Molecular evolution of thyroid peroxidase.

Thyroid peroxidase is a member of a family of mammalian peroxidases that includes myeloperoxidase, lactoperoxidase, eosinophil peroxidase, and salivary peroxidase. Protein sequences showing a high degree of sequence similarity with mammalian peroxidases have recently been observed in several invertebrate species. A multiple sequence alignment prepared with five mammalian and six invertebrate peroxidases shows complete conservation of amino acid residues considered to be important in the formation of peroxidase compound 1. These include the distal and proximal histidines, a catalytic arginine residue, and an asparagine residue hydrogen bonded to the proximal histidine. TPO-2, an alternatively spliced form of TPO, lacks the essential asparagine (Asn 579). It is now possible to speak more broadly of the family of animal peroxidases, rather than mammalian peroxidases. The animal peroxidases comprise a group of homologous proteins that differ markedly from the plant/fungal/bacterial peroxidases in primary, secondary and tertiary structure, but which share with them a common function. Animal peroxidases probably arose independently of the plant/fungal/bacterial peroxidase superfamily and most likely belong to a different gene family. The relationship between animal and non-animal peroxidases probably represents an example of convergent evolution to a common enzymatic mechanism.     

Catalytic mechanism of the glutathione peroxidase-type tryparedoxin peroxidase of Trypanosoma brucei

Trypanosoma brucei, the causative agent of African sleeping sickness, encodes three nearly identical genes for cysteine-homologues of the selenocysteine-containing glutathione peroxidases. The enzymes, which are essential for the parasites, lack glutathione peroxidase activity but catalyse the trypanothione/Tpx (tryparedoxin)-dependent reduction of hydroperoxides. Cys47, Gln82 and Trp137 correspond to the selenocysteine, glutamine and tryptophan catalytic triad of the mammalian selenoenzymes. Site-directed mutagenesis revealed that Cys47 and Gln82 are essential. A glycine mutant of Trp137 had 13% of wild-type activity, which suggests that the aromatic residue may play a structural role but is not directly involved in catalysis. Cys95, which is conserved in related yeast and plant proteins but not in the mammalian selenoenzymes, proved to be essential as well. In contrast, replacement of the highly conserved Cys76 by a serine residue resulted in a fully active enzyme species and its role remains unknown. Thr50, proposed to stabilize the thiolate anion at Cys47, is also not essential for catalysis. Treatment of the C76S/C95S but not of the C47S/C76S double mutant with H2O2 induced formation of a sulfinic acid and covalent homodimers in accordance with Cys47 being the peroxidative active site thiol. In the wild-type peroxidase, these oxidations are prevented by formation of an intramolecular disulfide bridge between Cys47 and Cys95. As shown by MS, regeneration of the reduced enzyme by Tpx involves a transient mixed disulfide between Cys95 of the peroxidase and Cys40 of Tpx. The catalytic mechanism of the Tpx peroxidase resembles that of atypical 2-Cys-peroxiredoxins but is distinct from that of the selenoenzymes.     

Reaction of horseradish peroxidase with azide and some implications for the heme environmental structure. NMR and kinetic studies.

The azide complex of horseradish peroxidase was studied by high resolution 1H and 15N NMR spectroscopy and by the temperature-jump method. The heme peripheral methyl proton peaks and the ligand 15N resonance were resolved to show that binding of azide by horseradish peroxidase occurs only in acidic solution below pH 6.5. It was also found that the chemical exchange rate of azide with the ferric enzyme was much faster on the 1H and 15N NMR time scale. This was further substantiated by kinetics of azide binding by horseradish peroxidase where the chemical exchange rate was confirmed to be in the microseconds range at pH 5.0 and 23 degrees C. This rate is salient in usual ligand exchange reactions in hemoproteins so far reported. pH dependences of the first order association and dissociation rate constants were also studied by the temperature-jump method to suggest a strong linkage of the azide binding with a proton uptake of an amino acid residue on the enzyme. These results were compared with the case of horse metmyoglobin and were interpreted to indicate that a heme-linked ionizable group on the enzyme facilitates the fast entry of the ligand to the coordination site. A histidyl residue is a possible candidate for the ionizable group of the enzyme.     

