Peroxidatic Activity Research Articles

Sleeve Gastrectomy Provides Cardioprotection from Oxidative Stress In Vitro Due to Reduction of Circulating Myeloperoxidase.

Bariatric surgery, including sleeve gastrectomy (SG), improves systolic and diastolic function, which is independent of weight loss in rodent models. The cause of weight loss-independent improvements in cardiac function are unknown but may originate from the gastrointestinal tract. In this study, we investigated whether a circulating blood factor is a mechanism for acute cardioprotection after SG by testing the utility of rodent SG plasma to reduce metabolic stress in vitro. For the initial experiment, obese male Zucker rats underwent SG, ad lib sham, or pair-fed sham surgeries (n = six SG, n = eight SH, n = eight PF). For all other studies, a second group of Zucker rats underwent SG or ad lib sham surgeries (n = eight SH, n = six SG). Six weeks following surgery, plasma was collected from each group, both in the fasting and post-prandial (pp) state. This plasma was then pooled per surgical group and nutrient state and tested in multiple in vitro cell culture and extra-cellular assays to determine the effect of SG on myotubular metabolic stress compared to the sham surgeries. Post-prandial SG plasma (ppSG), but not fasting SG, pp, or fasting sham plasma, reduced the metabolic stress of the H9c2 cells as measured by lactate dehydrogenase (LDH) release (p < 0.01). Unlike SG, weight reduction through pair-feeding did not prevent H9c2 metabolic stress. The PpSG plasma had the slowest rate of extracellular hydrogen peroxide consumption and peroxidatic activity compared to the pp sham, fasting SG, and fasting sham groups. Redox testing of plasma with aminiobenzoic acid hydrazide and edaravone suggested a pattern supporting myeloperoxidase (MPO), or other peroxidases, as the primary component responsible for reduced metabolic stress with ppSG plasma. The PpSG plasma contained 35% less circulating MPO protein as compared to the pp sham and fasting SG plasma. The plasma from an MPO global knockout rat also prevented metabolic stress of the H9c2 cells, compared to the significant increase in LDH release from the plasma of the WT controls (p < 0.01). The MPO global knockout plasma also had a rate of extracellular hydrogen peroxide consumption and peroxidatic activity comparable to the ppSG plasma. These studies suggest that one of the weight loss-independent mechanisms by which SG improves myocellular function could be a reduced pro-oxidative environment due to lower circulating levels of MPO. It appears that the gastrointestinal tract is of critical importance to these findings, as the MPO levels were only lowered after enteral, nutrient stimulation in the SG rats. If this surgical effect is confirmed in humans, SG may be a unique surgical treatment for multiple diseases with a pathogenesis of inflammation and oxidative damage, including obesity-associated heart failure with preserved ejection fraction.

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

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

Structural and mechanistic basis for redox sensing by the cyanobacterial transcription regulator RexT

Organisms have a myriad of strategies for sensing, responding to, and combating reactive oxygen species, which are unavoidable consequences of aerobic life. In the heterocystous cyanobacterium Nostoc sp. PCC 7120, one such strategy is the use of an ArsR-SmtB transcriptional regulator RexT that senses H2O2 and upregulates expression of thioredoxin to maintain cellular redox homeostasis. Different from many other members of the ArsR-SmtB family which bind metal ions, RexT has been proposed to use disulfide bond formation as a trigger to bind and release DNA. Here, we present high-resolution crystal structures of RexT in the reduced and H2O2-treated states. These structures reveal that RexT showcases the ArsR-SmtB winged-helix-turn-helix fold and forms a vicinal disulfide bond to orchestrate a response to H2O2. The importance of the disulfide-forming Cys residues was corroborated using site-directed mutagenesis, mass spectrometry, and H2O2-consumption assays. Furthermore, an entrance channel for H2O2 was identified and key residues implicated in H2O2 activation were pinpointed. Finally, bioinformatics analysis of the ArsR-SmtB family indicates that the vicinal disulfide “redox switch” is a unique feature of cyanobacteria in the Nostocales order, presenting an interesting case where an ArsR-SmtB protein scaffold has been evolved to showcase peroxidatic activity and facilitate redox-based regulation.

