Catalatic Activity Research Articles

Comparison of Fungal Thermophilic and Mesophilic Catalase-Peroxidases for Their Antioxidative Properties.

Catalase-peroxidases (KatGs) are unique bifunctional oxidoreductases that contain heme in their active centers allowing both the peroxidatic and catalatic reaction modes. These originally bacterial enzymes are broadly distributed among various fungi allowing them to cope with reactive oxygen species present in the environment or inside the cells. We used various biophysical, biochemical, and bioinformatics methods to investigate differences between catalase-peroxidases originating in thermophilic and mesophilic fungi from different habitats. Our results indicate that the architecture of the active center with a specific post-translational modification is highly similar in mesophilic and thermophilic KatG and also the peroxidatic acitivity with ABTS, guaiacol, and L-DOPA. However, only the thermophilic variant CthedisKatG reveals increased manganese peroxidase activity at elevated temperatures. The catalatic activity releasing molecular oxygen is comparable between CthedisKatG and mesophilic MagKatG1 over a broad temperature range. Two constructed point mutations in the active center were performed selectively blocking the formation of described post-translational modification in the active center. They exhibited a total loss of catalatic activity and changes in the peroxidatic activity. Our results indicate the capacity of bifunctional heme enzymes in the variable reactivity for potential biotech applications.

Application of metal complexes as biomimetic catalysts on glycerol oxidation

Two biomimetic complexes were evaluated as catalysts in the H2O2 mediated oxidation of glycerol, namely a peroxidase mimetic Fe(III) protoporphyrin complex (hematin) and the superoxide-dismutase mimetic complex of Mn(III) with 1,3-bis(5-sulphonatesalycilidenamino) propane (MnL−). Catalysis was targeted to glyceraldehyde since antimicrobial power was proved for it. Glyceraldehyde evolved at a higher rate than the uncatalyzed reaction only with hematin acid treated solutions and kinetics were typical of a radical mechanism. Nonetheless, glycerol conversions were low. H2O2 bleached hematin and the immobilization on a porous matrix could not prevent this. Meanwhile, the catalatic activity of hematin was high but its peroxidatic activity was inhibited at pH > 8. Thus, the coordination of hematin compound I to H2O2 over glycerol may be the preferred route with the accumulation of peroxy radicals, able to degrade the porphyrinic ring -with probable iron releasing- but also contributing to glycerol oxidation. On the other hand, a prompt decay with time of the catalatic and peroxidatic activities of MnL− was observed, which was improved by the addition of dimethylsulfoxide (DMSO), dimethylformamide (DMF) or acetone to the basic buffer system. Finally, EPR spectroscopy of MnL− supported the hypothesis of the formation of an inactive bis-oxo-bridged Mn(IV)Mn(IV) dimer upon addition of H2O2.

Horseradish peroxidase-mediated decolourization of Orange II: modelling hydrogen peroxide utilization efficiency at different pH values

Enzymatic decolourization of azo-dyes could be a cost-competitive alternative compared to physicochemical or microbiological methods. Stoichiometric and kinetic features of peroxidase-mediated decolourization of azo-dyes by hydrogen peroxide (P) are central for designing purposes. In this work, a modified version of the Dunford mechanism of peroxidases was developed. The proposed model takes into account the inhibition of peroxidases by high concentrations of P, the substrate-dependant catalatic activity of peroxidases (e.g. the decomposition of P to water and oxygen), the generation of oxidation products (OP) and the effect of pH on the decolourization kinetics of the azo-dye Orange II (OII). To obtain the parameters of the proposed model, two series of experiments were performed. In the first set, the effects of initial P concentration (0.01-0.12mM) and pH (5-10) on the decolourization degree were studied at a constant initial OII concentration (0.045mM). Obtained results showed that at pH9-10 and low initial P concentrations, the consumption of P was mainly to oxidize OII. From the proposed model, an expression for the decolourization degree was obtained. In the second set of experiments, the effect of the initial concentrations of OII (0.023-0.090mM), P (0.02-4.7mM), HRP (34-136mg/L) and pH (5-10) on the initial specific decolourization rate (q0) was studied. As a general rule, a noticeable increase in q0 was observed for pHs higher than 7. For a given pH, q0 increased as a function of the initial OII concentration. Besides, there was an inhibitory effect of high P concentrations on q0. To asses the possibility of reusing the enzyme, repeated additions of OII and P were performed. Results showed that the enzyme remained active after six reuse cycles. A satisfactory accordance between the change of the absorbance during these experiments and absorbances calculated using the proposed model was obtained. Considering that this set of data was not used during the fitting procedure of the model, the agreement between predicted and experimental absorbances provides a powerful validation of the model developed in the present work.

