A novel activity for fungal nitronate monooxygenase: detoxification of the metabolic inhibitor propionate-3-nitronate

Nitronate monooxygenase (NMO; E.C. 1.13.12.16) oxidizes alkyl nitronates to aldehydes and nitrite. Although the biochemistry of the enzyme from fungal sources has been studied extensively, the physiological role is unknown. The ability of NMO to detoxify propionate-3-nitronate was tested by measuring growth of recombinant Escherichia coli containing the gene encoding for the enzyme in either the absence or presence of the nitronate and its conjugate acid 3-nitropropionate. The mixture propionate-3-nitronate/3-nitropropionate is toxic to E. coli cells lacking expression of NMO, but the toxicity is overcome through either induction of the gene for NMO or through addition of exogenous enzyme to the cultures. Both Williopsis saturnus and Neurospora crassa were able to grow in the presence of 0.4mM propionate-3-nitronate and 19.6mM 3-nitropropionate, while a knockout mutant of N. crassa lacking NMO was inhibited by concentrations of propionate-3-nitronate and 3-nitropropionate >0.3 and 600μM, respectively. These results strongly support the conclusion that NMO functions to protect the fungi from the environmental occurrence of the metabolic toxin.     

The kinetic mechanism of beef kidney D-aspartate oxidase

The mechanism of action of the flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been investigated by steady-state and stopped flow kinetic studies using D-aspartate and O2 as substrates in 50 mM KPi, 0.3 mM EDTA, pH 7.4, 4 degrees C. Steady-state results indicate that a ternary complex containing enzyme, O2, and substrate (or product) is an obligatory intermediate in catalysis. The kinetic parameters are turnover number = 11.1 s-1, Km(D-Asp) = 2.2 x 10(-3) M, Km(O2) = 1.7 x 10(-4) M. Rapid reaction studies show that 1) the reductive half reaction is essentially irreversible with a maximum rate of reduction of 180 s-1; 2) the free reduced enzyme cannot be the species which is reoxidized during turnover since its reoxidation by oxygen (second order rate constant equal to 5.3 x 10(2) M-1 s-1) is too slow to be of relevance in catalysis; 3) reduced enzyme can bind a ligand rapidly and be reoxidized as a complex at a rate faster than that observed for the free reduced enzyme; 4) the rate of reoxidation of reduced enzyme by oxygen during turnover is dependent on both O2 and D-aspartate concentrations (second order rate constant of reaction between O2 and reduced enzyme-substrate complex equal to 6.2 x 10(4) M-1 s-1); and 5) the rate-limiting step in catalysis occurs after reoxidation of the enzyme and before its reduction in the following turnover. A mechanism involving reduction of enzyme by substrate, dissociation of product from reduced enzyme, binding of a second molecule of substrate to the reduced enzyme, and reoxidation of the reduced enzyme-substrate complex is proposed for the enzyme-catalyzed oxidation of D-aspartate.     

Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects

The reactions of nitric oxide (NO) with the turnover intermediates of cytochrome c oxidase were investigated by combining amperometric and spectroscopic techniques. We show that the complex of nitrite with the oxidized enzyme (O) is obtained by reaction of both the "peroxy" (P) and "ferryl" (F) intermediates with stoichiometric NO, following a common reaction pathway consistent with P being an oxo-ferryl adduct. Similarly to chloride-free O, NO reacted with P and F more slowly [k approximately (2-8) x 10(4) M(-1) s(-1)] than with the reduced enzyme (k approximately 1 x 10(8) M(-1) s(-1)). Recovery of activity of the nitrite-inhibited oxidase, either during turnover or after a reduction-oxygenation cycle, was much more rapid than nitrite dissociation from the fully oxidized enzyme (t(1/2) approximately 80 min). The anaerobic reduction of nitrite-inhibited oxidase produced the fully reduced but uncomplexed enzyme, suggesting that reversal of inhibition occurs in turnover via nitrite dissociation from the cytochrome a(3)-Cu(B) site: this finding supports the hypothesis that oxidase may have a physiological role in the degradation of NO into nitrite. Kinetic simulations suggest that the probability for NO to be transformed into nitrite is greater at low electron flux through oxidase, while at high flux the fully reduced (photosensitive) NO-bound oxidase is formed; this is fully consistent with our recent finding that light releases the inhibition of oxidase by NO only at higher reductant pressure [Sarti, P., et al. (2000) Biochem. Biophys. Res. Commun. 274, 183].     