Ascorbate Peroxidase Neofunctionalization at the Origin of APX-R and APX-L: Evidence from Basal Archaeplastida.

Ascorbate peroxidases (APX) are class I members of the Peroxidase-Catalase superfamily, a large group of evolutionarily related but rather divergent enzymes. Through mining in public databases, unusual subsets of APX homologs were identified, disclosing the existence of two yet uncharacterized families of peroxidases named ascorbate peroxidase-related (APX-R) and ascorbate peroxidase-like (APX-L). As APX, APX-R harbor all catalytic residues required for peroxidatic activity. Nevertheless, proteins of this family do not contain residues known to be critical for ascorbate binding and therefore cannot use it as an electron donor. On the other hand, APX-L proteins not only lack ascorbate-binding residues, but also every other residue known to be essential for peroxidase activity. Through a molecular phylogenetic analysis performed with sequences derived from basal Archaeplastida, the present study discloses the existence of hybrid proteins, which combine features of these three families. The results here presented show that the prevalence of hybrid proteins varies among distinct groups of organisms, accounting for up to 33% of total APX homologs in species of green algae. The analysis of this heterogeneous group of proteins sheds light on the origin of APX-R and APX-L and suggests the occurrence of a process characterized by the progressive deterioration of ascorbate-binding and catalytic sites towards neofunctionalization.

Cardiolipin interactions with cytochrome c increase tyrosine nitration yields and site-specificity

The interaction between cytochrome c and cardiolipin is a relevant process in the mitochondrial redox homeostasis, playing roles in the mechanism of electron transfer to cytochrome c oxidase and also modulating cytochrome c conformation, reactivity and function. Peroxynitrite is a widespread nitrating agent formed in mitochondria under oxidative stress conditions, and can result in the formation of tyrosine nitrated cytochrome c. Some of the nitro-cytochrome c species undergo conformational changes at physiological pH and increase its peroxidase activity. In this work we evaluated the influence of cardiolipin on peroxynitrite-mediated cytochrome c nitration yields and site-specificity. Our results show that cardiolipin enhances cytochrome c nitration by peroxynitrite and targets it to heme-adjacent Tyr67. Cytochrome c nitration also modifies the affinity of protein with cardiolipin. Using a combination of experimental techniques and computer modeling, it is concluded that structural modifications in the Tyr67 region are responsible for the observed changes in protein-derived radical and tyrosine nitration levels, distribution of nitrated proteoforms and affinity to cardiolipin. Increased nitration of cytochrome c in presence of cardiolipin within mitochondria and the gain of peroxidatic activity could then impact events such as the onset of apoptosis and other processes related to the disruption of mitochondrial redox homeostasis.

Cubic Liquid Crystalline Nanostructures Involving Catalase and Curcumin: BioSAXS Study and Catalase Peroxidatic Function after Cubosomal Nanoparticle Treatment of Differentiated SH-SY5Y Cells.

The development of nanomedicines for the treatment of neurodegenerative disorders demands innovative nanoarchitectures for combined loading of multiple neuroprotective compounds. We report dual-drug loaded monoolein-based liquid crystalline architectures designed for the encapsulation of a therapeutic protein and a small molecule antioxidant. Catalase (CAT) is chosen as a metalloprotein, which provides enzymatic defense against oxidative stress caused by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). Curcumin (CU), solubilized in fish oil, is co-encapsulated as a chosen drug with multiple therapeutic activities, which may favor neuro-regeneration. The prepared self-assembled biomolecular nanoarchitectures are characterized by biological synchrotron small-angle X-ray scattering (BioSAXS) at multiple compositions of the lipid/co-lipid/water phase diagram. Constant fractions of curcumin (an antioxidant) and a PEGylated agent (TPEG1000) are included with regard to the lipid fraction. Stable cubosome architectures are obtained for several ratios of the lipid ingredients monoolein (MO) and fish oil (FO). The impact of catalase on the structural organization of the cubosome nanocarriers is revealed by the variations of the cubic lattice parameters deduced by BioSAXS. The outcome of the cellular uptake of the dual drug-loaded nanocarriers is assessed by performing a bioassay of catalase peroxidatic activity in lysates of nanoparticle-treated differentiated SH-SY5Y human cells. The obtained results reveal the neuroprotective potential of the in vitro studied cubosomes in terms of enhanced peroxidatic activity of the catalase enzyme, which enables the inhibition of H2O2 accumulation in degenerating neuronal cells.