Theory Uncovers the Role of the Methionine-Tyrosine-Tryptophan Radical Adduct in the Catalase Reaction of KatGs: O2 Release Mediated by Proton-Coupled Electron Transfer.

Catalase-peroxidases (KatGs) are bifunctional enzymes exhibiting both peroxidase and substantial catalase activities. It is widely recognized from experiments that the catalatic activity of KatGs is correlated with a unique covalent adduct (M-Y-W) formed in the active site, but the exact role of this adduct was elusive up to now. Here, quantum mechanical/molecular mechanical (QM/MM) calculations and QM/MM metadynamics are employed to elucidate the molecular mechanism and the role of M-Y-W adduct in the catalase reaction. It is shown that O2 formation proceeds through a mechanism involving proton-coupled electron transfer (PCET). The M-Y-W cation radical adduct, which is close to the heme, His112 and the HOO. radical intermediate, acts as an electron sink during the PCET process. The present study also highlights the structural differences and functional similarities between KatGs and monofunctional catalases.

Inhibitory effects of a novel Val to Thr mutation on the distal heme of human catalase.

True catalases efficiently breakdown hydrogen peroxide, whereas the catalase-related enzyme allene oxide synthase (cAOS) is completely unreactive and instead metabolizes a fatty acid hydroperoxide. In cAOS a Thr residue adjacent to the distal His restrains reaction with H2O2 (Tosha et al. (2006) J. Biol. Chem. 281:12610; De Luna et al. (2013) J. Phys. Chem. B 117: 14635) and its mutation to the consensus Val of true catalases permits the interaction. Here we investigated the effects of the reciprocal experiment in which the Val74 of human catalase is mutated to Thr, Ser, Met, Pro, or Ala. The Val74Thr substitution decreased catalatic activity by 3.5-fold and peroxidatic activity by 3-fold. Substitution with Ser had similar negative effects (5- and 3-fold decreases). Met decreased catalatic activity 2-fold and eliminated peroxidatic activity altogether, whereas the Val74Ala substitution was well tolerated. (The Val74Pro protein lacked heme). We conclude that the conserved Val74 of true catalases helps optimize catalysis. There are rare substitutions of Val74 with Ala, Met, or Pro, but not with Ser of Thr, possibly due their hydrogen bonding affecting the conformation of His75, the essential distal heme residue for activity in catalases.

A density functional investigation of hydrogen peroxide activation by high-valent heme centers: implications for the catalase catalytic cycle

Catalases employ a tyrosinate-ligated ferric heme in order to catalyze the dismutation of hydrogen peroxide to O2 and water. In the first half of the catalytic cycle, H2O2 oxidizes Fe(III) to the formally Fe(V) state commonly referred to as Compound I. The second half of the cycle entails oxidation of a second hydrogen peroxide molecule by Compound I to dioxygen. The present study employs density functional (DFT) calculations to examine the nature of this second step of the catalatic reaction. In order to account for the unusual choice of tyrosinate as an axial ligand in catalases, oxidation of hydrogen peroxide by an imidazole-ligated Compound I is also examined, bearing in mind that imidazole-ligated hemoproteins such as myoglobin or horseradish peroxidase tend to display little, if any, catalatic activity. Furthermore, in order to gauge the importance of the cation radical of Compound I in peroxide activation, the performance of Compound II (the one-electron reduced version of Compound I, formally Fe(IV) ), is also examined. It is found that hydrogen peroxide oxidation occurs in a quasi-concerted manner, with two hydrogen-atom transfer reactions, and that the tyrosinate ligand is in no way required at this stage. We propose that the role of the tyrosinate is purely thermodynamic, in avoiding accumulation of the much less peroxide-reactive ferrous form in vivo – all in line with the predominantly thermodynamic role of the cysteinate ligands in enzymes such as cytochromes P450.