The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates

We have solved the x-ray structures of the binary horseradish peroxidase C-ferulic acid complex and the ternary horseradish peroxidase C-cyanide-ferulic acid complex to 2.0 and 1.45 A, respectively. Ferulic acid is a naturally occurring phenolic compound found in the plant cell wall and is an in vivo substrate for plant peroxidases. The x-ray structures demonstrate the flexibility and dynamic character of the aromatic donor binding site in horseradish peroxidase and emphasize the role of the distal arginine (Arg(38)) in both substrate oxidation and ligand binding. Arg(38) hydrogen bonds to bound cyanide, thereby contributing to the stabilization of the horseradish peroxidase-cyanide complex and suggesting that the distal arginine will be able to contribute with a similar interaction during stabilization of a bound peroxy transition state and subsequent O-O bond cleavage. The catalytic arginine is additionally engaged in an extensive hydrogen bonding network, which also includes the catalytic distal histidine, a water molecule and Pro(139), a proline residue conserved within the plant peroxidase superfamily. Based on the observed hydrogen bonding network and previous spectroscopic and kinetic work, a general mechanism of peroxidase substrate oxidation is proposed.     

Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.     

Free fatty acids enhance the oxidation of oxymyoglobin and inhibit the peroxidase activity of metmyoglobin

The effect of low concentrations of sodium oleate on the oxidation of oxymyoglobin to metmyoglobin has been examined. This long chain fatty acid results in a tripling of the initial rate (1.5-4.3 h-1) at which oxymyoglobin is converted to metmyoglobin and more than doubling of the rate of the long-term reaction (0.12-0.33 h-1). Examination of rate constant enhancement over a range of oleate concentrations (0-0.215 mM) has allowed an estimate of association constants for both phases of the reaction system. The peroxidase activity expressed by metmyoglobin towards hydrogen peroxide is inhibited by the presence of sodium oleate by a fivefold increase in the apparent Km value (0.33-1.77 mM). The observed changes in oxymyoglobin concentration over time are discussed in terms of competition between metmyoglobin, which acts as a peroxidase decreasing in situ concentrations of H2O2, and oxymyoglobin, which also is oxidized by the peroxide. It is shown that oleate can bind to metmyoglobin and azidometmyoglobin, but not oxymyoglobin. Catalase reduces the oxidation rates of oxymyoglobin in the presence or in the absence of oleate, substantiating the involvement of H2O2. The results are discussed in relation to the potential increase in tissue peroxidations in the presence of ischaemically elevated fatty acid concentrations.     

Magnetic circular dichroism studies on acid and alkaline forms of horseradish peroxidase.

The heme vicinities of the acid and alkaline forms of native (Fd(III)) horseradish peroxidase were investigated in terms of the magnetic circular dichroism (MCD) spectroscopy. The MCD spectrum of the acid form of native horseradish peroxidase was characteristic of a ferric high spin heme group. The resemblance in the MCD spectrum between the acid form and acetato-iron (III)protoporphyrin IX dimethyl ester suggests that the heme iron of the acid form has the electronic structure similar to that in a pentocoordinated heme complex. The MCD spectra of native horseradish peroxidase did not shown any substantial pH dependence in the pH range from 5.20 to 9.00. The MCD spectral change indicated the pK value for the equilibrium between the acid and alkaline forms to be 11.0 which agrees with the results from other methods. The alkaline form of native horseradish peroxidase at pH 12.01 exhibited the MCD spectrum of a low spin complex. The near infrared MCD spectrum suggests that the alkaline form of native horseradish peroxidase has a 6th ligand somehow different from a normal nitrogen ligand such as histidine or lysine. It implicates that the alkaline form has an overall ligand field strength of between the low spin component of metmyoglobin hydroxide and metmyoglobin azide.     

Inactivation of peroxidase by hydrogen peroxide and its protection by a reductant agent

Hydrogen peroxide, the oxidant substrate of peroxidase, is also an inactivating agent of this enzyme. The reductant substrates protect the enzyme from the inactivating process. A reaction mechanism is proposed, in which two competitive routes exist for Compound I of peroxidase; one catalytic and one inactivating. The analytical solution produced at the end of the reaction supports the proposed mechanism and shows the dependence between the number of turnovers of the enzyme (r) and the ratio of both substrates.     