Application of metal complexes as biomimetic catalysts on glycerol oxidation

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

Fe(III) Protoporphyrin IX Encapsulated in a Zinc Metal-Organic Framework Shows Dramatically Enhanced Peroxidatic Activity.

Two MOFs, [H2N(CH3)2][Zn3(TATB)2(HCOO)]·HN(CH3)2·DMF·6H2O (1) and Zn-HKUST-1 (2), were investigated as potential hosts to encapsulate Fe(III) heme (Fe(III) protoporphyrin IX = Fe(III)PPIX). Methyl orange (MO) adsorption was used as an initial model for substrate uptake. MOF 1 showed good adsorption of MO (10.3 ± 0.8 mg g-1) which could undergo in situ protonation upon exposure to aqueous HCl vapor. By contrast, MO uptake by 2 was much lower (2 ± 1 mg g-1), and PXRD indicated that structural instability on exposure to water was the likely cause. Two methods for Fe(III)PPIX-1 preparation were investigated: soaking and encapsulation. Encapsulation was verified by SEM-EDS and showed comparable concentrations of Fe(III)PPIX on exposed interior surfaces and on the original surface of fractured crystals. SEM-EDS results were consistent with ICP-OES data on bulk material (1.2 ± 0.1 mass % Fe). PXRD data showed that the framework in 1 was unchanged after encapsulation of Fe(III)PPIX. MO adsorption (5.8 ± 1.2 mg g-1) by Fe(III)PPIX-1 confirmed there is space for substrate diffusion into the framework, while the UV-vis spectrum of solubilized crystals confirmed that Fe(III)PPIX retained its integrity. A solid-state UV-vis spectrum of Fe(III)PPIX-1 indicated that Fe(III)PPIX was not in a μ-oxo dimeric form. Although single-crystal XRD data did not allow for full refinement of the encapsulated Fe(III)PPIX molecule owing to disorder of the metalloporphyrin, the Fe atom and pyrrole N atoms were located, enabling rigid-body modeling of the porphine core. Reaction of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) with H2O2, catalyzed by Fe(III)PPIX-1 and -2, showed that Fe(III)PPIX-1 is significantly more efficient than Fe(III)PPIX-2 and is superior to solid Fe(III)PPIX-Cl. Fe(III)PPIX-1 was used to catalyze the oxidation of hydroquinone, thymol, benzyl alcohol, and phenyl ethanol by tert-butyl-hydroperoxide with t1/2 values that increase with increasing substrate molecular volume.

Heightened Dynamics of the Oxidized Y48H Variant of Human Cytochrome c Increases Its Peroxidatic Activity

