Purification and characterization of a catalase-peroxidase from the photosynthetic bacterium Rhodopseudomonas capsulata

Catalase-peroxidase was isolated from aerobically grown Rhodopseudomonas capsulata. The enzyme resembles typical catalases in some of its physicochemical properties. It has an apparent molecular weight of 236,000 and is composed of four identical subunits. It shows a typical high spin ferric heme spectrum with absorption maxima at 403 and 635 nm and shoulders at 503 and 535 nm. Upon binding of cyanide, the enzyme is converted to the low spin state, as shown by the shift of the Soret maximum to 418 nm and the band at 532 nm. It has an isoelectric point at pH 4.5. The enzyme differs from typical catalases in also having a strong peroxidatic activity with dianisidine, pyrogallol, and diaminobenzidine as electron donors. Both the catalatic and the peroxidatic activities are similarly inactivated by treatment with 1 mM H2O2, heating to 50 degrees C, exposure to ethanol/chloroform, and photooxidative conditions. In contrast to typical catalases, but similarly to peroxidases, the enzyme is reduced by sodium dithionite. The pH optimum of the peroxidatic activity is 5-5.3 (in contrast to 6-6.5 of the catalatic activity). 50% of the apparent maximal activities are reached at 0.3 and 4.2 mM H2O2 for the peroxidatic and catalatic activities, respectively. Both enzymic activities are equally inhibited by cyanide, 50% inhibition being achieved with 2.2 X 10(-5) M KCN. Contrarily, the two activities differ in their response to hydroxylamine and azide. 50% inhibition of the catalatic activity is obtained with 1.5 X 10(-4) M azide or 2.15 X 10(-6) M hydroxylamine; 50% inhibition of the peroxidatic activity requires 7.3 X 10(-4) M azide or 7.8 X 10(-5) M hydroxylamine. The activation energies of the catalatic and the peroxidatic activities are 1.9 and 1.7 kcal/mol, respectively.     

Purification and characterization of a novel bromoperoxidase-catalase isolated from bacteria found in recycled pulp white water

A bacterial strain, Pseudomonad EF group 70B, containing a high catalase-like activity was found in process water (white water) from pulp using recycled fibers. The enzyme was purified and characterized, and found to be a hydroperoxidase. The active enzyme has an apparent molecular mass of about 153 kDa with two identical subunits and a pI value of 4.7. It has a rather sharp pH optimum for catalase activity at 6.0 but exhibits catalase, peroxidase and brominating activities over a broad pH range from 4 to 8. It was not inhibited by 3-amino-1,2,4-triazole. Peroxidase-like activity was found when adding o-dianisidine, pyrogallol, guaiacol and 4-aminoantipyrine. Brominating activity was noticed using monochlorodimedone as a substrate. The absorption spectrum exhibited a Soret band at 404 nm. Upon reduction with dithionite the Soret peak decreased and shifted to 436 nm. Pyridine hemochrome spectra indicated the presence of a protophorfyrin IX heme group and the enzyme was inhibited by the known heme ligands cyanide and azide. N-terminal amino acid analysis gave the sequence STEVKLPYAVAGGGTTILDAFPGE, which showed no homology with those of known catalases or peroxidases. It is concluded that the enzyme is a novel type of catalase-peroxidase or, more specifically, a bromoperoxidase-catalase, and that future developments of inhibitors of hydrogen peroxide-degrading activities in white water may be based on this enzyme and other catalase-peroxidases.     

Modulation of the activities of catalase-peroxidase HPI of Escherichia coli by site-directed mutagenesis

Catalase-peroxidases have a predominant catalatic activity but differ from monofunctional catalases in exhibiting a substantial peroxidatic reaction which has been implicated in the activation of the antitubercular drug isoniazid in Mycobacterium tuberculosis. Hydroperoxidase I of Escherichia coli encoded by katG is a catalase-peroxidase, and residues in its putative active site have been the target of a site directed-mutagenesis study. Variants of residues R102 and H106, on the distal side of the heme, and H267, the proximal side ligand, were constructed, all of which substantially reduced the catalatic activity and, to a lesser extent, the peroxidatic activity. In addition, the heme content of the variants was reduced relative to the wild-type enzyme. The relative ease of heme loss from HPI and a mixture of tetrameric enzymes with 2, 3, and 4 hemes was revealed by mass spectrometry analysis. Conversion of W105 to either an aromatic (F) or aliphatic (I) residue caused a 4-5-fold increase in peroxidatic activity, coupled with a >99% inhibition of catalatic activity. The peroxidatic-to-catalatic ratio of the W105F variant was increased 2800-fold such that compound I could be identified by both electronic and EPR spectroscopy as being similar to the porphyrin cation radical formed in other catalases and peroxidases. Compound I, when generated by a single addition of H(2)O(2), decayed back to the native or resting state within 1 min. When H(2)O(2) was generated enzymatically in situ at low levels, active compound I was evident for up to 2 h. However, such prolonged treatment resulted in conversion of compound I to a reversibly inactivated and, eventually, to an irreversibly inactivated species, both of which were spectrally similar to compound I.     