Evidence for a free radical mechanism of N-demethylation of N,N-dimethylaniline and an analog by hemeprotein--H2O2 systems.

Horseradish peroxidase and metmyoglobin catalyze the H₂O₂-supported N-demethylation of N,N-dimethylaniline and N,N-dimethyl-p-toluidine. The catalytic activities of horseradish peroxidase are more than 100-fold larger than those of metmyoglobin or those previously reported for liver microsomal cytochrome P-450. Distinct free radical species of these N-methyl substrates were detected with both catalysts. These findings establish the general validity of a recently proposed free radical mechanism of oxidative N-demethylation (Griffin, B. W., and Ting, P. L., Biochemistry (1978), 2206–2211), which is quite different from that previously suggested for the analogous cytochrome P-450-dependent reactions.     

Horseradish peroxidase mutants that autocatalytically modify their prosthetic heme group: insights into mammalian peroxidase heme-protein covalent bonds

The mammalian peroxidases, including myeloperoxidase and lactoperoxidase, bind their prosthetic heme covalently through ester bonds to two of the heme methyl groups. These bonds are autocatalytically formed. No other peroxidase is known to form such bonds. To determine whether features other than an appropriately placed carboxylic acid residue are important for covalent heme binding, we have introduced aspartate and/or glutamic acid residues into horseradish peroxidase, a plant enzyme that exhibits essentially no sequence identity with the mammalian peroxidases. Based on superposition of the horseradish peroxidase and myeloperoxidase structures, the mutated residues were Leu(37), Phe(41), Gly(69), and Ser(73). The F41E mutant was isolated with no covalently bound heme, but the heme was completely covalently bound upon incubation with H(2)O(2). As predicted, the modified heme released from the protein was 3-hydroxymethylheme. The S73E mutant did not covalently bind its heme but oxidized it to the 8-hydroxymethyl derivative. The hydroxyl group in this modified heme derived from the medium. The other mutations gave unstable proteins. The rate of compound I formation for the F41E mutant was 100 times faster after covalent bond formation, but the reduction of compound I to compound II was similar with and without the covalent bond. The results clearly establish that an appropriately situated carboxylic acid group is sufficient for covalent heme attachment, strengthen the proposed mechanism, and suggest that covalent heme attachment in the mammalian peroxidases relates to peroxidase biology or stability rather than to intrinsic catalytic properties.     

Role of oxygen during horseradish peroxidase turnover and inactivation

Horseradish peroxidase catalyzed oxidation of phenol has been reinvestigated to determine the requirements of facile enzyme autoinactivation. Turnover of this peroxidase was monitored spectrophotometrically at 400 nm and found dependent on the concentration of phenol and hydrogen peroxide. The inactivation of the peroxidase required both substrates, phenol and H2O2, but surprisingly was also potentiated by molecular oxygen. Exclusion of diffusible superoxide or hydroxyl radicals had slight effect on product formation or loss of catalytic activity. A mechanism is proposed to explain the unanticipated role of oxygen during enzyme inactivation.     

Molecular simulation studies of a selenium-containing scFv catalytic antibody that mimics glutathione peroxidase

GPX is a mammalian antioxidant selenoenzyme which protects biomembranes and other cellular components from oxidative damage by catalyzing the reduction of a variety of hydroperoxides (ROOH), using Glutathione (GSH) as the reducing substrate. The single-chain Fv fragment of the monoclonal antibody 2F3 (scFv2F3) can be converted into the selenium-containing Se-scFv2F3 by chemical modification of the serine. The new selenium-containing catalytic antibody Se-scFv2F3 acts as a glutathione peroxidase (GPX) mimic with high catalytic efficiency.In order to investigate which residue of scFv2F3 is converted into selenocysteine and to describe the proper reaction site of GSH to Se-scFv2F3, a three-dimensional structure of scFv2F3 is built by means of homology modeling. The 3D model is assessed by molecular dynamics (MD) simulation to determine its stability and by comparison with those of known protein structures. After the serine in the scFv2F3 is modified to selenocysteine, a catalytic antibody (abzyme) is obtained. From geometrical considerations, the solvent-accessible surface of the protein is examined. The computer-aided docking and energy minimization (EM) calculations of the abzyme–GSH complex are then carried out to explore the possible active site of the glutathione peroxidase mimic Se-scFv2F3. The structural information from the theoretically modeled complex can help us to further understand the catalytic mechanism of GPX.     