Proteins performing multiple biochemical functions are called "moonlighting proteins" or extreme multifunctional (EMF) proteins. Mitochondrial cytochrome c is an EMF protein that binds multiple partner proteins to act as a signaling molecule, transfers electrons in the respiratory chain, and acts as a peroxidase in apoptosis. Mutations in the cytochrome c gene lead to the disease thrombocytopenia, which is accompanied by enhanced apoptotic activity. The Y48H variant arises from one such mutation and is found in the 40-57 Ω-loop, the lowest-unfolding free energy substructure of the cytochrome c fold. A 1.36 Å resolution X-ray structure of the Y48H variant reveals minimal structural changes compared to the wild-type structure, with the axial Met80 ligand coordinated to the heme iron. Despite this, the intrinsic peroxidase activity is enhanced, implying that a pentacoordinate heme state is more prevalent in the Y48H variant, corroborated through determination of a Met80 "off rate" of >125 s-1 compared to a rate of ∼6 s-1 for the wild-type protein. Heteronuclear nuclear magnetic resonance measurements with the oxidized Y48H variant reveal heightened dynamics in the 40-57 Ω-loop and the Met80-containing 71-85 Ω-loop relative to the wild-type protein, illustrating communication between these substructures. Placed into context with the G41S cytochrome c variant, also implicated in thrombocytopenia, a dynamic picture associated with this disease relative to cytochrome c is emerging whereby increasing dynamics in substructures of the cytochrome c fold serve to facilitate an increased population of the peroxidatic pentacoordinate heme state in the following order: wild type < G41S < Y48H.

Interactive effects of zinc and nickel on the glutathione system state in Mimulus guttatus plants

To determine whether the enhanced stress tolerance of ZnSO4 with NiSO4-treated Mimulus guttatus Fischer ex DC. plants was associated with the glutathione (GR-GSH) system, we investigated the changes in glutathione redox state (reduced (GSH), oxidized (GSSG) forms, total reduced (GSHt) glutathione, and GSH/GSSG ratio) and in the enzymatic activities of glutathione reductase (GR) and peroxidatic glutathione S-transferases (GST). The 6-week-old plants were grown in water culture during 4 weeks on a modified Rorison’s medium with ZnSO4 (50, 100, and 200 μM) and NiSO4 (20 and 80 μM) in a condition of separate or simultaneous supply of the components. Dry biomass accumulations of roots and shoots were not influenced by the examined treatments. The positive correlations between the total external concentrations of ZnSO4 and NiSO4 and the total Zn and Ni contents in roots and leaves were found. It was determined that the MDA content was higher in the ZnSO4-treated plants than in the NiSO4-treated ones. The supplementation of the ZnSO4-treated plants with varied concentrations of NiSO4 decreased the Zn-induced increase in the MDA levels. The inverse proportionality between the MDA and pigment levels in leaves was found. The Zn-Ni interactions were shown to induce the decreases in the GR activity, the total peroxidatic GST activity, and the GSH/GSSG ratio in roots. However, in leaves, the GR activity and the GSH/GSSG ratio were significantly increased and the total peroxidatic GST activity was decreased. The supplementation of the ZnSO4-treated plants with varied concentrations of NiSO4 restored the Zn-induced reduction in the GSHt levels in roots and decreased the Zn-induced increase in the GSSG levels in leaves, which resulted in more reduced state of the intracellular environment. It was likely to cause a decrease of the MDA level. Thus, our studies on the Zn−Ni interactions identified the antagonizing role of Ni in Zn toxicity by the GR-GSH system.

Heterologous expression and characterization of a new heme-catalase in Bacillus subtilis 168.

Reactive oxygen species (ROS) is an inherent consequence to all aerobically living organisms that might lead to the cells being lethal and susceptible to oxidative stress. Bacillus pumilus is characterized by high-resistance oxidative stress that stimulated our interest to investigate the heterologous expression and characterization of heme-catalase as potential biocatalyst. Results indicated that recombinant enzyme significantly exhibited the high catalytic activity of 55,784U/mg expressed in Bacillus subtilis 168 and 98.097µmol/min/mg peroxidatic activity, the apparent K m of catalytic activity was 59.6±13mM with higher turnover rate (K cat=322.651×10(3)s(-1)). The pH dependence of catalatic and peroxidatic activity was pH 7.0 and pH 4.5 respectively with temperature dependence of 40°C and the recombinant heme-catalase exhibited a strong Fe(2+) preference. It was further revealed that catalase KatX2 improved the resistance oxidative stress of B. subtilis. These findings suggest that this B. pumilus heme-catalase can be considered among the industrially relevant biocatalysts due to its exceptional catalytic rate and high stability and it can be a potential candidate for the improvement of oxidative resistance of industrially produced strains.