Preconditioning with millimolar concentrations of vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide -- H(2)O(2), superoxide anion radical O(2)*- and hydroxyl radical *OH -- the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO* from sodium nitroprusside -- SNP), and peroxynitrite (ONOO(-) from 3-morpholinosydnonimine -- SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 microM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H(2)O(2), O(2)*-, *OH, ONOO(-) and NO*. Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H(2)O(2) and ONOO(-), while NO* additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate - ASC (0.01, 0.1, 1 mM), or N-acetylcysteine - NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.     

Inefficient spin trapping of superoxide in the presence of nitric-oxide: implications for studies on nitric-oxide synthase uncoupling

Uncoupling of nitric-oxide synthase (NOS) by deficiency of the substrate L-arginine or the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is known to generate the reactive oxygen species H2O2 and superoxide. Discrimination between these two compounds is usually achieved by spin trapping of superoxide. We measured superoxide formation by uncoupled rat neuronal NOS, which contained one equivalent of tightly bound BH4 per dimer, using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap. As expected, the Ca2+-stimulated enzyme exhibited reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity that was accompanied by generation of superoxide and H2O2 in the absence of added L-arginine and BH4. Addition of BH4 (10 microM) did not significantly affect the rate of H2O2 formation but almost completely inhibited the apparent formation of superoxide, suggesting direct formation of H2O2. Although L-arginine (0.1 mM) increased the rate of NADPH oxidation about two-fold, the substrate largely attenuated apparent formation of both superoxide and H2O2, indicating that the spin trap did not efficiently outcompete the reaction between NO and superoxide. The efficiency of DEPMPO to scavenge superoxide in the presence of NO was studied by measuring free NO with a Clark-type electrode under conditions of NO/superoxide cogeneration. Neuronal NOS half-saturated with BH4 and the donor compound 3-morpholinosydnonimine (SIN-1) were used as enzymatic and nonenzymatic sources of NO/superoxide, respectively. Neither of the two systems gave rise to considerable NO signals in the presence of 50-100 mM DEPMPO, and even at 400 mM the spin trap uncovered less than 50% of the NO release that was detectable in the presence of 5000 U/ml superoxide dismutase. These results indicate that DEPMPO and all other currently available superoxide spin traps do not efficiently outcompete the reaction with NO. In addition, the similar behavior of nNOS and SIN-1 provides further evidence for NO as initial product of the NOS reaction.     

Characterization of free radical generation by xanthine oxidase. Evidence for hydroxyl radical generation

Xanthine oxidase has been hypothesized to be an important source of biological free radical generation. The enzyme generates the superoxide radical, .O2- and has been widely applied as a .O2- generating system; however, the enzyme may also generate other forms of reduced oxygen. We have applied electron paramagnetic resonance (EPR) spectroscopy using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) to characterize the different radical species generated by xanthine oxidase along with the mechanisms of their generation. Upon reaction of xanthine with xanthine oxidase equilibrated with air, both DMPO-OOH and DMPO-OH radicals are observed. In the presence of ethanol or dimethyl sulfoxide, alpha-hydroxyethyl or methyl radicals are generated, respectively, indicating that significant DMPO-OH generation occurred directly from OH rather than simply from the breakdown of DMPO-OOH. Superoxide dismutase totally scavenged the DMPO-OOH signal but not the DMPO-OH signal suggesting that .O2- was not required for .OH generation. Catalase markedly decreased the DMPO-OH signal, while superoxide dismutase + catalase totally scavenged all radical generation. Thus, xanthine oxidase generates .OH via the reduction of O2 to H2O2, which in turn is reduced to .OH. In anaerobic preparations, the enzyme reduces H2O2 to .OH as evidenced by the appearance of a pure DMPO-OH signal. The presence of the flavin in the enzyme is required for both .O2- and .OH generation confirming that the flavin is the site of O2 reduction. The ratio of .O2- and .OH generation was affected by the relative concentrations of dissolved O2 and H2O2. Thus, xanthine oxidase can generate the highly reactive .OH radical as well as the less reactive .O2- radical. The direct production of .OH by xanthine oxidase in cells and tissues containing this enzyme could explain the presence of oxidative cellular damage which is not prevented by superoxide dismutase.     

