The influence of fluid mixing upon respiratory patterns for extended growth of a methylotroph in an air-lift fermenter

The influence of methanol dispersion and fluid mixing upon respiratory patterns observed during unlimited fedbatch growth of the methylotrophic bacterium Methylophilus methylotrophus has been investigated. A concentric tube air-lift fermenter was employed for which the mixing and fluid circulation patterns have been well characterized. Respiratory quotients showed a marked dependence upon the position in the vessel at which methanol was injected, the volumetric rate of such methanol addition, the fluid circulation time, and the local mixing behavior; the latter two factors of which are both determined by the air throughput. Such variations are discussed on the basis of simple mixing concepts and observations of fluid dispersion.     

-->H+/2e- stoichiometry in NADH-quinone reductase reactions catalyzed by bovine heart submitochondrial particles.

Tightly coupled bovine heart submitochondrial particles treated to activate complex I and to block ubiquinol oxidation were capable of rapid uncoupler-sensitive inside-directed proton translocation when a limited amount of NADH was oxidized by the exogenous ubiquinone homologue Q1. External alkalization, internal acidification and NADH oxidation were followed by the rapidly responding (t1/2 < or = 1 s) spectrophotometric technique. Quantitation of the initial rates of NADH oxidation and external H+ decrease resulted in a stoichiometric ratio of 4 H+ vectorially translocated per 1 NADH oxidized at pH 8.0. ADP-ribose, a competitive inhibitor of the NADH binding site decreased the rates of proton translocation and NADH oxidation without affecting -->H+/2e- stoichiometry. Rotenone, piericidin and thermal deactivation of complex I completely prevented NADH-induced proton translocation in the NADH-endogenous ubiquinone reductase reaction. NADH-exogenous Q1 reductase activity was only partially prevented by rotenone. The residual rotenone- (or piericidin-) insensitive NADH-exogenous Q1 reductase activity was found to be coupled with vectorial uncoupler-sensitive proton translocation showing the same -->H+/2e- stoichiometry of 4. It is concluded that the transfer of two electrons from NADH to the Q1-reactive intermediate located before the rotenone-sensitive step is coupled with translocation of 4 H+.     

Vanadate-stimulated NADH oxidation by xanthine oxidase: an intrinsic property

Vanadate-dependent oxidation of NADH by xanthine oxidase does not require the presence of xanthine and therefore is not due to cooxidation. Addition of NADH or xanthine had no effect on the oxidation of the other substrate. Oxidation of NADH was high at acid pH and oxidation of xanthine was high at alkaline pH. The specific activity was relatively very high with NADH. Concentration-dependent oxidation of NADH Concentration-dependent oxidation of NADH was obtained in the presence of the polymeric form of vanadate, but not orthovanadate or metavanadate. Both NADH and NADPH were oxidized, as in the nonenzymatic system. Oxidation of NADH, but not xanthine, was inhibited by KCN, ascorbate, MnCl2, cytochrome c, mannitol, Tris, epinephrine, norepinephrine, and triiodothyronine. Oxidation of NADH was accompanied by uptake of oxygen and generation of H2O2 with a stoichiometry of 1:1:1 for NADH:O2:H2O2. A 240-nm-absorbing species was formed during the reaction which was different from H2O2 or superoxide. A mechanism of NADH oxidation is suggested wherein Vv and O2 receive one electron each successively from NADH followed by VIV giving the second electron to superoxide and reducing it to H2O2.     

Activation by ATP of calcium-dependent NADPH-oxidase generating hydrogen peroxide in thyroid plasma membranes

An H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H2O2 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0.1 microM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hexokinase together with glucose to remove ATP. The Km value for NADPH was 35 microM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H2O2 production. The stoichiometry between the oxidation and the H2O2 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2 production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H2O2 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H2O2. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H2O2. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H2O2, a substrate for peroxidase.     

Bacterial respiration-linked proton translocation and its relationship to respiratory-chain composition.

