Structure of coenzyme F420H2 oxidase (FprA), a di-iron flavoprotein from methanogenic Archaea catalyzing the reduction of O2 to H2O

The di-iron flavoprotein F(420)H(2) oxidase found in methanogenic Archaea catalyzes the four-electron reduction of O(2) to 2H(2)O with 2 mol of reduced coenzyme F(420)(7,8-dimethyl-8-hydroxy-5-deazariboflavin). We report here on crystal structures of the homotetrameric F(420)H(2) oxidase from Methanothermobacter marburgensis at resolutions of 2.25 A, 2.25 A and 1.7 A, respectively, from which an active reduced state, an inactive oxidized state and an active oxidized state could be extracted. As found in structurally related A-type flavoproteins, the active site is formed at the dimer interface, where the di-iron center of one monomer is juxtaposed to FMN of the other. In the active reduced state [Fe(II)Fe(II)FMNH(2)], the two irons are surrounded by four histidines, one aspartate, one glutamate and one bridging aspartate. The so-called switch loop is in a closed conformation, thus preventing F(420) binding. In the inactive oxidized state [Fe(III)FMN], the iron nearest to FMN has moved to two remote binding sites, and the switch loop is changed to an open conformation. In the active oxidized state [Fe(III)Fe(III)FMN], both irons are positioned as in the reduced state but the switch loop is found in the open conformation as in the inactive oxidized state. It is proposed that the redox-dependent conformational change of the switch loop ensures alternate complete four-electron O(2) reduction and redox center re-reduction. On the basis of the known Si-Si stereospecific hydride transfer, F(420)H(2) was modeled into the solvent-accessible pocket in front of FMN. The inactive oxidized state might provide the molecular basis for enzyme inactivation by long-term O(2) exposure observed in some members of the FprA family.     

Stability of globular proteins in H2O and D2O

In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the stability of globular protein structure is determined in a systematic way. The differential scanning calorimetry technique is applied to allow for a thermodynamic analysis of two types of globular proteins: hen's egg lysozyme (LSZ) with relatively strong internal cohesion ("hard" globular protein) and bovine serum albumin (BSA), which is known for its conformational adaptability ("soft" globular protein). Both proteins tend to be more stable in D2O compared to H2O. We explain the increase of protein stability in D2O by the observation that D2O is a poorer solvent for nonpolar amino acids than H2O, implying that the hydrophobic effect is larger in D2O. In case of BSA the transitions between different isomeric forms, at low pH values the Nm and F forms, and at higher pH values Nm and B, were observed by the presence of a supplementary peak in the DSC thermogram. It appears that the pH-range for which the Nm form is the preferred one is wider in D2O than in H2O.     

Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2.

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.     

Irreversible cross-linking of heme to the distal tryptophan of stromal ascorbate peroxidase in response to rapid inactivation by H2O2

Ascorbate peroxidase (APX) isoforms localized in the stroma and thylakoid membrane of chloroplasts play a central role in scavenging reactive oxygen species generated by photosystems. These enzymes are inactivated within minutes by H2O2 when the reducing substrate, ascorbate, is depleted. We found that, when the enzyme is inactivated by H2O2, a heme at the catalytic site of a stromal APX isoform is irreversibly cross-linked to a tryptophan residue facing the distal cavity. Mutation of this tryptophan to phenylalanine abolished the cross-linking and increased the half-time for inactivation from <10 to 62 s. In contrast with H2O2-tolerant peroxidases, rapid formation of the cross-link in APXs suggests that a radical in the reaction intermediate tends to be located in the distal tryptophan so that heme is easily cross-linked to it. This is the first report of a mutation that improves the tolerance of chloroplast APXs to H2O2.     

Decrease of H2O2 plasma membrane permeability during adaptation to H2O2 in Saccharomyces cerevisiae

Contrary to what is widely believed, recent published results show that H2O2 does not freely diffuse across biomembranes. The fast removal of H2O2 by antioxidant enzymes is able to generate a gradient if H2O2 is produced in a different compartment from that containing the enzymes (Antunes, F., and Cadenas, E. (2000) FEBS Lett. 475, 121-126). In this work, we extended these studies and tested whether an active regulation of biomembranes permeability characteristics is part of the cell response to oxidative stress. Using Saccharomyces cerevisiae as a model, we showed that: (a) H2O2 gradients across the plasma membrane are formed upon exposure to external H2O2; (b) there is a correlation between the magnitude of the gradients and the resistance to H2O2; (c) there is not a correlation between the intracellular capacity to remove H2O2 and the resistance to H2O2; (d) the plasma membrane permeability to H2O2 decreases by a factor of two upon acquisition of resistance to this agent by pre-exposing cells either to nonlethal doses of H2O2 or to cycloheximide, an inhibitor of protein synthesis; and (e) erg3Delta and erg6Delta mutants, which have impaired ergosterol biosynthesis pathways, show higher plasma membrane permeability to H2O2 and are more sensitive to H2O2. Altogether, the regulation of the plasma membrane permeability to H2O2 emerged as a new mechanism by which cells respond and adapt to H2O2. The consequences of the results to cellular redox compartmentalization and to the origin and evolution of the eukaryotic cell are discussed.     

Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H2O2 generation and the influence of Mn2+.

Formation of H2O2 during the oxidation of three lignin-derived hydroquinones by the ligninolytic versatile peroxidase (VP), produced by the white-rot fungus Pleurotus eryngii, was investigated. VP can oxidize a wide variety of phenols, including hydroquinones, either directly in a manner similar to horseradish peroxidase (HRP), or indirectly through Mn3+ formed from Mn2+ oxidation, in a manner similar to manganese peroxidase (MnP). From several possible buffers (all pH 5), tartrate buffer was selected to study the oxidation of hydroquinones as it did not support the Mn2+-mediated activity of VP in the absence of exogenous H2O2 (unlike glyoxylate and oxalate buffers). In the absence of Mn2+, efficient hydroquinone oxidation by VP was dependent on exogenous H2O2. Under these conditions, semiquinone radicals produced by VP autoxidized to a certain extent producing superoxide anion radical (O2*-) that spontaneously dismutated to H2O2 and O2. The use of this peroxide by VP produced quinone in an amount greater than equimolar to the initial H2O2 (a quinone/H2O2 molar ratio of 1 was only observed under anaerobic conditions). In the presence of Mn2+, exogenous H2O2 was not required for complete oxidation of hydroquinone by VP. Reaction blanks lacking VP revealed H2O2 production due to a slow conversion of hydroquinone into semiquinone radicals (probably via autooxidation catalysed by trace amounts of free metal ions), followed by O2*- production through semiquinone autooxidation and O2*- reduction by Mn2+. This peroxide was used by VP to oxidize hydroquinone that was mainly carried out through Mn2+ oxidation. By comparing the activity of VP to that of MnP and HRP, it was found that the ability of VP and MnP to oxidize Mn2+ greatly increased hydroquinone oxidation efficiency.     

Inactivation in vivo of basic peroxidase and increased content of H2O2 in grapevine leaves post treatment with DTT and paraquat

Substantial differences in the in vivo effect of paraquat (Pq) and DTT on basic peroxidase (GBPx) activity and on H2O2 levels were found in grapevine leaves cv. Sultana. GBPx activity decreased and H2O2 levels increased in illuminated Pq treated leaf-discs. Inactivation of GBPx and accumulation of H2O2 depended on the duration and intensity of the illumination to which discs were exposed. Since GBPx was inactivated directly by H2O2 and not by Pq in leaf extracts, and since GBPx are cytosolic isoenzymes and H2O2 is a stable molecule that can easily permeate chloroplast membranes, we concluded that Pq inactivation of GBPx in vivo is mediated by H2O2. In contrast to the effect induced by Pq, DTT directly inactivated GBPx in leaf extracts. In leaf-discs, however, it reduced GBPx activity in the absence of light, although the levels of H2O2 increased only after exposure of the discs to high irradiance, suggesting that under excess of light, a significant fraction of the photosynthetically produced electrons are dissipated through the water-water cycle and H2O2 accumulates as a consequence of GBPx inactivation.     

Prevention of H2O2 generation by monoamine oxidase protects against CNS O2 toxicity.

Toxicity to the central nervous system (CNS) by hyperbaric oxygen (HBO) presumably relates to increased production of reactive oxygen species. The sites of generation of reactive oxygen species during HBO, however, have not been fully characterized in the brain. We investigated the relationship between regional generation of hydrogen peroxide (H2O2) in the brain in the presence of an irreversible inhibitor of catalase, aminotriazole (ATZ), and protection from CNS O2 toxicity by a monoamine oxidase (MAO) inhibitor, pargyline. At 6 ATA of oxygen, pargyline significantly protected rats from CNS O2 toxicity whereas ATZ enhanced O2 toxicity. In animals pretreated with ATZ, HBO inactivated 21-40% more catalase than air exposure in the six brain regions studied. Because ATZ-mediated inactivation of catalase was H2O2 dependent, the decrease in catalase activity during hyperoxia was proportional to the intracellular production of H2O2. Pargyline, administered 30 min before HBO, inhibited MAO by greater than 90%, prevented ATZ inhibition of catalase activity during HBO, and reversed the augmentation of CNS O2 toxicity by ATZ. These findings indicate that H2O2 generated by MAO during hyperoxia is important to the pathogenesis of CNS O2 toxicity in rats.     