1-Cysteine peroxiredoxin: A dual-function enzyme with peroxidase and acidic Ca-independent phospholipase A2 activities

Peroxiredoxins (Prx) are enzymes that catalyze the reduction of hydrogen peroxide and alkyl hydroperoxides. Prxs are ubiquitous enzymes with representatives found in Bacteria, Archaea and Eukarya. Many 1-cysteine peroxiredoxins (1-CysPrx) are dual-function enzyme with both peroxidase and acidic Ca²⁺-independent phospholipase A₂ (aiPLA₂) activities. The functions proposed for 1-CysPrx/aiPLA₂ include the protection of cell membrane phospholipids against oxidative damage (peroxidation) and the metabolism (hydrolysis) of phospholipids, such as those of lung surfactant. The peroxidase active site motif PVCTTE of 1-CysPrx contains the conserved catalytic cysteine residue, and the esterase (lipase) motif GXSXG of the enzyme contains the conserved catalytic serine residue. In addition to the classic lipase motif GXSXG, various 1-CysPrx/aiPLA₂s have closely related variant putative lipase motifs containing the catalytic serine residue. The PLA₂ moieties are prevalent and highly homologous in vertebrate and bacterial 1-CysPrx/aiPLA₂s that is consistent with a high degree evolutional conservation of the enzyme.     

Electron spin resonance study of peroxidase activity and kinetics.

An electron spin resonance (ESR) assay has been developed for peroxidase activity. The assay measures the formation of the paramagnetic nitroxide Tempol from the oxidation of its hydroxylamine derivative (TOLH) by short-lived radicals produced by peroxidase cycle intermediates, Compounds I and II. Using phenol as a peroxidase electron donor, the ESR approach is suitable for measurements of peroxidase activity ( > or = 0.003 U/ml) and micromolar quantities of H2O2 in sample sizes as small as 2 microliters. In addition, the ESR method can be used to continuously monitor activity in cell suspensions and other media that are susceptible to optical artifacts. The high membrane permeability of TOLH also makes it possible to estimate peroxidase activity in membrane-enclosed compartments, provided that TOLH oxidation rates can be stimulated with exogenous peroxidase reductants, e.g., phenol. Analysis of TOLH oxidation rates under conditions of low electron donor concentrations and high concentrations of H2O2 also shows clear indications of substrate-dependent inhibition and increased catalytic activity. Computer simulations indicate that the results obtained are consistent with the peroxidase reaction scheme proposed by Kohler et al. (1988, Arch. Biochem. Biophys. 264, 438-449) modified to correct for a nitroxide dependent stimulation of peroxidase catalytic activity.     

Conformational mobility in the active site of a heme peroxidase

Conformational mobility of the distal histidine residue has been implicated for several different heme peroxidase enzymes, but unambiguous structural evidence is not available. In this work, we present mechanistic, spectroscopic, and structural evidence for peroxide- and ligand-induced conformational mobility of the distal histidine residue (His-42) in a site-directed variant of ascorbate peroxidase (W41A). In this variant, His-42 binds "on" to the heme in the oxidized form, duplicating the active site structure of the cytochromes b but, in contrast to the cytochromes b, is able to swing "off" the iron during catalysis. This conformational flexibility between the on and off forms is fully reversible and is used as a means to overcome the inherently unreactive nature of the on form toward peroxide, so that essentially complete catalytic activity is maintained. Contrary to the widely adopted view of heme enzyme catalysis, these data indicate that strong coordination of the distal histidine to the heme iron does not automatically undermine catalytic activity. The data add a new dimension to our wider appreciation of structure/activity correlations in other heme enzymes.     