Investigation of the chemical basis of nitroalkane toxicity: tautomerism and decomposition of propane 1- and 2-nitronate under physiological conditions

Unlike primary nitroalkanes, such as 1-nitropropane, the secondary nitroalkane 2-nitropropane is geno- and hepatotoxic. Nitroalkanes exist in equilibrium with alkane nitronates. In order to investigate the relationship between nitroalkane toxicity and generation and stability of nitronates, propane 1- or 2-nitronate (4-6 mM) were incubated in buffer (pH 3.8 -7.4) in the absence or presence of cysteine. Equilibrium formation and degradation were studied by 1H-NMR spectroscopy and ion pair HPLC chromatography. Propane 1-nitronate generated 1-nitropropane rapidly and almost quantitatively. In the case of propane 2-nitronate equilibrium at pH 7.4 was reached within 8 h, when 48% of initial nitronate had tautomerised to 2-nitropropane. The pKa of the reaction 2-nitropropane less than--greater than propane 2-nitronate measured by HPLC was 7.63. Equilibrium formation, hydrolysis and reduction of nitronates were pH-dependent and, in the case of propane 2-nitronate, yielded mainly acetone, nitrite and acetone oxime, apart from 2-nitropropane. Hydrolysis of propane 2-nitronate (4 mM) to nitrite was modulated by cysteine (4 mM) and p-methoxyphenol (0.4 mM). At pH 7.4 they increased nitrite generation by 300 and 28%, respectively, at pH 4.8 they decreased nitrite formation by 91 and 82%, respectively, probably by scavenging radical intermediates. Differences between nitroalkanes in terms of content of nitronate tautomer at equilibrium are probably an important chemical determinant of their toxic potential.     

Two-step oxidation of glycerol to glyceric acid catalyzed by the Phanerochaete chrysosporium glyoxal oxidase

Glyoxal oxidase of P. chrysosporium is a radical copper oxidase that catalyzes oxidation of aldehydes to carboxylic acids coupled to dioxygen reduction to H(2)O(2). In addition to known substrates, glycerol is also found to be a substrate for glyoxal oxidase. During enzyme turnover, glyoxal oxidase undergoes a reversible inactivation, probably caused by loss of the active site free radical, resulting in short-lasting enzyme activities and undetectable substrate conversions. Enzyme activity could be extended by including two additional enzymes, horseradish peroxidase and catalase, in addition to a redox chemical activator, such as Mn(III) (or Mn(II)+H(2)O(2)) or hexachloroiridate. Using this three-enzyme system glycerol was converted in glyceric acid in a two-step reaction, with glyceraldehyde as intermediate. A possible operation mechanism is proposed in which the three enzymes would work coordinately allowing to maintain a sustained glyoxal oxidase activity. In the course of its catalytic cycle, glyoxal oxidase alternates between two functional and interconvertible reduced and oxidized forms resulting from a two-electron transfer process. However, glyoxal oxidase can also undergo an one-electron reduction to a catalytically inactive form lacking the active site free radical. Horseradish peroxidase could use glyoxal oxidase-generated H(2)O(2) to oxidize Mn(II) to Mn(III) which, in turn, would reoxidize and reactivate the inactive form of glyoxal oxidase. Catalase would remove the excess of H(2)O(2) generated during the reaction. In spite of the improvement achieved using the three-enzyme system, glyoxal oxidase inactivation still occurred, which resulted in low substrate conversions. Possible causes of inactivation, including end-product inhibition, are discussed.     

The co-oxidation of ammonia to nitrite during the aerobic xanthine oxidase reaction

The xanthine oxidase reaction causes a co-oxidation of NH3 to NO2-, which was inhibitable by superoxide dismutase, catalase, hydroxyl radical scavengers, or by the chelating agents, desferrioxamine or diethylene triaminepentaacetic acid. Hydroxylamine was oxidized to NO2- much more rapidly than was NH3, and in this case superoxide dismutase or the chelating agents inhibited but catalase or the HO. scavengers did not. Hydrazine was not detectably oxidized to NO2-, and NO2- was not oxidized to NO3-, by the xanthine oxidase reaction. These results are accommodated by a reaction scheme involving (a) the metal-catalyzed production of HO. from O2- + H2O2; (b) the oxidation of H3N to H2N. by OH.; (c) the coupling of H2N. with O2- to yield peroxylamine, which hydrolyzes to hydroxylamine plus H2O2; (d) the metal-catalyzed oxidation of HO-NH2 to (Formula: see text), which couples with O2- to yield (Formula: see text), which finally dehydrates to yield NO2-.     

