Divergent evolutionary lines of fungal cytochrome c peroxidases belonging to the superfamily of bacterial, fungal and plant heme peroxidases

Novel open reading frames coding for cytochrome c peroxidase (CcP) belonging to the superfamily of bacterial, fungal, and plant heme peroxidases were analyzed in the available fungal genomes. Multiple sequence alignment of 71 selected peroxidase genes revealed the presence of three conserved regions essential for their function: one on the distal and two on the proximal side of the prosthetic heme group. Conserved sequence motifs on the proximal heme side are peculiar for CcPs and are responsible for their reactivity. Phylogenetic analysis performed with the distance method as well as with the maximum likelihood method revealed the existence of three distinct subfamilies of fungal CcP and their relationship to other members of the peroxidase superfamily. These divergent CcP evolutionary lines apparently evolved from a single primordial heme peroxidase gene in parallel with the evolution of ascorbate peroxidase genes. Analyzed CcPs differ significantly in their N-terminal sequences. Only subfamily I did not exhibit a presence of any signal sequence. Subfamily II members possess a well defined signal sequence allowing processing and release into mitochondrion and also in subfamily III a signal sequence was detected. Several here analyzed peroxidase genes mainly from Candida albicans and from Rhizopus oryzae can be considered interesting for the investigation of the structure-function relationship of novel CcPs revealing differences to the well documented properties of cytochrome c peroxidase from Saccharomyces cerevisiae.     

Redox properties of heme peroxidases

Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the reduction potentials of three redox couples, namely Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺. In particular, the oxidative power of heme peroxidases is controlled by the (high) reduction potential of the latter two couples. Moreover, the rapid H₂O₂-mediated two-electron oxidation of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on the reduction potential of the Fe³⁺/Fe²⁺ couple. The understanding of the molecular determinants of the reduction potentials of the above redox couples is crucial for the comprehension of the molecular determinants of the catalytic properties of heme peroxidases.This review provides an overview of the data available on the redox properties of Fe³⁺/Fe²⁺, Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺ couples in native and mutated heme peroxidases. The influence of the electron donor properties of the axial histidine and of the polarity of the heme environment is analyzed and the correlation between the redox properties of the heme group with the catalytic activity of this important class of metallo-enzymes is discussed.     

Reactions of heme peroxidases with peroxynitrite

The interaction of peroxynitrite, produced by ozonation of azide, with two heme peroxidases (horseradish peroxidase and lactoperoxidase) was studied. Enzymes retained full activity after incubation with peroxynitrite at neutral pH. Lactoperoxidase alone was found to catalyze peroxynitrite decomposition, whereas horseradish peroxidase accelerated peroxynitrite decomposition only in the presence of certain substrates. For example, in the presence of guaiacol the catalyzing effect was clear, but in the presence of trolox was only noticeable.     

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.     

Mechanisms of compound I formation in heme peroxidases

The formation of compound I is the first step in the reaction mechanism of plant heme peroxidases. This intermediate stores two oxidizing equivalents from hydrogen peroxide as an oxyferryl iron center and a radical, either on the porphyrin ring or on a tryptophan residue. Site-directed mutagenesis has proved to be a most useful tool for the identification of the intermediates involved and the resulting nature of the compound I formed. Although there is no doubt that an acid-base mechanism operates in heme peroxidase during the formation of compound I, the roles of several distal pocket residues are currently the subject of intensive research. It is now generally accepted that the conserved distal histidine in the active site of heme peroxidases is the acid-base catalyst that promotes the heterolytic cleavage of hydrogen peroxide. Other residues, such as the distal arginine and asparagine, participate in a range of roles assisting catalysis by the distal histidine. Recent advances in the elucidation of the mechanism at the molecular level are discussed. Another aspect related to the nature of compound I is the location of the radical center. Novel radical species have been detected in the reactions of ascorbate peroxidase, lignin peroxidase and several mutants of horseradish peroxidase. Detailed kinetic and spectroscopic studies of these radical species have provided important insights about the factors that control porphyrin-protein radical exchange. The wide range of data being obtained on compound I will lead to an understanding of its vital function in peroxidase catalysis and the physiological roles played by these enzymes.     

