Yeast cytochrome c peroxidase: mechanistic studies via protein engineering

Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme that catalyzes the reduction of hydrogen peroxide to water by ferrocytochrome c. It was the first heme enzyme to have its crystallographic structure determined and, as a consequence, has played a pivotal role in developing ideas about structural control of heme protein reactivity. Genetic engineering of the active site of CcP, along with structural, spectroscopic, and kinetic characterization of the mutant proteins has provided considerable insight into the mechanism of hydrogen peroxide activation, oxygen-oxygen bond cleavage, and formation of the higher-oxidation state intermediates in heme enzymes. The catalytic mechanism involves complex formation between cytochrome c and CcP. The cytochrome c/CcP system has been very useful in elucidating the complexities of long-range electron transfer in biological systems, including protein-protein recognition, complex formation, and intracomplex electron transfer processes.     

Geobacter sulfurreducens cytochrome c peroxidases: electrochemical classification of catalytic mechanisms

Bacterial cytochrome c peroxidase (CcP) enzymes are diheme redox proteins that reduce hydrogen peroxide to water. They are canonically characterized by a peroxidatic (called L, for "low reduction potential") active site heme and a secondary heme (H, for "high reduction potential") associated with electron transfer, and an enzymatic activity that exists only when the H-heme is prereduced to the Fe(II) oxidation state. The prereduction step results in a conformational change at the active site itself, where a histidine-bearing loop will adopt an "open" conformation allowing hydrogen peroxide to bind to the Fe(III) of the L-heme. Notably, the enzyme from Nitrosomonas europaea does not require prereduction. Previously, we have shown that protein film voltammetry (PFV) is a highly useful tool for distinguishing the electrocatalytic mechanisms of the Nitromonas type of enzyme from other CcPs. Here, we apply PFV to the recently described enzyme from Geobacter sulfurreducens and the Geobacter S134P/V135K double mutant, which have been shown to be similar to members of the canonical subclass of peroxidases and the Nitrosomonas subclass of enzymes, respectively. Here we find that the wild-type Geobacter CcP is indeed similar electrochemically to the bacterial CcPs that require reductive activation, yet the S134P/V135K mutant shows two phases of electrocatalysis: one that is low in potential, like that of the wild-type enzyme, and a second, higher-potential phase that has a potential dependent upon substrate binding and pH yet is at a potential that is very similar to that of the H-heme. These findings are interpreted in terms of a model in which rate-limiting intraprotein electron transfer governs the catalytic performance of the S134P/V135K enzyme.     

pH Dependence of heme iron coordination, hydrogen peroxide reactivity, and cyanide binding in cytochrome c peroxidase(H52K)

Replacement of the distal histidine, His-52, in cytochrome c peroxidase (CcP) with a lysine residue produces a mutant cytochrome c peroxidase, CcP(H52K), with spectral and kinetic properties significantly altered compared to those of the wild-type enzyme. Three spectroscopically distinct forms of the enzyme are observed between pH 4.0 and 8.0 with two additional forms, thought to be partially denatured forms, making contributions to the observed spectra at the pH extremes. CcP(H52K) exists in at least three, slowly interconverting conformational states over most of the pH range that was investigated. The side chain epsilon-amino group of Lys-52 has an apparent pK(a) of 6.4 +/- 0.2, and the protonation state of Lys-52 affects the spectral properties of the enzyme and the reactions with both hydrogen peroxide and HCN. In its unprotonated form, Lys-52 acts as a base catalyst facilitating the reactions of both hydrogen peroxide and HCN with CcP(H52K). The major form of CcP(H52K) reacts with hydrogen peroxide with a rate approximately 50 times slower than that of wild-type CcP but reacts with HCN approximately 3 times faster than does the wild-type enzyme. The major form of the mutant enzyme has a higher affinity for HCN than does native CcP.     