Reaction of superoxide and nitric oxide with peroxynitrite. Implications for peroxynitrite-mediated oxidation reactions in vivo

Peroxynitrite (ONOO(-)/ONOOH), the product of the diffusion-limited reaction of nitric oxide (*NO) with superoxide (O(-*)(2)), has been implicated as an important mediator of tissue injury during conditions associated with enhanced *NO and O(-*)(2) production. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized peroxynitrite, others have proposed that the relative rates of *NO and production may be critical in determining the reactivity of peroxynitrite formed in situ (Miles, A. M., Bohle, D. S., Glassbrenner, P. A., Hansert, B., Wink, D. A., and Grisham, M. B. (1996) J. Biol. Chem. 271, 40-47). In the present study, we examined the mechanisms by which excess O(-*)(2) or *NO production inhibits peroxynitrite-mediated oxidation reactions. Peroxynitrite was generated in situ by the co-addition of a chemical source of *NO, spermineNONOate, and an enzymatic source of O(-*)(2), xanthine oxidase, with either hypoxanthine or lumazine as a substrate. We found that the oxidation of the model compound dihydrorhodamine by peroxynitrite occurred via the free radical intermediates OH and NO(2), formed during the spontaneous decomposition of peroxynitrite and not via direct reaction with peroxynitrite. The inhibitory effect of excess O(-*)(2) on the oxidation of dihydrorhodamine could not be ascribed to the accumulation of the peroxynitrite scavenger urate produced from the oxidation of hypoxanthine by xanthine oxidase. A biphasic oxidation profile was also observed upon oxidation of NADH by the simultaneous generation of *NO and O(-*)(2). Conversely, the oxidation of glutathione, which occurs via direct reaction with peroxynitrite, was not affected by excess production of *NO. We conclude that the oxidative processes initiated by the free radical intermediates formed from the decomposition of peroxynitrite are inhibited by excess production of *NO or O(-*)(2), whereas oxidative pathways involving a direct reaction with peroxynitrite are not altered. The physiological implications of these findings are discussed.     

Ca2+-dependent and independent mitochondrial damage in HepG2 cells that overexpress CYP2E1

The effect of detergents on electron and proton transfer in bovine cytochrome c oxidase was studied using steady-state and transient-state methods. Cytochrome c oxidase in lauryl maltoside has high maximal turnover (TN(max)=400 s(-1)), whereas activity is low (TN(max)=10 s(-1)) in Triton X-100. Single turnover studies of intramolecular electron transfer show similar rates in either detergent. Transient proton uptake experiments show the oxidase in lauryl maltoside consumes 1.8+/-0.3 H(+)/aa(3) during either partial reduction of the oxidase or reaction of fully reduced enzyme with O(2). However, the oxidase in Triton X-100 consumes 2.6+/-0.4 H(+)/aa(3) during partial reduction and 1.0+/-0.2 H(+)/aa(3) in the O(2) reaction. Absorption spectra recorded during turnover show that the enzyme undergoes activation in lauryl maltoside, but does not activate in Triton X-100. We propose that cytochrome c oxidase in different detergents allows access to different sites of protonation, which in turn influences steady-state activity.     

Inhibition and inactivation of horseradish peroxidase by thiourea

The kinetics of horseradish peroxidase (EC 1.11.1.7)-catalyzed oxidation of o-dianisidine by hydrogen peroxide in the presence of thiourea were studied. At the first, fast step of this process thiourea acts as a competitive reversible inhibitor with respect to o-dianisidine (Ki = 0.22 mM). The formation of a thiourea-peroxidase complex was determined by the increase in the absorbance at A495 and A638 of the enzyme. The dissociation constant for the peroxidase-thiourea complex is equal to 2.0-2.7 mM. Thiourea is not a specific substrate of peroxidase during the oxidation reaction by H2O2, but is an oxidase substrate (although not a very active one) of peroxidase. The irreversible inactivation of the enzyme during its incubation with thiourea was studied. The first-order inactivation rate constant (kin) was shown to increase with a fall in the enzyme concentration. The curve of the dependence of kin on the initial concentration of thiourea shows a maximum at 5-7 mM. The enzyme inactivation is due to its modification by intermediate free radical products of thiourea oxidation. The inhibitors of the free radical reactions (o-dianisidine) protect the enzyme against inactivation. The degree of inactivation depends on concentrations and ratio of thiourea and peroxidase. A possible mechanism of peroxidase interaction with thiourea is discussed.     

Preparation and kinetic properties of 5-ethylphenazine-lactate-dehydrogenase-NAD+ conjugate, a semisynthetic lactate oxidase showing a hide-and-seek effect.

5-Ethylphenazine-lactate-dehydrogenase-NAD+ conjugate (EP(+)-LDH-NAD+) was prepared by linking poly(ethylene glycol)-bound 5-ethylphenazine and poly(ethylene glycol)-bound NAD+ to lactate dehydrogenase. The average number of the ethylphenazine moieties bound per molecule of enzyme subunit was 0.46, and that of the NAD+ moieties was 0.32. This conjugate is a semisynthetic enzyme having lactate oxidase activity using oxygen or 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) as an electron acceptor; to make such conjugates seems to be a general method for artificially converting a dehydrogenase into an oxidase. When the concentration of oxygen or MTT is varied, the oxidase activity fits the Michaelis-Menten equation with the following kinetic constants: for the reaction system with oxygen, the turnover number per subunit is 2.3 min-1 and Km for oxygen is 1.91 mM; and for the system with MTT, the turnover number is 0.25 min-1 and Km for MTT is 0.076 mM. At the initial steady state of the oxidase reaction, only 2.1% of the NAD+ moieties of the conjugate are in the free state (i.e. not bound in the coenzyme-binding site of the lactate dehydrogenase moiety) and the rest are hidden in the coenzyme site; almost all the NAD+ moieties are in the reduced state. The apparent intramolecular rate constant for the reaction between a free NADH moiety and an oxidized ethylphenazine moiety is 2.3 s-1 and 2.1 s-1 for the systems with oxygen and with MTT, respectively. The apparent effective concentration of the free NADH moiety for the ethylphenazine moiety is 5.5 microM and is much smaller than that (0.34 mM) of the ethylphenazine moiety for the free NADH moiety; this difference is due to the effect of hiding the NADH moiety in the binding site, as the hidden NADH moiety cannot react with the ethylphenazine moiety.     

Inactivation of copper-containing amine oxidases by turnover products.

For bovine serum amine oxidase, two different mechanisms of substrate-induced inactivation have been proposed. One consists of a slow oxidation by H2O2 of a conserved residue in the reduced enzyme after the fast turnover phase [Pietrangeli, P., Nocera, S., Fattibene, P., Wang, X.T., Mondovì, B. & Morpurgo, L. (2000) Biochem. Biophys. Res. Commun.267, 174-178] and the other of the oxidation by H2O2 of the dihydrobenzoxazole in equilibrium with the product Schiff base, during the catalytic cycle [Lee, Y., Shepard, E., Smith, J., Dooley, D.M. & Sayre, L.M. (2001) Biochemistry40, 822-829]. To discriminate between the two mechanisms, the inactivation was studied using Lathyrus cicera (red vetchling) amine oxidase. This, in contrast to bovine serum amine oxidase, formed the Cu+-semiquinolamine radical with a characteristic UV-vis spectrum when oxygen was exhausted by an excess of any tested amine in a closed cuvette. The inactivation, lasting about 90 min, was simultaneous with the radical decay and with the formation of a broad band (shoulder) at 350 nm. No inactivation occurred when a thousand-fold excess of amine was rapidly oxidized in an L. cicera amine oxidase solution stirred in open air. Thus, the inactivation is a slow reaction of the reduced enzyme with H2O2, following the turnover phase. Catalase protected L. cicera amine oxidase from inactivation. This effect was substrate-dependent, varying from full protection (benzylamine) to no protection (putrescine). In the absence of H2O2, a specific inactivating reaction, without formation of the 350 nm band, was induced by some aldehydes, notably putrescine. Some mechanisms of inactivation are proposed.     

An investigation by e.p.r. and optical spectroscopy of cytochrome oxidase during turnover.

Cytochrome oxidase (EC 1.9.3.1; ferrocytochrome c:oxygen oxidoreductase) was studied during steady-state by optical and e.p.r. methods. Starting with either the 'resting' or the 'pulsed' enzyme, oxidase, cytochrome c, ascorbate and O2 were mixed and the reaction monitored optically. Tetramethylphenylenediamine was used as mediator to poise the steady-state to the desired reduction level. After mixing, the reaction was quenched by the used of rapid-freeze techniques. The e.p.r. spectra of samples captured at increasing tetramethylphenylenediamine concentrations (i.e. higher electron flux) show decreasing g = 2 (Cu A) and g = 3 (cytochrome a) signals. No Cu B or g = 6 signals (high-spin cytochrome a3) could be found during the reaction. Also, the signal with peaks at g = 1.69, 1.78 and 5 as well as the g = 12 signal was hardly detectable at higher turnover rates. The only new signal appearing during turnover is a radical signal, which is discussed in terms of a protein radical. Finally, a scheme is presented, proposing a catalytic cycle for cytochrome oxidase with respect to the O2 binding Cu B-cytochrome a3 unit.     