H2O2-Induced Inhibition of Photosynthetic O2 Evolution by Anabaena variabilis Cells

Hydrogen peroxide inhibits photosynthetic O2 evolution. It has been shown that H2O2 destroys the function of the oxygen-evolving complex (OEC) in some chloroplast and Photosystem (PS) II preparations causing release of manganese from the OEC. In other preparations, H2O2 did not cause or caused only insignificant release of manganese. In this work, we tested the effect of H2O2 on the photosynthetic electron transfer and the state of OEC manganese in a native system (intact cells of the cyanobacterium Anabaena variabilis). According to EPR spectroscopy data, H2O2 caused an increase in the level of photooxidation of P700, the reaction centers of PS I, and decreased the rate of their subsequent reduction in the dark by a factor larger than four. Combined effect of H2O2, CN-, and EDTA caused more than eight- to ninefold suppression of the dark reduction of P700+. EPR spectroscopy revealed that the content of free (or loosely bound) Mn2+ in washed cyanobacterial cells was ~20% of the total manganese pool. This content remained unchanged upon the addition of CN- and increased to 25-30% after addition of H2O2. The content of the total manganese decreased to 35% after the treatment of the cells with EDTA. The level of the H2O2-induced release of manganese increased after the treatment of the cells with EDTA. Incubation of cells with H2O2 for 2 h had no effect on the absorption spectra of the photosynthetic pigments. More prolonged incubation with H2O2 (20 h) brought about degradation of phycobilins and chlorophyll a and lysis of cells. Thus, H2O2 causes extraction of manganese from cyanobacterial cells, inhibits the OEC activity and photosynthetic electron transfer, and leads to the destruction of the photosynthetic apparatus. H2O2 is unable to serve as a physiological electron donor in photosynthesis.     

Salicylic acid-induced inactivation of creatine kinase in the presence of lactoperoxidase and H2O2

To clarify one mechanism of aspirin-induced gastric mucosal damage, inactivation of creatine kinase (CK) by salicylic acid that is easily produced from aspirin in vivo was examined in the presence of lactoperoxidase (LPO) and H2O2 (LPO-H2O2). Salicylic acid inactivated CK (rabbit muscle) during its interaction with LPO-H2O2. CK activity in gastric mucosal homogenate decreased dependent on the concentration of salicylic acid in the presence of LPO-H2O2. Oxygen radical scavengers did not prevent the inactivation of CK. Direct detection of free radicals of salicylic acid by electron spin resonance was unsuccessful. However, glutathionyl radicals were formed during the interaction of salicylic acid with LPO-H2O2 in the presence of reduced glutathione and 5,5-dimethyl-1-pyrroline oxide as a spin trap agent. Among salicylic acid-related drugs, salsalate, but not aspirin and ethenzamide, inactivated CK, indicating the phenolic hydroxyl group is oxidized by LPO-H2O2. During oxidation of salicylic acid by LPO-H2O2, the sulfhydryl group in CK markedly decreased, and salicylic acid bound to CK. These results indicate that CK was inactivated through loss of the sulfhydryl group and binding of salicylic acid.     