The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation

We report the characterization of the diheme cytochrome c peroxidase (CcP) from Shewanella oneidensis (So) using UV-visible absorbance, electron paramagnetic resonance spectroscopy, and Michaelis-Menten kinetics. While sequence alignment with other bacterial diheme cytochrome c peroxidases suggests that So CcP may be active in the as-isolated state, we find that So CcP requires reductive activation for full activity, similar to the case for the canonical Pseudomonas type of bacterial CcP enzyme. Peroxide turnover initiated with oxidized So CcP shows a distinct lag phase, which we interpret as reductive activation in situ. A simple kinetic model is sufficient to recapitulate the lag-phase behavior of the progress curves and separate the contributions of reductive activation and peroxide turnover. The rates of catalysis and activation differ between MBP fusion and tag-free So CcP and also depend on the identity of the electron donor. Combined with Michaelis-Menten analysis, these data suggest that So CcP can accommodate electron donor binding in several possible orientations and that the presence of the MBP tag affects the availability of certain binding sites. To further investigate the structural basis of reductive activation in So CcP, we introduced mutations into two different regions of the protein that have been suggested to be important for reductive activation in homologous bacterial CcPs. Mutations in a flexible loop region neighboring the low-potential heme significantly increased the activation rate, confirming the importance of flexible loop regions of the protein in converting the inactive, as-isolated enzyme into the activated form.     

Ascorbate peroxidase – a hydrogen peroxide-scavenging enzyme in plants

Ascorbate peroxidase is a hydrogen peroxide-scavenging enzyme that is specific to plants and algae and is indispensable to protect chloroplasts and other cell constituents from damage by hydrogen peroxide and hydroxyl radicals produced from it. In this review, first, the participation of ascorbate peroxidase in the scavenging of hydrogen peroxide in chloroplasts is briefly described. Subsequently, the phylogenic distribution of ascorbate peroxidase in relation to other hydrogen peroxide-scavenging peroxidases using glutathione, NADH and cytochrome c is summarized. Chloroplastic and cytosolic isozymes of ascorbate peroxidase have been found, and show some differences in enzymatic properties. The basic properties of ascorbate peroxidases, however, are very different from those of the guaiacol peroxidases so far isolated from plant tissues. Amino acid sequence and other molecular properties indicate that ascorbate peroxidase resembles cytochrome c peroxidase from fungi rather than guaiacol peroxidase from plants, and it is proposed that the plant and yeast hydrogen peroxide-scavenging peroxidases have the same ancestor.     

Characterisation of Fasciola hepatica cytochrome c peroxidase as an enzyme with potential antioxidant activity in vitro.

Cytochrome c peroxidase oxidises hydrogen peroxide using cytochrome c as the electron donor. This enzyme is found in yeast and bacteria and has been also described in the trematodes Fasciola hepatica and Schistosoma mansoni. Using partially purified cytochrome c peroxidase samples from Fasciola hepatica we evaluated its role as an antioxidant enzyme via the investigation of its ability to protect against oxidative damage to deoxyribose in vitro. A system containing FeIII-EDTA plus ascorbate was used to generate reactive oxygen species superoxide radical, H2O2 as well as the hydroxyl radical. Fasciola hepatica cytochrome c peroxidase effectively protected deoxyribose against oxidative damage in the presence of its substrate cytochrome c. This protection was proportional to the amount of enzyme added and occurred only in the presence of cytochrome c. Due to the low specific activity of the final partially purified sample the effects of ascorbate and calcium chloride on cytochrome c peroxidase were investigated. The activity of the partially purified enzyme was found to increase between 10 and 37% upon reduction with ascorbate. However, incubation of the partially purified enzyme with 1 mM calcium chloride did not have any effect on enzyme activity. Our results showed that Fasciola hepatica CcP can protect deoxyribose from oxidative damage in vitro by blocking the formation of the highly toxic hydroxyl radical (.OH). We suggest that the capacity of CcP to inhibit .OH-formation, by efficiently removing H2O2 from the in vitro oxidative system, may extend the biological role of CcP in response to oxidative stress in Fasciola hepatica.     

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: mutations near the high-affinity cytochrome c binding site

Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c-CcP complex [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1-ferrocytochrome c(C102T) as a substrate at pH 7.5. Some of the charge-reversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta- and hexacoordinate heme species in the enzyme. An increase in the amount of hexacoordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the 15 mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c-CcP complex observed in the crystal is the dominant catalytically active complex in solution.     