Purification and characterization of a catalase-peroxidase and a typical catalase from the bacterium Klebsiella pneumoniae

The bacterium Klebsiella pneumoniae synthesizes three different types of catalase: a catalase-peroxidase, a typical catalase and an atypical catalase, designated KpCP, KpT and KpA, respectively (Goldberg, I. and Hochman, A. (1989) Arch. Biochem. Biophys. 268, 124-128). KpCP, but not the other two enzymes, in addition to the catalatic activity, catalyzes peroxidatic activities with artificial electron donors, as well as with NADH and NADPH. Both KpCP and KpT are tetramers, with heme IX as a prosthetic group, and they show a typical high-spin absorption spectrum which is converted to low-spin when a cyanide complex is formed. The addition of dithionite to KpCP causes a shift in the absorption maxima typical of ferrous heme IX. KpCP has a pH optimum of 6.3 for the catalatic activity and 5.2-5.7 for the peroxidatic activity, and relatively low 'Km' values: 6.5 mM and 0.65 H2O2 for the catalatic and peroxidatic activities, respectively. The activity of the catalase-peroxidase is inhibited by azide and cyanide, but not by 3-amino-1,2,4-triazole. KpT has wide pH optimum: 5-10.5 and a 'Km' of 50 mM H2O2, it is inhibited by incubation with 3-amino-1,2,4-triazole and by the acidic forms of cyanide and azide. A significant distinction between the typical catalase and the catalase-peroxidase is the stability of their proteins: KpT is more stable than KpCP to H2O2, temperature, pH and urea.     

Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.     

Occurrence and biochemistry of hydroperoxidases in oxygenic phototrophic prokaryotes (cyanobacteria)

Blue-green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant-type photosynthetic organisms. They were the first which produced molecular oxygen as a byproduct of photosynthetic activity. Also today they live in habitats with potentially damaging photooxidative conditions due to high irradiation and oxygen concentrations. Therefore, the cells must have evolved protective mechanisms to cope with reactive oxygen species produced by incomplete reduction of molecular oxygen via electron transport processes to prevent damage of biologically important macromolecules. Hydrogen peroxide and organic peroxides can be removed by enzymes called hydroperoxidases which on the one hand disproportionate it (catalases and catalase-peroxidases) and on the other hand use electron donors to reduce it to water or the corresponding alcohols. Until now the sequenced or partially sequenced genomes of six cyanobacteria are available in databases. Based on similarity searches and multiple sequence alignments, several cyanobacterial hydroperoxidases can be detected. All the cyanobacteria possess peroxiredoxins which use thioredoxin or other reduced thiols to get rid of hydrogen peroxide and lipid peroxides. Nearly all cyanobacteria contain an NADPH-dependent glutathione peroxidase-like protein which uses NADPH to reduce unsaturated fatty acid hydroperoxides. The best analyzed cyanobacterial antioxidative enzyme is the hemoprotein catalase-peroxidase which has a high catalase activity but concerning the sequence it is a typical peroxidase. Two species seem to encode a manganese-containing catalase and Nostoc punctiforme could use a monofunctional catalase. There are as well additional peroxidases encoded in cyanobacteria whose physiological relevance is unknown.     

Thirty years of heme catalases structural biology

About thirty years ago the crystal structures of the heme catalases from Penicillium vitale (PVC) and, a few months later, from bovine liver (BLC) were published. Both enzymes were compact tetrameric molecules with subunits that, despite their size differences and the large phylogenetic separation between the two organisms, presented a striking structural similarity for about 460 residues. The high conservation, confirmed in all the subsequent structures determined, suggested a strong pressure to preserve a functional catalase fold, which is almost exclusively found in these mono-functional heme catalases. However, 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 ten years ago from the halophilic archaebacterium Haloarcula marismortui (HmCPx) and from the bacterium Burkholderia pseudomallei (BpKatG), showed a heme pocket closely related to that of plant peroxidases, though with a number of unique modifications that enable the catalase reaction. Despite the wealth of structural information already available, for both monofunctional catalases and catalase-peroxidases, a number of unanswered major questions require continuing structural research with truly innovative approaches.     