1. The relationship between chain composition and the efficiency of respiration-linked proton translocation was studied in nine bacterial species of widely differing taxonomic and ecological status. 2. All the bacteria investigated contained respiratory chain dehydrogenases, ubiquinone and/or menaquinone, cytochrome b and cytochrome oxidase aa3 and/or o. In addition, some of these organisms also contained pyridine nucleotide transhydrogenase and/or cytochrome c. 3. leads to H+/O ratios of whole cell suspensions oxidising endogenous substrates were in the approximate range 4-8 mol H+ translocated per g-atom oxygen consumed. It was concluded from the observed leads to H+/O ratios of cells loaded with specific substrates that proton-translocating loops 1 and 2 were present in all of the organisms investigated, but that loops 0 and 3 were dependent upon the presence of pyridine nucleotide transhydrogenase and cytochrome c respectively. 4. The wide range in energy conservation efficiency which was observed in these organisms is discussed in relation to their respiratory chain composition and natural habitat.     

Chorion peroxidase-mediated NADH/O(2) oxidoreduction cooperated by chorion malate dehydrogenase-catalyzed NADH production: a feasible pathway leading to H(2)O(2) formation during chorion hardening in Aedes aegypti mosquitoes

A specific chorion peroxidase is present in Aedes aegypti and this enzyme is responsible for catalyzing chorion protein cross-linking through dityrosine formation during chorion hardening. Peroxidase-mediated dityrosine cross-linking requires H(2)O(2), and this study discusses the possible involvement of the chorion peroxidase in H(2)O(2) formation by mediating NADH/O(2) oxidoreduction during chorion hardening in A. aegypti eggs. Our data show that mosquito chorion peroxidase is able to catalyze pH-dependent NADH oxidation, which is enhanced in the presence of Mn(2+). Molecular oxygen is the electron acceptor during peroxidase-catalyzed NADH oxidation, and reduction of O(2) leads to the production of H(2)O(2), demonstrated by the formation of dityrosine in a NADH/peroxidase reaction mixture following addition of tyrosine. An oxidoreductase capable of catalyzing malate/NAD(+) oxidoreduction is also present in the egg chorion of A. aegypti. The cooperative roles of chorion malate/NAD(+)oxidoreductase and chorion peroxidase on generating H(2)O(2) with NAD(+) and malate as initial substrates were demonstrated by the production of dityrosine after addition of tyrosine to a reaction mixture containing NAD(+) and malate in the presence of both malate dehydrogenase fractions and purified chorion peroxidase. Data suggest that chorion peroxidase-mediated NADH/O(2) oxidoreduction may contribute to the formation of the H(2)O(2) required for chorion protein cross-linking mediated by the same peroxidase, and that the chorion associated malate dehydrogenase may be responsible for the supply of NADH for the H(2)O(2) production.     

Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata

A homogeneous Mn-dependent peroxidase (MnP) was purified from the extracellular culture fluid of the lignin-degrading white rot fungus Phlebia radiata by anion exchange chromatography. The enzyme had a molecular weight of 49,000 and pI 3.8. It was a glycoprotein, containing carbohydrate moieties accounting for 10% of the molecular weight. Mn-peroxidase was capable of oxidizing phenolic compounds in the presence of H2O2, whereas the effect on nonphenolic lignin model compounds was insignificant. MnP contained protoporphyrin IX as a prosthetic group. During enzymatic reactions H2O2 converted the native MnP to compound II. Mn2+ was essential in completing the catalytic cycle by returning the enzyme to its native state. The oxidation of ultimate substrates was dependent on superoxide radicals, O2- and probably on Mn3+ generated during the catalytic cycle. MnP exhibited high activity of NADH oxidation without exogenously added H2O2. It was shown to produce H2O2 at the expense of NADH.     

Electron transfer flavoprotein from Methylophilus methylotrophus: properties, comparison with other electron transfer flavoproteins, and regulation of expression by carbon source.

When grown on methylated amines as a carbon source, Methylophilus methylotrophus synthesizes an electron transfer flavoprotein (ETF) which is the natural electron acceptor of trimethylamine dehydrogenase. It is composed of two dissimilar subunits of 38,000 and 42,000 daltons and 1 mol of flavin adenine dinucleotide. It was reduced by trimethylamine dehydrogenase to a stable anionic semiquinone form, which could not be converted, either enzymatically or chemically, to the fully reduced dihydroquinone. This ETF exhibited spectral properties which were nearly identical to ETFs from bacterium W3A1, Paracoccus denitrificans, and pig liver mitochondria. M. methylotrophus ETF cross-reacted immunologically and enzymatically with the ETF of bacterium W3A1 but not with the other two ETFs. In M. methylotrophus and bacterium W3A1, ETF and trimethylamine dehydrogenase were each expressed during growth on trimethylamine and were each absent during growth on methanol.     

The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation

The involvement of reactive oxygen species (ROS) in cardiac ischemia-reperfusion injuries is well-established, but the deleterious effects of hydrogen peroxide (H(2)O(2)), hydroxyl radical (HO*) or superoxide anion (O(2)*(-) ) on mitochondrial function are poorly understood. Here, we report that incubation of rat heart mitochondria with each of these three species resulted in a decline of the ADP-stimulated respiratory rate but not substrate-dependent respiration. These three species reduced oxygen consumption induced by an uncoupler without alteration of the respiratory chain complexes, but did not modify mitochondrial membrane permeability. HO* slightly decreased F1F0-ATPase activity and HO* and O(2)*(-) partially inhibited the activity of adenine nucleotide translocase; H(2)O(2) failed to alter these targets. They inhibited NADH production by acting specifically on aconitase for O(2)*(-) and alpha-ketoglutarate dehydrogenase for H(2)O(2) and HO*. Our results show that O(2)*(-), H(2)O(2) and HO* act on different mitochondrial targets to alter ATP synthesis, mostly through inhibition of NADH production.     

Energy transduction in the mitochondrionlike bacterium Paracoccus denitrificans during carbon- or sulphate-limited aerobic growth in continuous culture.

Paracoccus denitrificans was grown in carbon-limited aerobic continuous culture (critical dilution rate (Dc) = 0.48 h-1). The molar growth yield for carbon (succinate or malate) was constant at about 60 over a broad dilution range (growth rate) from 0.10 to 0.48 h-1. Measurements of the stoichiometry of proton translocation associated with the oxidation of endogenous substrates yielded a ratio of protons ejected from the cell per atom of oxygen consumed(leads to H+:O) of 8.55 which decreased to 5.85 in the presence of piericidin A (PA), a specific inhibitor of NADH dehydrogenase (EC 1.6.99.3). With starved cells, the observed leads to H+:O associated with the oxidation of added succinate in the presence of PA was 5.61. These observed leads to H:O's represent an underestimation since no correction was made for proton backflow during the short interval of respiratory activity. Aerobic growth of Pc. denitrificans in the chemostat becomes sulphate limited at entering concentrations of sulphate less than 300 is microM. Neither the maximum specific growth rate (measured at Dc) nor the observed molar growth yield for succinate decreased under sulphate limitation. The NADH oxidase in electron transport particles prepared from sulphate-limited cells was completely inhibited by PA. The stoichiometry of proton translocation associated with malate oxidation was similarly unaffected by sulphate limitation. It is concluded that (a) the respiratory chain of aerobic, heterotrophically grown Pc. denitrificans possesses three sites of energy conservation, including site III, (b) the number of protons ejected during the transfer of one pair of reducing equivalents along a region of the electron transport chain equivalent to a single energy-coupling site is 3, and (c) that sulphate limitation does not lead to a loss of proton translocation associated with the cytochrome-independent region of the respiratory chain.     

Hydrogen peroxide formation and iron ion oxidoreduction linked to NADH oxidation in radish plasmalemma vesicles

Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.     

Re-evaluation of the kinetics of lactate dehydrogenase-catalyzed chain oxidation of nicotinamide adenine dinucleotide by superoxide radicals in the presence of ethylenediaminetetraacetate.

The chain oxidation of lactate dehydrogenase-bound NADH initiated by superoxide radicals and propagated by oxygen was studied with pulse radiolysis. The kinetic parameters were re-evaluated in a system with carefully purified reagents (water and other chemicals) and in the presence of EDTA. The rate constant for the oxidation of the enzyme-bound NADH by O2- is calculated from the observed pseudo-first order disappearance of NADH and the chain length (molecules of NADH oxidized per O2- anion generated in the pulse). It is (1.0 +/- 0.2) X 10(5) M-1 S-1, consistent within a 13-fold variation in lactate dehydrogenase. NADH complex concentration and with varying chain length up to 6.1. Based on experiments with varying pH values from 4.5 to 9.0, the rate constant for oxidation of enzyme-bound NADH by HO2 is estimated to be 2.0 X 10(6) M-1 S-1.     