Role of glutathione in the adaptive tolerance to H2O2

Endogenous antioxidant defense systems are enhanced by various physiological stimuli including sublethal oxidative challenges, which induce tolerance to subsequent lethal oxidative injuries. We sought to evaluate the contributions of catalase and the glutathione system to the adaptive tolerance to H2O2. For this purpose, H9c2 cells were stimulated with 100 microM H2O2, which was the maximal dose at which no significant acute cell damage was observed. Twenty-four hours after stimulation, control and pretreated cells were challenged with a lethal concentration of H2O2 (300 microM). Compared with the control cells, pretreated cells were significantly tolerant of H2O2, with reduced cell lysis and improved survival rate. In pretreated cells, glutathione content increased to 48.20 +/- 6.38 nmol/mg protein versus 27.59 +/- 2.55 nmol/mg protein in control cells, and catalase activity also increased to 30.82 +/- 2.64 versus 15.46 +/- 1.29 units/mg protein in control cells, whereas glutathione peroxidase activity was not affected. Increased glutathione content was attributed to increased gamma-glutamylcysteine synthetase activity, which is known as the rate-limiting enzyme of glutathione synthesis. To elucidate the relative contribution of the glutathione system and catalase to tolerance of H2O2, control and pretreated cells were incubated with specific inhibitors of gamma-glutamyl cysteine synthetase (L-buthionine sulfoximine) or catalase (3-amino-1,2,4-triazole), and challenged with H2O2. Cytoprotection by the low-dose H2O2 pretreatment was almost completely abolished by L-buthionine sulfoximine, while it was preserved after 3-amino-1,2,4-triazole treatment. From these results, it is concluded that both the glutathione system and catalase can be enhanced by H2O2 stimulation, but increased glutathione content rather than catalase activity was operative in the tolerance of lethal oxidative stress.     

Mechanism of horseradish peroxidase-catalyzed conversion of iodine to iodide in the presence of EDTA and H2O2

EDTA not only blocks the horseradish peroxidase (HRP)-catalyzed iodide oxidation to I-3 but also causes an enzymatic conversion of oxidized iodine species to iodide (Banerjee, R. K., De, S. K., Bose, A. K., and Datta, A. G. (1986) J. Biol. Chem. 261, 10592-10597). The EDTA effect on both of these reactions can be withdrawn with a higher concentration of iodide and not with H2O2. Spectral studies indicate a possible interaction of EDTA with HRP as evidenced by the formation of modified compound 1 with H2O2 at 416 nm instead of 412 nm in the absence of EDTA. EDTA causes a hypochromic effect on HRP at 402 nm which undergoes the bathochromic red shift to 416 nm by H2O2. The addition of iodide to the 416 nm complex causes the reappearance of the Soret band of HRP at 402 nm. Among various EDTA analogues tested, N-N-N'-N'-tetramethylethylenediamine (TEMED) is 80% as effective as EDTA in the conversion of I-3 to iodide and produces a spectral shift of HRP similar to EDTA. Interaction of EDTA with HRP is further indicated by the hyperchromic effect of HRP and H2O2 on the absorption of EDTA at 212 nm. The addition of oxidized iodine species produces a new peak at 230 nm due to formation of iodide. EDTA at a higher concentration can effectively displace radioiodide specifically bound to HRP indicating its interaction at the iodide-binding site. The enzyme, after radioiodide displacement with EDTA, shows a characteristic absorption maximum at 416 nm on the addition of H2O2, indicating that EDTA is bound with the enzyme. Both positive and negative circular dichroism spectra of HRP and the HRP.H2O2 complex, characteristic of heme absorption, are altered by EDTA, suggesting an EDTA-induced conformational change at or near the heme region. This is associated with a change of affinity of heme toward H2O2 and azide. It is postulated that EDTA interacts at the iodide-binding site of the HRP inducing a new conformation that blocks iodide oxidation but is suitable to convert iodine to iodide by a redox reaction with H2O2.     

Laccase-catalyzed oxidation of Mn(2+) in the presence of natural Mn(3+) chelators as a novel source of extracellular H(2)O(2) production and its impact on manganese peroxidase

A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn(2+) to Mn(3+), as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn(3+) complexes were produced at 0.15, 0.05, and 0.10 micromol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H(2)O(2) formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H(2)O(2) production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn(2+) oxidation and abiotic decomposition of these organic chelators by the resulting Mn(3+), which leads to formation of superoxide and its subsequent reduction to H(2)O(2). A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn(3+) complexes in assays containing 1 mM Mn(2+) and 100 mM oxalate or malonate, but omitting an additional H(2)O(2) source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn(2+) oxidation in providing H(2)O(2) for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.     

Hemoglobin autoxidation and regulation of endogenous H2O2 levels in erythrocytes

Red cells from mice deficient in glutathione peroxidase-1 were used to estimate the hemoglobin autoxidation rate and the endogenous level of H2O2 and superoxide. Methemoglobin and the rate of catalase inactivation by 3-amino-2,4,5-triazole (3-AT) were determined. In contrast with iodoacetamide-treated red cells, catalase was not inactivated by 3-AT in glutathione peroxidase-deficient erythrocytes. Kinetic models incorporating reactions known to involve H2O2 and superoxide in the erythrocyte were used to estimate H2O2, superoxide, and methemoglobin levels. The experimental data could not be modeled unless the intraerythrocytic concentration of Compound I is very low. Two additional models were tested. In one, it was assumed that a rearranged Compound I, termed Compound II*, does not react with 3-AT. However, experiments with an NADPH-generating system provided evidence that this mechanism does not occur. A second model that explicitly includes peroxiredoxin II can fit the experimental findings. Insertion of the data into the model predicted a hemoglobin autoxidation rate constant of 4.5 x 10(-7) s(-1) and an endogenous H2O2 and superoxide concentrations of 5 x 10(-11) and 5 x 10(-13) M, respectively, lower than previous estimates.     