Role of fungal peroxidases in biological ligninolysis

The degradation of lignin by filamentous fungi is a major route for the recycling of photosynthetically fixed carbon, and the oxidative mechanisms employed have potential biotechnological applications. The lignin peroxidases (LiPs), manganese peroxidases (MnPs), and closely related enzymes of white rot basidiomycetes are likely contributors to fungal ligninolysis. Many of them cleave lignin model compounds to give products consistent with those found in residual white-rotted lignin, and at least some depolymerize synthetic lignins. However, none has yet been shown to delignify intact lignocellulose in vitro. The likely reason is that the peroxidases need to act in concert with small oxidants that can penetrate lignified tissues. Recent progress in the dissolution and NMR spectroscopy of plant cell walls may allow new inferences about the nature of the oxidants involved. Furthermore, increasing knowledge about the genomes of ligninolytic fungi may help us decide whether any of the peroxidases has an essential role.     

Substrate oxidation by the heme edge of fungal peroxidases. Reaction of Coprinus macrorhizus peroxidase with hydrazines and sodium azide

The peroxidase from Coprinus macrorhizus is inactivated by phenylhydrazine or sodium azide in the presence of H2O2. Inactivation by phenylhydrazine results in formation of the delta-meso-phenyl and 8-hydroxymethyl derivatives of the prosthetic heme group and covalent binding of the phenyl moiety to the protein but not in the detectable formation of Fe-phenyl- or N-phenylheme adducts. Alkylhydrazines are catalytically oxidized but do not inactivate the enzyme. Catalytic oxidation of sodium azide produces the azidyl radical and results in its addition to the delta-meso position of the prosthetic heme group. Comparison of the heme adducts obtained with C. macrorhizus peroxidase with those generated by horseradish peroxidase shows that the regiochemistry of the addition reactions is the same in both cases. The results suggest that substrates interact primarily or exclusively with the heme edge rather than the ferryl oxygen of C. macrorhizus peroxidase and indicate that the interaction occurs with the same sector of the heme edge as in horseradish peroxidase. The active-site topologies of this pair of plant and fungal peroxidases thus appear to be similar, although the observation that alkylhydrazines add to the heme edge of horseradish but not C. macrorhizus peroxidase clearly shows that there are significant differences in the two active sites.     

Interaction of hypohalous acids and heme peroxidases with unsaturated phosphatidylcholines

The formation of chlorohydrins, bromohydrins, and iodohydrins from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by the myeloperoxidase-hydrogen peroxide-halide system was evaluated by means of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry. This approach allows to detect different kinds of the halogenation reaction even in one mass spectrum. Using a mixture of Cl-, Br-, I-, and SCN- at physiological concentrations, a bromination of POPC dominates by the MPO-hydrogen peroxide-halide system. Hypothiocyanite does apparently not react with the double bond of POPC, but increasing amounts of SCN- cause a decrease of the bromohydrin peaks. An interconversion between different hypohalous acids produced by the myeloperoxidase-hydrogen peroxide-halide system determines the pattern of halogenohydrins in POPC. Especially, hypochlorous acid is able to oxidise Br- to hypobromous acid.     

Interrogation of heme pocket environment of mammalian peroxidases with diatomic ligands

Recent studies demonstrate that myeloperoxidase (MPO), eosinophil peroxidase (EPO), and lactoperoxidase (LPO), homologous members of the mammalian peroxidase superfamily, can all serve as catalysts for generating nitric oxide- (nitrogen monoxide, NO) derived oxidants. These enzymes contain heme prosthetic groups that are ligated through a histidine nitrogen and use H(2)O(2) as the electron acceptor in the catalysis of oxidative reactions. Here we show that heme reduction of these peroxidases results in distinct electronic and/or conformational changes in their heme pockets using a combination of rapid kinetics measurements, optical absorbance, and diatomic ligand binding studies. Addition of reducing agent to each peroxidase at ground state [Fe(III) state] causes immediate buildup of the corresponding Fe(II) complexes. Spectral changes indicate that two LPO-Fe(II) species are present in solution at equilibrium. Analyses of stopped-flow traces collected when EPO, MPO, or LPO solutions rapidly mixed with NO were accurately fit by single-exponential functions. Plots of the apparent rate constants as a function of NO concentration for all Fe(III) and Fe(II) forms were linear with positive intercepts, consistent with NO binding to each form in a simple reversible one-step mechanism. Fe(II) forms of MPO and LPO, but not EPO, displayed significantly lower affinity toward NO compared to Fe(III) forms, suggesting that heme reduction causes a dramatic change in the heme pocket electronic environment that alters the affinity and/or accessibility of heme iron toward NO. Optical absorbance spectra indicate that CO binds to the Fe(II) forms of both LPO and EPO, but not with MPO, and generates their respective low-spin six-coordinate complexes. Kinetic analyses indicate that the binding of CO to EPO is monophasic while CO binding to LPO is biphasic. Collectively, these results illustrate for the first time functional differences in the heme pocket environments of Fe(II) forms of EPO, LPO, and MPO toward binding of diatomic ligands. Our results suggest that, upon reduction, the heme pocket of MPO collapses, LPO adopts two spectroscopically and kinetically distinguishable forms (one partially open and the other relatively closed), and EPO remains open.     