Excess no predisposes mitochondrial succinate-cytochrome c reductase to produce hydroxyl radical

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O(2)(?-) generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate-cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/(?)OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron-H(2)O(2)-dependent Fenton reaction or O(2)(?-)-dependent Haber-Weiss mechanism. Direct EPR measurement of SCR at 77K indicated the formation of a nonheme iron-NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce (?)OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.     

Spin traps inhibit formation of hydrogen peroxide via the dismutation of superoxide: implications for spin trapping the hydroxyl free radical.

To enhance the sensitivity of EPR spin trapping for radicals of limited reactivity, high concentrations (10-100 mM) of spin traps are routinely used. We noted that in contrast to results with other hydroxyl radical detection systems, superoxide dismutase (SOD) often increased the amount of hydroxyl radical-derived spin adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) produced by the reaction of hypoxanthine, xanthine oxidase and iron. One possible explanation for these results is that high DMPO concentrations (approximately 100 mM) inhibit dismutation of superoxide (O2.-) to hydrogen peroxide (H2O2). Therefore, we examined the effect of DMPO on O2.- dismutation to H2O2. Lumazine +/- 100 mM DMPO was placed in a Clark oxygen electrode following which xanthine oxidase was added. The amount of H2O2 formed in this reaction was determined by introducing catalase and measuring the amount of generated via O2.- dismutation as compared to direct divalent O2 reduction. In the presence of 100 mM DMPO, H2O2 generation decreased 43%. DMPO did not scavenge H2O2 nor alter the rate of O2.- production. The effect of DMPO was concentration-dependent with inhibition of H2O2 production observed at [DMPO] greater than 10 mM. Inhibition of H2O2 production by DMPO was not observed if SOD was present or if the rate of O2.- formation increased. The spin trap 2-methyl-2-nitroso-propane (MNP, 10 mM) also inhibited H2O2 formation (81%). However, alpha-phenyl-N-tert-butylnitrone (PBN, 10 mM), 3,3,5,5 tetramethyl-1-pyrroline N-oxide (M4PO, 100 mM), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN, 100 mM) had no effect. These data suggest that in experimental systems in which the rate of O2.- generation is low, formation of H2O2 and thus other H2O2-derived species (e.g., OH) may be inhibited by commonly used concentrations of some spin traps. Thus, under some experimental conditions spin traps may potentially prevent production of the very free radical species they are being used to detect.     

Nitric oxide reacts with the single-electron reduced active site of cytochrome c oxidase

The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.     

Nitric oxide and mitochondrial complex IV

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl- [a3(2+) -NO] or a nitrite- [a3(3+) -NO2-] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k' approximately equal to 0.01 s(-1) (37 degrees C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one.     

Cytochrome c oxidase and nitric oxide in action: molecular mechanisms and pathophysiological implications

BACKGROUND: The reactions between Complex IV (cytochrome c oxidase, CcOX) and nitric oxide (NO) were described in the early 60's. The perception, however, that NO could be responsible for physiological or pathological effects, including those on mitochondria, lags behind the 80's, when the identity of the endothelial derived relaxing factor (EDRF) and NO synthesis by the NO synthases were discovered. NO controls mitochondrial respiration, and cytotoxic as well as cytoprotective effects have been described. The depression of OXPHOS ATP synthesis has been observed, attributed to the inhibition of mitochondrial Complex I and IV particularly, found responsible of major effects. SCOPE OF REVIEW: The review is focused on CcOX and NO with some hints about pathophysiological implications. The reactions of interest are reviewed, with special attention to the molecular mechanisms underlying the effects of NO observed on cytochrome c oxidase, particularly during turnover with oxygen and reductants. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: The NO inhibition of CcOX is rapid and reversible and may occur in competition with oxygen. Inhibition takes place following two pathways leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) of the enzyme reduced, or a more labile nitrite-derivative (CcOX-NO(2)(-)) of the enzyme oxidized, and during turnover. The pathway that prevails depends on the turnover conditions and concentration of NO and physiological substrates, cytochrome c and O(2). All evidence suggests that these parameters are crucial in determining the CcOX vs NO reaction pathway prevailing in vivo, with interesting physiological and pathological consequences for cells.