Direct detection of Fe(IV)[double bond]O intermediates in the cytochrome aa3 oxidase from Paracoccus denitrificans/H2O2 reaction

We report the first evidence for the formation of the "607- and 580-nm forms" in the cytochrome oxidase aa3/H2O2 reaction without the involvement of tyrosine 280. The pKa of the 607-580-nm transition is 7.5. The 607-nm form is also formed in the mixed valence cytochrome oxidase/O2 reaction in the absence of tyrosine 280. Steady-state resonance Raman characterization of the reaction products of both the wild-type and Y280H cytochrome aa3 from Paracoccus denitrificans indicate the formation of six-coordinate low spin species, and do not support, in contrast to previous reports, the formation of a porphyrin pi-cation radical. We observe three oxygen isotope-sensitive Raman bands in the oxidized wild-type aa3/H2O2 reaction at 804, 790, and 358 cm-1. The former two are assigned to the Fe(IV)[double bond]O stretching mode of the 607- and 580-nm forms, respectively. The 14 cm-1 frequency difference between the oxoferryl species is attributed to variations in the basicity of the proximal to heme a3 His-411, induced by the oxoferryl conformations of the heme a3-CuB pocket during the 607-580-nm transition. We suggest that the 804-790 cm-1 oxoferryl transition triggers distal conformational changes that are subsequently communicated to the proximal His-411 heme a3 site. The 358 cm-1 mode has been found for the first time to accumulate with the 804 cm-1 mode in the peroxide reaction. These results indicate that the mechanism of oxygen reduction must be reexamined.     

New dimanganese(III) complexes of pentadentate (N2O3) Schiff base ligands with the [Mn2(mu-OAc)(mu-OR)2]3+ core: synthesis, characterization and mechanistic studies of H2O2 disproportionation

Two new diMn(III) complexes [Mn(2)(III)L(1)(mu-AcO)(mu-MeO)(methanol)(2)]Br (1) and [Mn(2)(III)L(2)(mu-AcO)(mu-MeO)(methanol)(ClO(4))] (2) (L(1)H(3)=1,5-bis(2-hydroxybenzophenylideneamino)pentan-3-ol; L(2)H(3)=1,5-bis(2-hydroxynaphtylideneamino)pentan-3-ol) were synthesized and structurally characterized. Structural studies evidence that these complexes have a bis(mu-alkoxo)(mu-carboxylato) triply bridged diMn(III) core in the solid state and in solution, with two substitution-labile sites--one on each Mn ion--in cis-position. The two complexes show catalytic activity toward disproportionation of H(2)O(2), with saturation kinetics on [H(2)O(2)], in methanol and dimethyl formamide at 25 degrees C. Spectroscopic monitoring of the H(2)O(2) disproportionation reaction suggests that (i) complexes 1 and 2 dismutate H(2)O(2) by a mechanism involving redox cycling between Mn(2)(III) and Mn(2)(IV), (ii) the complexes retain the dinuclearity during catalysis, (iii) the active form of the catalyst contains bound acetate, and (iv) protons favors the formation of inactive Mn(II) species. Comparison to other dimanganese complexes of the same family shows that the rate of catalase reaction is not critically dependent on the redox potential of the catalyst, that substitution of phenolate by naphtolate in the Schiff base ligand favors formation of the catalyst-substrate adduct, and that, in the non-protic solvent, the bulkier substituent at the imine proton position hampers the binding to the substrate.     

Superoxide dismutases enhance H2O2-induced DNA damage and alter its site specificity

Superoxide dismutases (SODs) are involved in the protection of cells from oxygen toxicity. However, several papers have reported that the overexpression of CuZn-SOD causes oxidative damage to cells. We investigated a mechanism by which an excess of SODs accelerates oxidative stress. The presence of CuZn-SOD, Mn-SOD or Mn(II) enhanced the frequency of DNA damage induced by hydrogen peroxide (H2O2) and Cu(II), and altered the site specificity of the latter: H2O2 induced Cu(II)-dependent DNA damage with high frequency at the 5'-guanine of poly G sequences; when SODs were added, the frequency of cleavages at thymine and cytosine residues increased. SODs also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by H2O2 and Cu(II). We conclude that SODs may increase carcinogenic risks, e.g. of tumors in Down syndrome.     