Probing hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG) and selected variants

Catalase-peroxidases (KatGs) are unique bifunctional heme peroxidases that exhibit peroxidase and substantial catalase activities. Nevertheless, the reaction pathway of hydrogen peroxide dismutation, including the electronic structure of the redox intermediate that actually oxidizes H 2O 2, is not clearly defined. Several mutant proteins with diminished overall catalase but wild-type-like peroxidase activity have been described in the last years. However, understanding of decrease in overall catalatic activity needs discrimination between reduction and oxidation reactions of hydrogen peroxide. Here, by using sequential-mixing stopped-flow spectroscopy, we have investigated the kinetics of the transition of KatG compound I (produced by peroxoacetic acid) to its ferric state by trapping the latter as cyanide complex. Apparent bimolecular rate constants (pH 6.5, 20 °C) for wild-type KatG and the variants Trp122Phe (lacks KatG-typical distal adduct), Asp152Ser (controls substrate access to the heme cavity) and Glu253Gln (channel entrance) are reported to be 1.2 × 10 4 M − 1 s − 1 , 30 M − 1 s − 1 , 3.4 × 10 3 M − 1 s − 1 , and 8.6 × 10 3 M − 1 s − 1 , respectively. These findings are discussed with respect to steady-state kinetic data and proposed reaction mechanism(s) for KatG. Assets and drawbacks of the presented method are discussed.

The structure and peroxidase activity of a 33-kDa catalase-related protein from Mycobacterium avium ssp. paratuberculosis.

True catalases are tyrosine-liganded, usually tetrameric, hemoproteins with subunit sizes of approximately 55-84 kDa. Recently characterized hemoproteins with a catalase-related structure, yet lacking in catalatic activity, include the 40-43 kDa allene oxide synthases of marine invertebrates and cyanobacteria. Herein, we describe the 1.8 A X-ray crystal structure of a 33 kDa subunit hemoprotein from Mycobacterium avium ssp. paratuberculosis (annotated as MAP-2744c), that retains the core elements of the catalase fold and exhibits an organic peroxide-dependent peroxidase activity. MAP-2744c exhibits negligible catalatic activity, weak peroxidatic activity using hydrogen peroxide (20/s) and strong peroxidase activity (approximately 300/s) using organic hydroperoxides as co-substrate. Key amino acid differences significantly impact prosthetic group conformation and placement and confer a distinct activity to this prototypical member of a group of conserved bacterial "minicatalases". Its structural features and the result of the enzyme assays support a role for MAP-2744c and its close homologues in mitigating challenge by a variety of reactive oxygen species.

Unusual Properties of Catalase A (KatA) of Pseudomonas aeruginosa PA14 Are Associated with Its Biofilm Peroxide Resistance