Extracellular heme peroxidases in actinomycetes: a case of mistaken identity

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.     

Induction of peroxidases in cucumber hypocotyls by wounding and fungal infection

The induction of peroxidases (EC 1.11.1.7) during elicitation of lignification by α-1,4-linked oligogalacturonides in cucumber hypocotyl segments ( Cucumis sativus L. cv, Wisconsin SMR 58) was investigated. The wounding associated with the preparation of hypocotyl segments induced a 19-fold increase in peroxidase activity during the following 72 h. The increase was partially due to an increase in activity of a constitutive peroxidase with a pI of 8.9 and partially due to the expression of new peroxidase isozymes with pIs of 3.8, 5.4, 6.2, 9.1 and 9.4. The oligogalacturonides did not induce any peroxidase activity in addition to the wound-induced activity. These results suggest that either the constitutive peroxidase isozyme (pI 8.9) of intact hypocotyls or some of the wound-induced peroxidases are involved in the oligogalacturonide-induced lignification.
Induction of the peroxidases by wounding was inhibited by cycloheximide. This indicates that they accumulate as a result of de novo protein synthesis. Actinomycin D caused only a modest inhibition of the wound-induction peroxidases, indicating that the process is regulated at the level of translation.
Peroxidase activity increased more rapidly in resistant than in susceptible cucumber hypocotyls after inoculation with the pathogen Cladosporium cucumerinum Ellis & Arthur. The pattern of isozymes which was induced by fungal infection of resistant hypocotyls was similar to the pattern of isozymes induced by wounding. This suggests that similar induction mechanisms may be involved in the two processes.     

Selective oxygen transfer catalysed by heme peroxidases: synthetic and mechanistic aspects

The synthetic and mechanistic aspects of the use of heme peroxidases as functional mimics of the cytochrome P450 monooxygenases in oxygen-transfer reactions have been described. The chloroperoxidase from Caldariomyces fumago (CPO) is the catalyst of choice in sulfoxidation, hydroxylation and epoxidation on account of its high activity and enantioselectivity. Other heme peroxidases were less active by orders of magnitude; protein engineering has resulted in impressive improvements but even the most active mutant was still at least an order of magnitude less active than CPO. The 'oxygen-rebound' mechanisms of oxygen transfer mediated by heme enzymes - as originally conceived - have proved to be untenable. Dual pathway mechanisms, via oxoferryl species that insert oxygen as well as iron hydroperoxide species that insert OH(+), have been proposed that accommodate all of the known experimental data.     

Lignocellulose biotransformation with immobilized cellulase,d-glucose oxidase and fungal peroxidases

Three enzymes, cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], d-glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) immobilized on glass beads, have been incubated with lignocellulose. Fungal peroxidases from Trametes versicolor and Inonotus radiatus when mixed with cellulase and d-glucose oxidase were able to liberate phenolic compounds and d-glucose from lignocellulose. Three lignin monomers were identified. When the immobilized enzymes were incubated individually with lignocellulose they did not degrade lignin.     

Studies on the production of fungal peroxidases in Aspergillus niger

To get insight into the limiting factors existing for the efficient production of fungal peroxidase in filamentous fungi, the expression of the Phanerochaete chrysosporium lignin peroxidase H8 (lipA) and manganese peroxidase (MnP) H4 (mnp1) genes in Aspergillus niger has been studied. For this purpose, a protease-deficient A. niger strain and different expression cassettes have been used. Northern blotting experiments indicated high steady-state mRNA levels for the recombinant genes. Manganese peroxidase was secreted into the culture medium as an active protein. The recombinant protein showed specific activity and a spectrum profile similar to those of the native enzyme, was correctly processed at its N terminus, and had a slightly lower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant MnP production could be increased up to 100 mg/liter upon hemoglobin supplementation of the culture medium. Lignin peroxidase was also secreted into the extracellular medium, although the protein was not active, presumably due to incorrect processing of the secreted enzyme. Expression of the lipA and mnp1 genes fused to the A. niger glucoamylase gene did not result in improved production yields.     