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: evidence for a single, catalytically active, cytochrome c binding domain

Forty-six charge-reversal mutants of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutants include the conversion of all 20 glutamate residues and 24 of the 25 aspartate residues in CcP, one at a time, to lysine residues. In addition, two positive-to-negative charge-reversal mutants, R31E and K149D, are included in the study. The mutants have been characterized by absorption spectroscopy and hydrogen peroxide reactivity at pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c (C102T) as substrate at pH 7.5. Many of the charge-reversal mutations cause detectable changes in the absorption spectrum of the enzyme reflecting increased amounts of hexacoordinate heme compared to wild-type CcP. The increase in hexacoordinate heme in the mutant enzymes correlates with an increase in H 2O 2-inactive enzyme. The maximum velocity of the mutants decreases with increasing hexacoordination of the heme group. Steady-state velocity studies indicate that 5 of the 46 mutations (R31E, D34K, D37K, E118K, and E290K) cause large increases in the Michaelis constant indicating a reduced affinity for cytochrome c. Four of the mutations occur within the cytochrome c binding site identified in the crystal structure of the 1:1 complex of yeast cytochrome c and CcP [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] while the fifth mutation site lies outside, but near, the crystallographic site. These data support the hypothesis that the CcP has a single, catalytically active cytochrome c binding domain, that observed in the crystal structures of the cytochrome c/CcP complex.     

Oxidation of the His-52 --> Leu mutant of cytochrome c peroxidase by p-nitroperoxybenzoic acid: role of the distal histidine in hydroperoxide activation

Both cytochrome c peroxidase (CcP) and a mutant cytochrome c peroxidase in which the distal histidine has been replaced by leucine, CcP(H52L), are converted to hydroxy-ligated derivatives at alkaline pH. In CcP, the hydroxy-ligated derivative is subsequently converted to a bis-imidazole species prior to protein denaturation while the initial hydroxy-ligated CcP(H52L) is converted to a second, spectroscopically distinct hydroxy-ligated species prior to denaturation. The spectra of the alkaline forms of CcP and CcP(H52L) have been determined between 310 and 700 nm. The pH dependence of the rate of reaction between CcP(H52L) and hydrogen peroxide has been extended to pH 10. The hydroxy-ligated form of CcP(H52L) reacts with hydrogen peroxide 4 times more rapidly than the pentacoordinate, high-spin form of CcP(H52L) that exists at neutral pH. The rate of the reaction between p-nitroperoxybenzoic acid and CcP(H52L) has been measured between pH 4 and pH 8. Neutral p-nitroperoxybenzoic acid reacts with CcP(H52L) 10(5) times more slowly than with CcP while the negatively charged p-nitroperoxybenzoate reacts with CcP(H52L) 10(3) times more slowly than with CcP. These data indicate that the role of the distal histidine during the initial formation of the peroxy anion/heme iron complex is not simply base catalysis.     

Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.     

Crystal structure of guaiacol and phenol bound to a heme peroxidase

Guaiacol is a universal substrate for all peroxidases, and its use in a simple colorimetric assay has wide applications. However, its exact binding location has never been defined. Here we report the crystal structures of guaiacol bound to cytochrome c peroxidase (CcP). A related structure with phenol bound is also presented. The CcP-guaiacol and CcP-phenol crystal structures show that both guaiacol and phenol bind at sites distinct from the cytochrome c binding site and from the δ-heme edge, which is known to be the binding site for other substrates. Although neither guaiacol nor phenol is seen bound at the δ-heme edge in the crystal structures, inhibition data and mutagenesis strongly suggest that the catalytic binding site for aromatic compounds is the δ-heme edge in CcP. The functional implications of these observations are discussed in terms of our existing understanding of substrate binding in peroxidases [Gumiero A et al. (2010) Arch Biochem Biophys 500, 13-20].     