Pseudomonas aeruginosa is a ubiquitous environmental bacterium whose major catalase (KatA) is highly stable, extracellularly present, and required for full virulence as well as for peroxide resistance in planktonic and biofilm states. Here, we dismantled the function of P. aeruginosa KatA (KatA(Pa)) by comparing its properties with those of two evolutionarily related (clade 3 monofunctional) catalases from Bacillus subtilis (KatA(Bs)) and Streptomyces coelicolor (CatA(Sc)). We switched the coding region for KatA(Pa) with those for KatA(Bs) and CatA(Sc), expressed the catalases under the potential katA-regulatory elements in a P. aeruginosa PA14 katA mutant, and verified their comparable protein levels by Western blot analysis. The activities of KatA(Bs) and CatA(Sc), however, were less than 40% of the KatA(Pa) activity, suggestive of the difference in intrinsic catalatic activity or efficiency for posttranslational activity modulation in P. aeruginosa. Furthermore, KatA(Bs) and CatA(Sc) were relatively susceptible to proteinase K, whereas KatA(Pa) was highly stable upon proteinase K treatment. As well, KatA(Bs) and CatA(Sc) were undetectable in the extracellular milieu. Nevertheless, katA(Bs) and catA(Sc) fully rescued the peroxide sensitivity and osmosensitivity of the katA mutant, respectively. Both catalase genes rescued the attenuated virulence of the katA mutant in mouse acute infection and Drosophila melanogaster models. However, the peroxide susceptibility of the katA mutant in a biofilm growth state was rescued by neither katA(Bs) nor catA(Sc). Based on these results, we propose that the P. aeruginosa KatA is highly stable compared to the two major catalases from gram-positive bacteria and that its unique properties involving metastability and extracellular presence may contribute to the peroxide resistance of P. aeruginosa biofilm and presumably to chronic infections.

Modulation of Frankia alni ACN14a oxidative stress response: activity, expression and phylogeny of catalases

The finding that an oxidative burst was a general phenomenon in the response of hosts challenged by various symbiotic or pathogenic microbes has stimulated increasing interest in investigating the enzymology of antioxidant protection in bacteria. The question of whether the antioxidant mechanisms have been a decisive evolutionary driving force for the nitrogen‐fixing microsymbiont Frankia spp. is still open. In this study, Frankia alni strain ACN14a, which is a mid‐range infective strain, was used as a model to explore the basal oxidative stress response in terms of enzymatic activity, relative expression and phylogeny. Non‐denaturing polyacrylamide gels stained for catalatic activity revealed the presence of two monofunctional catalases KatA and KatB (EC number 1.11.1.6), which activity was shown to increase when the cells were challenged with the oxidants H2O2 and methyl viologen. Real‐time RT‐PCR targeting the two genes showed a consistent increase in the number of transcripts when the RNA was extracted from oxidative stress‐induced cells. Comparison between the expression and the activity data suggests that posttranscriptional mechanisms might be involved in the regulation of catalases in F. alni. Furthermore, upstream of katA, a fur gene coding for a ferric uptake regulator was found, forming a gene pair that functionally resembles the conserved actinobacterial synton fur–katG, which contains a gene coding for a catalase–peroxidase. Phylogenetic analysis of the F. alni fur–katA synton suggests that this genomic organization was the result of genome reshuffling events.

Identification of Trp106 as the tryptophanyl radical intermediate in Synechocystis PCC6803 catalase-peroxidase by multifrequency Electron Paramagnetic Resonance spectroscopy

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.

Immobilization of bovine catalase in sol–gels

Enzyme immobilization offers numerous advantages in terms of practical applications spanning biosensors to reactors. Bovine liver catalase (BLC [EC 1.11.1.6.]) was immobilized in tetraethoxyorthosilicate (TEOS) based sol–gels. TEOS sol–gels were found to be suitable for entrapping BLC with a high degree of immobilization. Kinetic constants for the catalatic mode of action of the immobilized enzyme were determined. Immobilized BLC retained less than 1% catalatic but 50% peroxidatic activity compared to its free counterpart. Addition of various additives did not improve the catalatic performance. Structural integrity of the immobilized enzyme and availability of active sites was confirmed by UV–vis-spectroscopy suggesting that the relatively low catalatic activity is due to effects of the sol–gel matrix on oxygen yielding reactions and dissolution into aqueous media. These data also imply that there may be significant mass transfer resistance due to the matrix depending on the nature of the substrate and product. Spectroscopic studies also indicated that the sol–gel matrix protects the catalase from formation of the inactive compound II. Heat inactivation studies showed a significantly improved thermostability of the immobilized preparation with a doubling of the inactivation energy when compared to the free enzyme. Sol–gel immobilized BLC was found to be stable for 17 consecutive reactor batch cycles with no apparent loss in activity.