Electrostatic modulation of electron transfer in the active site of heme peroxidases

 The heme enyzmes cytochrome c peroxidase (CCP) and pea cytosolic ascorbate peroxidase (APX) show a high level of sequence identity. The main difference near the active sites is the presence of a cation binding site in APX located about 1 nm from the Trp-179 side chain, which is hydrogen-bonded to Asp-208. It is possible that this difference in electrostatics provided by the protein environment is an essential determinant of the stabilization of the ion-pair or neutral form of the Trp...Asp couple in APX and CCP. Semiempirical molecular orbital calculations support the hypothesis that the position of the moving proton inside the couple influences the location of the free electron, leading to radical formation either on the heme or on the Trp side chain of these enzymes. Received, accepted: 26 November 1996     

Heme-protein covalent bonds in peroxidases and resistance to heme modification during halide oxidation

Plant peroxidases, as typified by horseradish peroxidase (HRP), primarily catalyze the one-electron oxidation of phenols and other low oxidation potential substrates. In contrast, the mammalian homologues such as lactoperoxidase (LPO) and myeloperoxidase primarily oxidize halides and pseudohalides to the corresponding hypohalides (e.g., Br(-) to HOBr, Cl(-) to HOCl). A further feature that distinguishes the mammalian from the plant and fungal enzymes is the presence of two or more covalent bonds between the heme and the protein only in the mammalian enzymes. The functional roles of these covalent links in mammalian peroxidases remain uncertain. We have previously reported that HRP can oxidize chloride and bromide ions, but during oxidation of these ions undergoes autocatalytic modification of its heme vinyl groups that virtually inactivates the enzyme. We report here that autocatalytic heme modification during halide oxidation is not unique to HRP but is a general feature of the oxidation of halide ions by fungal and plant peroxidases, as illustrated by studies with Arthromyces ramosus and soybean peroxidases. In contrast, LPO, a prototypical mammalian peroxidase, is protected from heme modification and its heme remains intact during the oxidation of halide ions. These results support the hypothesis that the covalent heme-protein links in the mammalian peroxidases protect the heme from modification during the oxidation of halide ions.     

Influence of Igepal reverse micellar and micellar systems on activity and stability of heme peroxidases

The activities of horseradish peroxidase (HRP) and lactoperoxidase (LPO) entrapped in reverse micelles of Igepal CO-520 in cyclohexane were studied. When the molar ratio of water to surfactant, w ₀ was ≥13, the activity of HRP encapsulated in the water pool of the reverse micelle was comparable with that measured in buffer. For LPO, however, lower activity was observed after its incorporation into the same system.

The activity of the investigated peroxidases was also measured in an aqueous solution of Igepal CO-720 or after incubation with this surfactant. The enzymes became inactivated in an aqueous micellar solution of Igepal CO-720, although this process was reversible.

The stability of HRP and LPO at 37 or 50°C was lower in the micellar systems than in buffer with the exception for HRP in reverse micelles at 50°C.     

Influence of Igepal reverse micellar and micellar systems on activity and stability of heme peroxidases

The activities of horseradish peroxidase (HRP) and lactoperoxidase (LPO) entrapped in reverse micelles of Igepal CO-520 in cyclohexane were studied. When the molar ratio of water to surfactant, w₀ was ≥13, the activity of HRP encapsulated in the water pool of the reverse micelle was comparable with that measured in buffer. For LPO, however, lower activity was observed after its incorporation into the same system.

The activity of the investigated peroxidases was also measured in an aqueous solution of Igepal CO-720 or after incubation with this surfactant. The enzymes became inactivated in an aqueous micellar solution of Igepal CO-720, although this process was reversible.

The stability of HRP and LPO at 37 or 50°C was lower in the micellar systems than in buffer with the exception for HRP in reverse micelles at 50°C.     

Detection of the halogenating activity of heme peroxidases in leukocytes by aminophenyl fluorescein

Abstract

The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively.