activity of KatG.     

Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.     

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.     

Vital roles of an interhelical insertion in catalase-peroxidase bifunctionality

The loop connecting the F and G helices of catalase-peroxidases contains a approximately 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(DeltaFG)). Absorption spectra indicated the heme environment of KatG(DeltaFG) 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(DeltaFG) as well as pH dependence studies suggested that the FG insertion supports hydrogen-bonded networks critical for reactions involving H2O2. The structure also appears to regulate access of electron donors to the active site.     

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.     

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.     

Specific Function of the Met-Tyr-Trp Adduct Radical and Residues Arg-418 and Asp-137 in the Atypical Catalase Reaction of Catalase-Peroxidase KatG

Catalase activity of the dual-function heme enzyme catalase-peroxidase (KatG) depends on several structural elements, including a unique adduct formed from covalently linked side chains of three conserved amino acids (Met-255, Tyr-229, and Trp-107, Mycobacterium tuberculosis KatG numbering) (MYW). Mutagenesis, electron paramagnetic resonance, and optical stopped-flow experiments, along with calculations using density functional theory (DFT) methods revealed the basis of the requirement for a radical on the MYW-adduct, for oxyferrous heme, and for conserved residues Arg-418 and Asp-137 in the rapid catalase reaction. The participation of an oxyferrous heme intermediate (dioxyheme) throughout the pH range of catalase activity is suggested from our finding that carbon monoxide inhibits the activity at both acidic and alkaline pH. In the presence of H₂O₂, the MYW-adduct radical is formed normally in KatG[D137S] but this mutant is defective in forming dioxyheme and lacks catalase activity. KatG[R418L] is also catalase deficient but exhibits normal formation of the adduct radical and dioxyheme. Both mutants exhibit a coincidence between MYW-adduct radical persistence and H₂O₂ consumption as a function of time, and enhanced subunit oligomerization during turnover, suggesting that the two mutations disrupting catalase turnover allow increased migration of the MYW-adduct radical to protein surface residues. DFT calculations showed that an interaction between the side chain of residue Arg-418 and Tyr-229 in the MYW-adduct radical favors reaction of the radical with the adjacent dioxyheme intermediate present throughout turnover in WT KatG. Release of molecular oxygen and regeneration of resting enzyme are thereby catalyzed in the last step of a proposed catalase reaction.     

Carbon monoxide adducts of KatG and KatG(S315T) as probes of the heme site and isoniazid binding

KatG, the catalase peroxidase from Mycobacterium tuberculosis, is important in the activation of the antitubercular drug, isoniazid. About 50% of isoniazid-resistant clinical isolates contain a mutation in KatG wherein the serine at position 315 is substituted with threonine, KatG(S315T). The heme pockets of KatG and KatG(S315T) and their interactions with isoniazid are probed using resonance Raman (rR) spectroscopy to characterize their ferrous CO complexes. Three vibrational modes, C-O and Fe-C stretching and Fe-CO bending, are assigned using 12CO and 13CO isotope shifts. Two conformers are observed for KatG-CO and KatG(S315T)-CO. Resonance Raman features assigned to form I are consistent with it having a neutral proximal histidine ligand and the Fe-C-O moiety hydrogen bonded to a distal residue. The nu(C-O) band for form I is sharp, consistent with a conformationally homogeneous Fe-CO unit. Form II also has a neutral proximal histidine ligand but is not hydrogen bonded. This appears to result in a conformationally disordered Fe-CO unit, as evidenced by a comparatively broad C-O stretching band. The 13CO-sensitive bands assigned to form II are predominant in the KatG(S315T)-CO rR spectrum. Isoniazid binding is apparent from the resonance Raman signatures of both WT KatG-CO and KatG(S315T)-CO. Moreover, isoniazid binding elicits an increase in the form I population of wild-type KatG-CO while having little, if any, effect on the already low population of form I of KatG(S315T)-CO. Since oxyKatG (compound III) also contains a low-spin diatomic ligand-heme adduct (heme-O2), it is reasonable to suggest that it too would exist as a mixture of conformers. Because the small form I population of KatG(S315T)-CO correlates with its inability to activate INH, we hypothesize that form I plays a role in INH activation.     

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.     

Catalase-peroxidase active site restructuring by a distant and "inactive" domain

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 catalytic activity, and is separated from the active site by >30 A. Nevertheless, without the C-terminal domain, the N-terminal domain exhibits neither catalase nor peroxidase activity due to the apparent coordination of the distal histidine to the heme iron. Here we report the ability of the separately expressed and isolated C-terminal domain (KatG(C)) to restructure the N-terminal domain (KatG(N)) to its bifunctional conformation. Addition of equimolar KatG(C) to KatG(N) decreased the hexacoordinate low-spin heme complex and increased the high-spin species (pentacoordinate and hexacoordinate). EPR spectra of the domain mixture showed a distribution between high-spin species nearly identical to that of wild-type KatG. The CD spectrum for the 1:1 physical mixture of the domains was identical to an arithmetic composite of individual spectra for KatG(N) and KatG(C). Both physical and arithmetic mixtures were nearly identical to the spectrum for wild-type KatG, suggesting that major shifts in secondary structure did not accompany active site reconfiguration. With the shift in heme environment, the parallel return of catalase and peroxidase activity was observed. Inclusion of bovine serum albumin instead of KatG(C) produced no activity, indicating that specific interdomain interactions were required to reestablish the bifunctional active site. Apparent constants for reactivation (k(react) approximately 4 x 10(-3) min(-1)) indicate that a slow process like movement of established structural elements may precede the restructuring of the heme environment and return of catalytic activity.     