The ferric-hydroperoxo complex of chloroperoxidase

The hydroperoxo-ferric complex, or Compound 0 (Cpd 0), is an unstable transient intermediate common for oxygen activating heme enzymes such as the cytochromes P450, nitric oxide synthases, and heme oxygenases, as well as the peroxidases and catalases which utilize hydrogen peroxide as a source of oxygen and reducing equivalents. Detailed understanding of the mechanism of oxygen activation and formation of the higher valent catalytically active intermediates in heme enzyme catalysis requires the structural and spectroscopic characterization of this immediate precursor, Cpd 0. Using the method of cryoradiolytic reduction of the oxy-ferrous heme complex, we have prepared and characterized hydroperoxo-ferric complex in chloroperoxidase (CPO) and compared this to the same intermediate generated in cytochrome P450 CYP101. Optical absorption spectrum of Cpd 0 in CPO has a Soret band at 449 nm and poorly resolved α, β bands at 576 and 546 nm.     

Oxidative stress-induced expression of catalases in Comamonas terrigena

When grown under oxidative stress, catalatic as well as peroxidatic activity is increased in the Gram-negative bacteriumComamonas terrigena N3H. Two distinct hydroperoxidases were demonstrated by a specific staining. Based on their molar masses and their sensitivity toward 3-amino-1,2,4-triazole and high temperatures, they were identified as dimeric catalase-1 (Cat-1; 150 kDa), and as a tetrameric catalase-2 (Cat-2; 240 kDa) with enhanced peroxidatic activity, respectively. These two catalases differ in their expression during the bacterial growth; whereas the expression of the smaller enzyme (Cat-1) is induced by 0.5 mmol/L peroxides in the medium, and to a lesser degree by 25 mg/L Cd²⁺, Cat-2 (typical catalase) is almost specifically induced with cadmium ions.     

Detection and purification of a catalase-peroxidase from Mycobacterium sp. Pyr-1

A catalase-peroxidase from Mycobacterium sp. Pyr-1, a strain capable of growth on pyrene, was purified to homogeneity by anion exchange and hydroxyapatite column chromatography. The enzyme, like the M. tuberculosis T-catalase, reduced nitroblue tetrazolium in the presence of isoniazid (INH) and H2O2. It also oxidized 3,3',5,5'-tetramethylbenzidine and other substrates of the catalase-peroxidase of M. tuberculosis in the presence of either tert-butyl hydroperoxide or H2O2. It had a UV/ visible absorption spectrum (Soret peak at 406 nm) similar to that of the catalase-peroxidase of M. tuberculosis (Soret peak at 408 nm) and identical to that of the catalase-peroxidase of M. smegmatis. After electrophoresis on non-denaturing gels the enzyme showed one single protein band with both catalase and peroxidase activity, which were lost after electrophoresis on SDS-PAGE. The enzyme was inhibited by sodium azide, glutathione, 2-mercaptoethanol, and isoniazid, but not by isonicotinic acid. The optimum enzyme activity was obtained at pH 4.5 and at 25 degrees C.     

Purification of the o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities.

Extracts of aerobically grown Escherichia coli B exhibit both catalase and dianisidine peroxidase activities. Polyacrylamide gel electrophoresis demonstrates two distinct catalases which have been designated hydroperoxidases I and II (HP-I and HP-II) in order of increasing anodic mobility. HP-I has been purified to essential homogeneity and found to be composed of four subunits of equal size. Its molecular weight is 337,000, and it contains two molecules of protoheme IX per tetramer. Its amino acid composition is unusual, for so large a protein, in lacking half-cystine. HP-I is a very efficient catalase with an activity optimum at pH 7.5, a Km for H2O2 of 3.9 mM, and a turnover number of 9.8 x 10(5) per min. It is also a broad specificity peroxidase capable of acting upon dianisidine, guaiacol, p-phenylenediamine, and pyrogallol. Dianisidine acted as a powerful reversible inhibitor of the catalatic activity of HP-I and as a suicide substrate when HP-I functioned in its peroxidatic mode.     

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

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 (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 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 Fe^III 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.     

Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases.