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.

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.

Single-site mutations on the catalase–peroxidase from Sinorhizobium meliloti: role of the distal Gly and the three amino acids of the putative intrinsic cofactor

KatB is the only catalase-peroxidase identified so far in Sinorhizobium meliloti. It plays a housekeeping role, as it is expressed throughout all the growth phases of the free-living bacterium and also during symbiosis. This paper describes the functional and structural characterization of the KatB mutants Gly303Ser, Trp95Ala, Trp95Phe, Tyr217Leu, Tyr217Phe and Met243Val carried out by optical and electron spin resonance spectroscopy. The aim of this work was to investigate the involvement of these residues in the catalatic and/or peroxidatic reaction and falls in the frame of the open dispute around the factors that influence the balance between catalatic and peroxidatic activity in heme enzymes. The Gly303 residue is not conserved in any other protein of this family, whereas the Trp95, Tyr217 and Met243 residues are thought to form an intrinsic cofactor that is likely to play a role in intramolecular electron transfer. Spectroscopic investigations show that the Gly303Ser mutant is almost similar to the wild-type KatB and should not be involved in substrate binding. Mutations on Trp95, Tyr217 and Met243 clear out the catalatic activity completely, whereas the peroxidatic activity is maintained or even increased with respect to that of the wild-type enzyme. The k (cat) values obtained for these mutants suggest that Trp95 and Tyr217 form a huge delocalized system that provides a pathway for electron transfer to the heme. Conversely, Met243 is likely to be placed close to the binding site of the organic molecules and plays a crucial role in substrate docking.

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.

Effect of Cysticercus cellulosae fractions on the respiratory burst of pig neutrophils

Neutrophils, eosinophils and macrophages are cells that interact with invading parasites and naive hosts have been shown to have anti-parasitic activity. The initial reaction of these leukocytes is the generation of reactive oxygen species (ROS) to play in parasite expulsion. The present work was carried out to study the effect of total extract, scolex and membrane fractions from Cysticercus cellulosae on respiratory burst by pig neutrophils. Hydrogen peroxide (H2O2) production by neutrophils incubated with metacestode fractions from C. cellulosae showed an increase of: 190% (total extract), 120% (scolex) and 44% (membrane). High antioxidant catalatic activity (33%, 28%, 28% by total extract, scolex and membrane, respectively) was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. Scolex and membrane fractions increased the phagocytic capacity of neutrophils (44% and 28%, respectively). On the other hand, total cysticerci did not alter the phagocytosis, possibly due to modifications in membrane function, caused by high ROS production from neutrophils in the presence of total cysticerci. Total fraction from C. cellulosae is toxic for neutrophils as shown by the decrease in phagocytic capacity, probably caused by high levels of ROS formation. The difference in toxicity of total extract, scolex and membrane fractions on neutrophils can be explained by the presence of an antigenic effect of the vesicular fluid in the total extract of C. cellulosae.

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.

The structure of coral allene oxide synthase reveals a catalase adapted for metabolism of a fatty acid hydroperoxide

8R-Lipoxygenase and allene oxide synthase (AOS) are parts of a naturally occurring fusion protein from the coral Plexaura homomalla. AOS catalyses the production of an unstable epoxide (an allene oxide) from the fatty acid hydroperoxide generated by the lipoxygenase activity. Here, we report the structure of the AOS domain and its striking structural homology to catalase. Whereas nominal sequence identity between the enzymes had been previously described, the extent of structural homology observed was not anticipated, given that this enzyme activity had been exclusively associated with the P450 superfamily, and conservation of a catalase fold without catalase activity is unprecedented. Whereas the heme environment is largely conserved, the AOS heme is planar and the distal histidine is flanked by two hydrogen-bonding residues. These critical differences likely facilitate the switch from a catalatic activity to that of a fatty acid hydroperoxidase.

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.