Radical formation at Tyr39 and Tyr153 following reaction of yeast cytochrome c peroxidase with hydrogen peroxide

The formation of yeast cytochrome c peroxidase (CcP) compound I has been recognized for many years to be associated with formation of two protein-centered radicals. One of these radical sites is located at Trp191 and is directly involved in catalytic oxidation of ferrocytochrome c (Sivaraja, M., Goodin, D. B., Smith, M., Hoffman, B. M. (1989) Science 245, 738-740). The second radical has been proposed to arise from one or more tyrosyl residues of CcP. However, the tyrosyl residue (or residues) capable of forming this radical has not been identified, and the functional role of this radical remains poorly understood. In the present work, this issue has been addressed through the combined use of the spin-trapping reagent 2-methyl-2-nitrosopropane and peptide mapping by electrospray mass spectrometry to identify Tyr39 and Tyr153 as two tyrosyl residues that are capable of forming radical centers upon reaction of CcP with hydrogen peroxide. The implications of this observation to the catalytic mechanism of CcP are addressed with reference to the three-dimensional structure of CcP.     

Characterization of a covalently linked yeast cytochrome c-cytochrome c peroxidase complex: evidence for a single, catalytically active cytochrome c binding site on cytochrome c peroxidase

A covalent complex between recombinant yeast iso-1-cytochrome c and recombinant yeast cytochrome c peroxidase (rCcP), in which the crystallographically defined cytochrome c binding site [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] is blocked, was synthesized via disulfide bond formation using specifically engineered cysteine residues in both yeast iso-1-cytochrome c and yeast cytochrome c peroxidase [Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580]. Previous studies on similar covalent complexes, those that block the Pelletier-Kraut crystallographic site, have demonstrated that samples of the covalent complexes have detectable activities that are significantly lower than those of wild-type yCcP, usually in the range of approximately 1-7% of that of the wild-type enzyme. Using gradient elution procedures in the purification of the engineered peroxidase, cytochrome c, and covalent complex, along with activity measurements during the purification steps, we demonstrate that the residual activity associated with the purified covalent complex is due to unreacted CcP that copurifies with the covalent complex. Within experimental error, the covalent complex that blocks the Pelletier-Kraut site has zero catalytic activity in the steady-state oxidation of exogenous yeast iso-1-ferrocytochrome c by hydrogen peroxide, demonstrating that only ferrocytochrome c bound at the Pelletier-Kraut site is oxidized during catalytic turnover.     

Effect of active site and surface mutations on the reduction potential of yeast cytochrome c peroxidase and spectroscopic properties of the oxidized and reduced enzyme

The reduction potentials of 22 yeast cytochrome c peroxidase (CcP) mutants were determined at pH 7.0 in order to determine the effect of both heme pocket and surface mutations on the Fe(III)/Fe(II) redox couple of CcP, as well as to determine the range in redox potentials that could be obtained through point mutations in the enzyme. Spectroscopic properties of the Fe(III) and Fe(II) forms of the mutant enzymes are also reported. The mutations include variants in the distal and proximal heme pockets as well as on the enzyme surface and involve single, double, and triple point mutations. A spectrochemical redox titration technique used in this study gave an E(0') value of -189 mV for yeast CcP compared to a previously reported value of -194 mV determined by potentiometry [C.W. Conroy, P. Tyma, P.H. Daum, J.E. Erman, Biochim. Biophys. Acta 537 (1978) 62-69]. Both positive and negative shifts in the reduction potential from that of the wild-type enzyme were observed, spanning a range of 113 mV. The His-52-->Asn mutation gave the most negative potential, -259 mV, while a triple mutant in which the three distal pocket residues, Arg-48, Trp-51, and His-52, were all converted to leucine residues gave the most positive potential, -146 mV.     

Cloning, overproduction and characterization of cytochrome c peroxidase from the purple phototrophic bacterium Rhodobacter capsulatus.

The bacterial cytochrome c peroxidase (BCCP) from Rhodobacter capsulatus was purified as a recombinant protein from an Escherichia coli clone over-expressing the BCCP structural gene. BCCP from Rb. capsulatus oxidizes the Rhodobacter cytochrome c2 and reduces hydrogen peroxide, probably functioning as a detoxification mechanism. The enzyme binds two haem c groups covalently. The gene encoding BCCP from Rb. capsulatus was cloned through the construction of a 7-kb subgenomic clone. In comparison with the protein sequence, the sequence deduced from the gene has a 21-amino-acid N-terminal extension with the characteristics of a signal peptide. The purified recombinant enzyme showed the same physico-chemical properties as the native enzyme. Spectrophotometric titration established the presence of a high-potential (Em=+270 mV) and a low-potential haem (between -190 mV and -310 mV) as found in other BCCPs. The enzyme was isolated in the fully oxidized but inactive form. It binds calcium tightly and EGTA treatment of the enzyme was necessary to show calcium activation of the mixed valence enzyme. This activation is associated with the formation of a high-spin state at the low-potential haem. BCCP oxidizes horse ferrocytochrome c better than the native electron donor, cytochrome c2; the catalytic activities ('turnover number') are 85 800 min(-1) and 63 600 min(-1), respectively. These activities are the highest ever found for a BCCP.     