Effect of the distal histidine on the peroxidatic activity of monomeric cytoglobin

The reaction of hydrogen peroxide with ferric human cytoglobin and a number of distal histidine variants were studied. The peroxidase activity of the monomeric wildtype protein with an internal disulfide bond, likely to be the form of the protein in vivo, exhibits a high peroxidase-like activity above that of other globins such as myoglobin. Furthermore, the peroxidatic activity of wildtype cytoglobin shows increased resistance to radical-based degradation compared to myoglobin. The ferryl form of wildtype cytoglobin is unstable, but is able to readily oxidize substrates such as guaiacol. In contrast distal histidine mutants of cytoglobin (H81Y and H81V) show very low peroxidase activity but enhanced radical-induced degradation. Therefore, the weakly bound distal histidine appears to modulate ferryl stability and limit haem degradation. These data are consistent with a role of a peroxidase activity of cytoglobin in cell stress response mechanisms.

The Corynebacterium glutamicum mycothiol peroxidase is a reactive oxygen species-scavenging enzyme that shows promiscuity in thiol redox control.

Cysteine glutathione peroxidases (CysGPxs) control oxidative stress levels by reducing hydroperoxides at the expense of cysteine thiol (-SH) oxidation, and the recovery of their peroxidatic activity is generally accomplished by thioredoxin (Trx). Corynebacterium glutamicum mycothiol peroxidase (Mpx) is a member of the CysGPx family. We discovered that its recycling is controlled by both the Trx and the mycothiol (MSH) pathway. After H2 O2 reduction, a sulfenic acid (-SOH) is formed on the peroxidatic cysteine (Cys36), which then reacts with the resolving cysteine (Cys79), forming an intramolecular disulfide (S-S), which is reduced by Trx. Alternatively, the sulfenic acid reacts with MSH and forms a mixed disulfide. Mycoredoxin 1 (Mrx1) reduces the mixed disulfide, in which Mrx1 acts in combination with MSH and mycothiol disulfide reductase as a biological relevant monothiol reducing system. Remarkably, Trx can also take over the role of Mrx1 and reduce the Mpx-MSH mixed disulfide using a dithiol mechanism. Furthermore, Mpx is important for cellular survival under H2 O2 stress, and its gene expression is clearly induced upon H2 O2 challenge. These findings add a new dimension to the redox control and the functioning of CysGPxs in general.

Effect of the distal histidine on the peroxidatic activity of monomeric cytoglobin.

The reaction of hydrogen peroxide with ferric human cytoglobin and a number of distal histidine variants were studied. The peroxidase activity of the monomeric wildtype protein with an internal disulfide bond, likely to be the form of the protein in vivo, exhibits a high peroxidase-like activity above that of other globins such as myoglobin. Furthermore, the peroxidatic activity of wildtype cytoglobin shows increased resistance to radical-based degradation compared to myoglobin. The ferryl form of wildtype cytoglobin is unstable, but is able to readily oxidize substrates such as guaiacol. In contrast distal histidine mutants of cytoglobin (H81Y and H81V) show very low peroxidase activity but enhanced radical-induced degradation. Therefore, the weakly bound distal histidine appears to modulate ferryl stability and limit haem degradation. These data are consistent with a role of a peroxidase activity of cytoglobin in cell stress response mechanisms.

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 (kcat = 339,000 s(-1) ). In addition, the enzyme supported a much slower (kcat = 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.