Ligational effects on reduction of myoglobin and horseradish peroxidase by inorganic reagents

Previous studies of the reduction of metmyoglobin and adducts by dithionite have been extended to horseradish peroxidase and its complexes. In addition, the reduction of metmyoglobin, horseradish peroxidase and adducts by a much bulkier reactant, cobalt(II) sepulchrate has been studied. Similar patterns of kinetic behavior were observed, namely, direct reduction of cyanide and imidazole adducts of the iron(III) proteins and indirect (via dissociation) reduction of the fluoride adduct. In the reduction of horseradish ferriperoxidase by cobalt(II) sepulchrate, three steps are observed and the spectral properties of the intermediate(s) and their kinetic behavior delineated. The final product is ferroperoxidase confirmed by spectral properties and its behavior on oxygenation. Reduction of cytochrome c(III) and Hipip by cobalt(II) sepulchrate appears to be a uniphasic reaction and second-order rate constants have been determined.     

Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms

Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.     

Oligomerization dynamics and functionality of Trypanosoma cruzi cytosolic tryparedoxin peroxidase as peroxidase and molecular chaperone

BackgroundTrypanosoma cruzi cytosolic tryparedoxin peroxidase (c-TXNPx) is a 2-Cys peroxiredoxin that plays an important role in coping with host cell oxidative response during the infection process, for which it has been described as a virulence factor.MethodsFour residues corresponding to c-TXNPx catalytic and solvent-exposed cysteines were individually mutated to serine by site-specific mutagenesis. Susceptibility to redox treatments and oligomeric dynamics were investigated by western-blot and gel filtration chromatography. Chaperone and peroxidase activities were determined.ResultsIn this study we demonstrated that c-TXNPx exists as different oligomeric forms, from decameric to high molecular mass aggregates. Moreover, c-TXNPx functions as a dual-function protein acting both as a peroxidase and as a molecular chaperone. Its chaperone function was shown to be independent of the presence of catalytic cysteines, even in the reduced and decameric forms, although it is enhanced when the protein is overoxidized leading to the formation of high molecular mass aggregates.Conclusionsc-TXNPx has chaperone activity which does not depend on the redox state. c-TXNPx does not undergo the dimer-decamer transition in the oxidized state described for other peroxiredoxins. Overoxidized c-TXNPx exists as different oligomeric forms from decamer to high molecular mass aggregates which are in a very slow dynamic equilibrium. The non-catalytic C57 residue may have a role in the maintenance of the decameric form, but seems not to have an alternative C_P and C_R role.General significanceThis study provides novel insights into some key aspects of the oligomerization dynamics and function of c-TXNPx.     

Oxidation of NAD dimers by horseradish peroxidase.

Horseradish peroxidase catalyses the oxidation of NAD dimers, (NAD)2, to NAD+ in accordance with a reaction that is pH-dependent and requires 1 mol of O2 per 2 mol of (NAD)2. Horseradish peroxidase also catalyses the peroxidation of (NAD)2 to NAD+. In contrast, bacterial NADH peroxidase does not catalyse the peroxidation or the oxidation of (NAD)2. A free-radical mechanism is proposed for both horseradish-peroxidase-catalysed oxidation and peroxidation of (NAD)2.     

Human catalytic antibodies with glutathione peroxidase activity

In order to generate catalytic antibodies with glutathione peroxidase (GPx) activity, we prepared GSH-S-DNP butyl ester and GSH-S-DNP benzyl ester as the haptens. Two ScFvs that bound specifically to the haptens were selected from the human phage-displayed antibody library. The two ScFv genes were highly homologous, consisting of 786 bps and belonging to the same VH family-DP25. In the premise of maintaining the amino acid sequence, mutated plasmids were constructed by use of the mutated primers in PCR, and they were over-expressed in E. coli. After the active site serine was converted into selenocysteine with the chemical modifying method, we obtained two human catalytic antibodies with GPx activity of 72.2U/micromol and 28.8U/micromol, respectively. With the aid of computer mimicking, it can be assumed that the antibodies can form dimers and the mutated selenocysteine residue is located in the binding site. Furthermore, the same Ping-Pong mechanism as the natural GPx was observed when the kinetic behavior of the antibody with the higher activity was studied.