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.     

Analysis of heme structural heterogeneity in Mycobacterium tuberculosis catalase-peroxidase (KatG)

Mycobacterium tuberculosis catalase-peroxidase (KatG) is a heme enzyme considered important for virulence, which is also responsible for activation of the anti-tuberculosis pro-drug isoniazid. Here, we present an analysis of heterogeneity in KatG heme structure using optical, resonance Raman, and EPR spectroscopy. Examination of ferric KatG under a variety of conditions, including enzyme in the presence of fluoride, chloride, or isoniazid, and at different stages during purification in different buffers allowed for assignment of spectral features to both five- and six-coordinate heme. Five-coordinate heme is suggested to be representative of "native" enzyme, since this species was predominant in the enzyme examined immediately after one chromatographic protocol. Quantum mechanically mixed spin heme is the most abundant form in such partially purified enzyme. Reduction and reoxidation of six-coordinate KatG or the addition of glycerol or isoniazid restored five-coordinate heme iron, consistent with displacement of a weakly bound distal water molecule. The rate of formation of KatG Compound I is not retarded by the presence of six-coordinate heme either in wild-type KatG or in a mutant (KatG[Y155S]) associated with isoniazid resistance, which contains abundant six-coordinate heme. These results reveal a number of similarities and differences between KatG and other Class I peroxidases.     

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.     

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.     

Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase-peroxidase (KatG)

The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.     

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.     

Role of the main access channel of catalase-peroxidase in catalysis

Catalase-peroxidases (KatG) are bifunctional heme peroxidases with an overwhelming catalatic activity. The structures show that the buried heme b is connected to the exterior of the enzyme by a main channel built up by KatG-specific loops named large loop LL1 and LL2, the former containing the highly conserved sequence Met-Gly-Leu-Ile-Tyr-Val-Asn-Pro-Glu-Gly. LL1 residues Ile248, Asn251, Pro252, and Glu253 of KatG from Synechocystis are the focus of this study because of their exposure to the solute matrix of the access channel. In particular, the I248F, N251L, P252A, E253Q, and E253D mutants have been analyzed by UV-visible and resonance Raman spectroscopies in combination with steady-state and presteady-state kinetic analyses. Exchange of these residues did not alter the kinetics of cyanide binding or the overall peroxidase activity. Moreover, the kinetics of compound I formation and reduction by one-electron donors was similar in the variants and the wild-type enzyme. However, the turnover numbers of the catalase activity of I248F, N251L, E253Q, and E253D were only 12.3, 32.6, 25, and 42% of the wild-type activity, respectively. These findings demonstrate that the oxidation reaction of hydrogen peroxide (not its reduction) was affected by these mutations. The altered kinetics allowed us to monitor the spectral features of the dominating redox intermediate of E253Q in the catalase cycle. Resonance Raman data and structural analysis demonstrated the existence of a very rigid and ordered structure built up by the interactions of these residues with distal side and also (via LL1) proximal side amino acids, with the heme itself, and with the solute matrix in the channel. The role of Glu253 and the other investigated channel residues in maintaining an ordered matrix of oriented water dipoles, which guides hydrogen peroxide to its site of oxidation, is discussed.     

Spectroscopic comparison of the heme active sites in WT KatG and its S315T mutant

KatG, the catalase-peroxidase from Mycobacterium tuberculosis, has been characterized by resonance Raman, electron spin resonance, and visible spectroscopies. The mutant KatG(S315T), which is found in about 50% of isoniazid-resistant clinical isolates, is also spectroscopically characterized. The electron spin resonance spectrum of ferrous nitrosyl KatG is consistent with a proximal histidine ligand. The Fe-His stretching vibration observed at 244 cm(-1) for ferrous wild-type KatG and KatG(S315T) confirms the imidazolate character of the proximal histidine in their five-coordinate high-spin complexes. The ferrous forms of wild-type KatG and KatG(S315T) are mixtures of six-coordinate low-spin and five-coordinate high-spin hemes. The optical and resonance Raman signatures of ferric wild-type KatG indicate that a majority of the heme exists in a five-coordinate high-spin state, but six-coordinate hemes are also present. At room temperature, more six-coordinate low-spin heme is observed in ferrous and ferric KatG(S315T) than in the WT enzyme. While the nature of the sixth ligand of LS ferric wild-type KatG is not completely clear, visible, resonance Raman, and electron spin resonance data of KatG(S315T) indicate that its sixth ligand is a neutral nitrogen donor. Possible effects of these differences on enzyme activity are discussed.     

Properties of catalase-peroxidase lacking its C-terminal domain

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.     

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.