A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is sensitive to reversible oxidative inactivation by hydrogen peroxide (H2O2). Here we show that H2O2 reactivity of the active site thiolate (C152) is catalyzed by a previously unrecognized mechanism based on a dedicated proton relay promoting leaving group departure. Disruption of the peroxidatic reaction mechanism does not affect the glycolytic activity of GAPDH. Therefore, specific and separate mechanisms mediate the reactivity of the same thiolate nucleophile toward H2O2 and glyceraldehyde 3-phosphate, respectively. The generation of mutants in which the glycolytic and peroxidatic activities of GAPDH are comprehensively uncoupled allowed for a direct assessment of the physiological relevance of GAPDH H2O2 sensitivity. Using yeast strains in which wild-type GAPDH was replaced with H2O2-insensitive mutants retaining full glycolytic activity, we demonstrate that H2O2 sensitivity of GAPDH is a key component of the cellular adaptive response to increased H2O2 levels.

Independent evolution of four heme peroxidase superfamilies

Four heme peroxidase superfamilies (peroxidase–catalase, peroxidase–cyclooxygenase, peroxidase–chlorite dismutase and peroxidase–peroxygenase superfamily) arose independently during evolution, which differ in overall fold, active site architecture and enzymatic activities. The redox cofactor is heme b or posttranslationally modified heme that is ligated by either histidine or cysteine. Heme peroxidases are found in all kingdoms of life and typically catalyze the one- and two-electron oxidation of a myriad of organic and inorganic substrates. In addition to this peroxidatic activity distinct (sub)families show pronounced catalase, cyclooxygenase, chlorite dismutase or peroxygenase activities. Here we describe the phylogeny of these four superfamilies and present the most important sequence signatures and active site architectures. The classification of families is described as well as important turning points in evolution. We show that at least three heme peroxidase superfamilies have ancient prokaryotic roots with several alternative ways of divergent evolution. In later evolutionary steps, they almost always produced highly evolved and specialized clades of peroxidases in eukaryotic kingdoms with a significant portion of such genes involved in coding various fusion proteins with novel physiological functions.

Specific methionine oxidation of cytochrome c in complexes with zwitterionic lipids by hydrogen peroxide: potential implications for apoptosis.

Cytochrome c (Cyt-c) has been previously shown to participate in cardiolipin (CL) oxidation and, therefore, in mitochondrial membrane permeabilization during the early events of apoptosis. The gain in this function has been ascribed to specific CL/Cyt-c interactions. Here we report that the cationic protein Cyt-c is also able to interact electrostatically with the main lipid components of the mitochondrial membranes, the zwitterionic lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE), through the mediation of phosphate anions that bind specifically to amino groups in the surfaces of protein and model membranes. In these complexes, Cyt-c reacts efficiently with H2O2 at submillimolar levels, which oxidizes the sulfur atom of the axial ligand Met80. The modified protein is stable and presents significantly enhanced peroxidatic activity. Based on these results, we postulate that the rise of H2O2 concentrations to the submillimolar levels registered during initiation of the apoptotic program may represent one signaling event that triggers the gain in peroxidatic function of the Cyt-c molecules bound to the abundant PE and PC membrane components. As the activated protein is a chemically stable species, it can potentially bind and oxidize important targets, such as CL.

Structural insight into the function and anti-TB pro-drug activation by KatG

Catalase-peroxidase (KatG) is a dual functional enzyme with both catalase and peroxidase activity. KatGs protect aerobic microorganisms from oxidative damage through their high catalase activity, and in Mycobacterium tuberculosis (M.tb.) KatG's peroxidatic activity is central in converting the anti-tuberculosis (TB) pro-drug isoniazid (INH) into an active bactericidal molecule in vivo. There are several antibiotics currently in use to treat TB with INH being one of the first anti-TB agents. A central goal is to understand the catalytic function of M.tb. KatG in drug activation and how mutations in KatG found in INH-resistant strains interfere with this process. One of the most common INH-resistant M.tb. strains has the KatG[Ser315Thr] mutation, previously reported to cause narrowing of a substrate access channel. We have now solved the structure of another mutant [Asp137Ser], which shows enhanced INH-activation ascribed to an enlarged access channel. This study demonstrates that altering the dimensions of the bottleneck in the substrate access channel in KatG can impede or enhance INH peroxidation rates relative to the WT enzyme.

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

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