Molecular phylogeny among catalase-peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed. Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected. Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity. The N-terminal and C-terminal domains of catalase-peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history. The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase-peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily. From the results, it is obvious that the duplication event in the gene for catalase-peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed. Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution.     

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.     

Interaction of bd-type quinol oxidase from Escherichia coli and carbon monoxide: Heme d binds CO with high affinity

Comparative studies on the interaction of the membrane-bound and detergent-solubilized forms of the enzyme in the fully reduced state with carbon monoxide at room temperature have been carried out. CO brings about a bathochromic shift of the heme d band with a maximum at 644 nm and a minimum at 624 nm, and a peak at 540 nm. In the Soret band, CO binding to cytochrome bd results in absorption decrease and minima at 430 and 445 nm. Absorption perturbations in the Soret band and at 540 nm occur in parallel with the changes at 630 nm and reach saturation at 3-5 microM CO. The peak at 540 nm is probably either beta-band of the heme d-CO complex or part of its split alpha-band. In both forms of cytochrome bd, CO reacts predominantly with heme d. Addition of high CO concentrations to the solubilized cytochrome bd results in additional spectral changes in the gamma-band attributable to the reaction of the ligand with 10-15% of low-spin heme b558. High-spin heme b595 does not bind CO even at high concentrations of the ligand. The apparent dissociation constant values for the heme d-CO complex of the membrane-bound and detergent-solubilized forms of the fully reduced enzyme are about 70 and 80 nM, respectively.     

Mechanisms of catalase activity of heme peroxidases

In the absence of exogenous electron donors monofunctional heme peroxidases can slowly degrade hydrogen peroxide following a mechanism different from monofunctional catalases. This pseudo-catalase cycle involves several redox intermediates including Compounds I, II and III, hydrogen peroxide reduction and oxidation reactions as well as release of both dioxygen and superoxide. The rate of decay of oxyferrous complex determines the rate-limiting step and the enzymes’ resistance to inactivation. Homologous bifunctional catalase-peroxidases (KatGs) are unique in having both a peroxidase and high hydrogen dismutation activity without inhibition reactions. It is demonstrated that KatGs follow a similar reaction pathway as monofunctional peroxidases, but use a unique post-translational distal modification (Met⁺-Tyr-Trp adduct) in close vicinity to the heme as radical site that enhances turnover of oxyferrous heme and avoids release of superoxide. Similarities and differences between monofunctional peroxidases and bifunctional KatGs are discussed and mechanisms of pseudo-catalase activity are proposed.     

Characterization of the katG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis.

The isoniazid susceptibility of Mycobacterium tuberculosis is mediated by the product of the katG gene which encodes the heme-containing enzyme catalase-peroxidase. In this study, the chromosomal location of katG has been established and its nucleotide sequence has been determined so that the primary structure of catalase-peroxidase could be predicted. The M. tuberculosis enzyme is an 80,000-dalton protein containing several motifs characteristic of peroxidases and shows strong similarity to other bacterial catalase-peroxidases. Expression of the katG gene in M. tuberculosis, M. smegmatis, and Escherichia coli was demonstrated by Western blotting (immunoblotting). Homologous genes were detected in other mycobacteria, even those which are naturally insensitive to isoniazid.     

Purification and characterization of spore-specific catalase-2 from Bacillus subtilis

Catalase-2, the catalase found in spores of Bacillus subtilis, has been purified to homogeneity from a nonsporulating strain. The apparent native molecular weight is 504,000. The enzyme appears to be composed of six identical protomers with a molecular weight of 81,000 each. The amino acid composition is similar to the composition of other catalases. Like most catalases, catalase-2 exhibits a broad pH optimum from pH 4 to pH 12 and is sensitive to cyanide, azide, thiol reagents, and amino triazole. The apparent Km for H2O2 is 78 mM. The enzyme exhibits extreme stability, losing activity only slowly at 93 degrees C and remaining active in 1% SDS-7 M urea. The green-colored enzyme exhibits a spectrum like heme d with a Soret absorption at 403 nm and a molar absorptivity consistent with one heme per subunit. The heme cannot be extracted with acetone-HCl or ether, suggesting that it is covalently bound to the protein.     

Calcium-dependent conformation of a heme and fingerprint peptide of the diheme cytochrome c peroxidase from Paracoccus pantotrophus

The structural changes in the heme macrocycle and substituents caused by binding of Ca(2+) to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca(2+)-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca(2+) or Mg(2+). This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca(2+) binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca(2+) binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.