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.     

Oxidative titrations of reduced cytochrome aa3: influence of cytochrome c and carbon monoxide on the midpoint potential values.

Oxidative titrations were performed on the electrostatic complex formed between cytochrome c and cytochrome aa3 at low ionic strength. Midpoint potentials of the redox centers in the proteins in 1:1 and 2:1 complexes were compared with those in mixtures of the cytochromes at high ionic strength. Computer simulations of all titrations yielded midpoint potentials for the components of cytochrome aa3 which were consistent with literature values for isolated cytochrome aa3 or mixture of cytochromes c and aa3. However, the unequal heme extinction coefficients observed previously (Schroedl, N.A., and Hartzell, C.R. (1977), Biochemistry 16, 1327) during oxidative titrations of cytochrome aa3 became equal in magnitude under these experimental conditions. The binding of cytochrome c to cytochrome aa3 changed the midpoint potentials of cytochrome aa3 by 15-20 mV, while the midpoint potentials for cytochrome c were altered by 50-60 mV. Careful analysis of these titrations including computer simulation revealed that cytochrome c was able to bind to cytochrome aa3 only after cytochrome aL2+ had become oxidized. When bound to cytochrome aa3, the midpoint potential of cytochrome c was 210 7V. Titrations performed under a carbon monoxide atmosphere revealed cytochrome aa3 midpoint potentials unchanged from reported values. Cytochrome c again exhibited a midpoint potential of 210 mV after binding to cytochrome aa3.     

Protective effect of L-carnitine on hyperammonemia

The diheme cytochrome c-554 which participates in ammonia oxidation in the chemoautotroph , Nitrosomonas europaea has been studied by Soret excitation resonance Raman spectroscopy. The Raman spectrum of reduced cytochrome c-554 at neutral pH is similar classical 6-coordinate low-spin ferrous mammalian cytochrome c. In contrast, the spectrum of ferric cytochrome c-554 suggests a 5-coordinate state which is unusual for c hemes. The oxidized spectrum closely resemble that of horseradish peroxidase (HRP) or cytochrome c peroxidase (CcP) at pH 6.4. The narrow linewidth of the heme core-size vibrations indicates that both heme irons of c-554 have similar geometries.     

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.     

Prokaryotic origins of the non-animal peroxidase superfamily and organelle-mediated transmission to eukaryotes

Members of the superfamily of plant, fungal, and bacterial peroxidases are known to be present in a wide variety of living organisms. Extensive searching within sequencing projects identified organisms containing sequences of this superfamily. Class I peroxidases, cytochrome c peroxidase (CcP), ascorbate peroxidase (APx), and catalase peroxidase (CP), are known to be present in bacteria, fungi, and plants, but have now been found in various protists. CcP sequences were detected in most mitochondria-possessing organisms except for green plants, which possess only ascorbate peroxidases. APx sequences had previously been observed only in green plants but were also found in chloroplastic protists, which acquired chloroplasts by secondary endosymbiosis. CP sequences that are known to be present in prokaryotes and in Ascomycetes were also detected in some Basidiomycetes and occasionally in some protists. Class II peroxidases are involved in lignin biodegradation and are found only in the Homobasidiomycetes. In fact class II peroxidases were identified in only three orders, although degenerate forms were found in different Pezizomycota orders. Class III peroxidases are specific for higher plants, and their evolution is thought to be related to the emergence of the land plants. We have found, however, that class III peroxidases are present in some green algae, which predate land colonization. The presence of peroxidases in all major phyla (except vertebrates) makes them powerful marker genes for understanding the early evolutionary events that led to the appearance of the ancestors of each eukaryotic group.