On the molecular mechanism of lactoperoxidase-catalyzed H2O2 metabolism and irreversible enzyme inactivation

Lactoperoxidase-catalyzed H2O2 metabolism proceeds through one of three different pathways, depending on the nature and the concentration of the second substrate as an e- donor and/or on pH conditions. In the lactoperoxidase (LPO)-H2O2 system, at low H2O2 concentrations and/or alkaline conditions the peroxidatic cycle involves ferric LPO----compound I----compound II----ferric LPO conversion, whereas high H2O2 concentrations and/or acidic conditions favor the ferric LPO----compound I----compound II----compound III----ferrous LPO----ferric LPO pathway. The compound III/ferroperoxidase states are associated with irreversible enzyme inactivation by cleavage of the heme moiety and liberation of iron. It is likely that either singlet oxygen or superoxide and hydroxyl radicals are involved in the attack on heme iron, because inactivation correlates with oxygen production and can be decreased to a certain degree by scavengers such as ethanol, 1-propanol, 2-propanol, or mannitol. In the LPO-H2O2-I- system, the enzyme may also be inactivated by I2 generated in the course of enzymatic I- oxidation (i.e. during ferric LPO----compound I----ferric LPO cycles).     

Feedback control of adrenal steroidogenesis via H2O2-dependent, reversible inactivation of peroxiredoxin III in mitochondria

Certain members of the peroxiredoxin (Prx) family undergo inactivation through hyperoxidation of the catalytic cysteine to sulfinic acid during catalysis and are reactivated by sulfiredoxin; however, the physiological significance of this reversible regulatory process is unclear. We now show that PrxIII in mouse adrenal cortex is inactivated by H(2)O(2) produced by cytochrome P450 enzymes during corticosterone production stimulated by adrenocorticotropic hormone. Inactivation of PrxIII triggers a sequence of events including accumulation of H(2)O(2), activation of p38 mitogen-activated protein kinase, suppression of steroidogenic acute regulatory protein synthesis, and inhibition of steroidogenesis. Interestingly, levels of inactivated PrxIII, activated p38, and sulfiredoxin display circadian oscillations. Steroidogenic tissue-specific ablation of sulfiredoxin in mice resulted in the persistent accumulation of inactive PrxIII and suppression of the adrenal circadian rhythm of corticosterone production. The coupling of CYP11B1 activity to PrxIII inactivation provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis.     

Significance of catalase in peroxisomal fatty acyl-CoA beta-oxidation: NADH oxidation by acetoacetyl-CoA and H2O2

To clarify the significance of catalase in peroxisomes, we have examined the effect of aminotriazole treatment of rats on the activity of beta-hydroxybutyryl-CoA dehydrogenase in liver peroxisomes. When the effect of H2O2 on the dehydrogenase activity was examined using an extract of liver peroxisomes from aminotriazole-treated rats, the acetoacetyl-CoA-dependent oxidation of NADH was found to increase considerably on the addition of dilute H2O2. Such an effect of H2O2 was not seen on the beta-hydroxybutyryl-CoA-dependent reduction of NAD nor with extracts from untreated animals. We then noticed that similar NADH oxidation was caused non-enzymatically by a mixture of acetoacetyl-CoA and H2O2. The oxidation was dependent on both acetoacetyl-CoA and H2O2, and was blocked by scavengers of oxyradicals such as ascorbate and ethanol. Degradation products formed during the reaction of acetoacetyl-CoA with H2O2 had no NADH oxidizing activity, indicating that effective oxidant(s) were generated during the reaction of H2O2 with acetoacetyl-CoA. No other fatty acyl-CoA so far examined nor acetoacetate could replace acetoacetyl-CoA in this reaction. Therefore, if H2O2 were to be accumulated in peroxisomes, it would decrease both NADH and acetoacetyl-CoA, thus affecting the fatty acyl-CoA beta-oxidation system. These results, together with our previous finding that peroxisomal thiolase was significantly inactivated by H2O2 [Hashimoto, F. & Hayashi, H. (1987) Biochim. Biophys. Acta 921, 142-150] suggest that the role of catalase in peroxisomes is at least in part to protect the fatty acyl-CoA beta-oxidation system from the deleterious action of H2O2.