X-ray scattering studies of Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein. Evidence for multiple conformational states and an induced fit mechanism for assembly with trimethylamine dehydrogenase

Small angle x-ray solution scattering has been used to generate a low resolution, model-independent molecular envelope structure for electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W(3)A(1)). Analysis of both the oxidized and 1-electron-reduced (anionic flavin semiquinone) forms of the protein revealed that the solution structures of the protein are similar in both oxidation states. Comparison of the molecular envelope of ETF from the x-ray scattering data with previously determined structural models of the protein suggests that ETF samples a range of conformations in solution. These conformations correspond to a rotation of domain II with respect to domains I and III about two flexible "hinge" sequences that are unique to M. methylotrophus ETF. The x-ray scattering data are consistent with previous models concerning the interaction of M. methylotrophus ETF with its physiological redox partner, trimethylamine dehydrogenase. Our data reveal that an "induced fit" mechanism accounts for the assembly of the trimethylamine dehydrogenase-ETF electron transfer complex, consistent with spectroscopic and modeling studies of the assembly process.     

Electrogenic malate uptake and improved growth energetics of the malolactic bacterium Leuconostoc oenos grown on glucose-malate mixtures.

Growth of the malolactic bacterium Leuconostoc oenos was improved with respect to both the specific growth rate and the biomass yield during the fermentation of glucose-malate mixtures as compared with those in media lacking malate. Such a finding indicates that the malolactic reaction contributed to the energy budget of the bacterium, suggesting that growth is energy limited in the absence of malate. An energetic yield (YATP) of 9.5 g of biomass.mol ATP-1 was found during growth on glucose with an ATP production by substrate-level phosphorylation of 1.2 mol of ATP.mol of glucose-1. During the period of mixed-substrate catabolism, an apparent YATP of 17.7 was observed, indicating a mixotrophy-associated ATP production of 2.2 mol of ATP.mol of glucose-1, or more correctly an energy gain of 0.28 mol of ATP.mol of malate-1, representing proton translocation flux from the cytoplasm to the exterior of 0.56 or 0.84 H+.mol of malate-1(depending on the H+/ATP stoichiometry). The growth-stimulating effect of malate was attributed to chemiosmotic transport mechanisms rather than proton consumption by the malolactic enzyme. Lactate efflux was by electroneutral lactate -/H+ symport having a constant stoichiometry, while malate uptake was predominantly by a malate -/H+ symport, though a low-affinity malate- uniport was also implicated. The measured electrical component (delta psi) of the proton motive force was altered, passing from -30 to -60 mV because of this translocation of dissociated organic acids when malolactic fermentation occurred.     

Effect of diazoxide on flavoprotein oxidation and reactive oxygen species generation during ischemia-reperfusion: a study on Langendorff-perfused rat hearts using optic fibers

This study analyzed the oxidant generation during ischemia-reperfusion protocols of Langendorff-perfused rat hearts, preconditioned with a mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) opener (i.e., diazoxide). The autofluorescence of mitochondrial flavoproteins, and that of the total NAD(P)H pool on the one hand and the fluorescence of dyes sensitive to H(2)O(2) or O(2)(*-) [i.e., the dihydrodichlorofluoroscein (H(2)DCF) and dihydroethidine (DHE), respectively] on the other, were noninvasively measured at the surface of the left ventricular wall by means of optic fibers. Isolated perfused rat hearts were subjected to an ischemia-reperfusion protocol. Opening mitoK(ATP) with diazoxide (100 microM) 1) improved the recovery of the rate-pressure product after reperfusion (72 +/- 2 vs. 16.8 +/- 2.5% of baseline value in control group, P < 0.01), and 2) attenuated the oxidant generation during both ischemic (-46 +/- 5% H(2)DCF oxidation and -40 +/- 3% DHE oxidation vs. control group, P < 0.01) and reperfusion (-26 +/- 2% H(2)DCF oxidation and -23 +/- 2% DHE oxidation vs. control group, P < 0.01) periods. All of these effects were abolished by coperfusion of 5-hydroxydecanoic acid (500 microM), a mitoK(ATP) blocker. During the preconditioning phase, diazoxide induced a transient, reversible, and 5-hydroxydecanoic acid-sensitive flavoprotein and H(2)DCF (but not DHE) oxidation. In conclusion, the diazoxide-mediated cardioprotection is supported by a moderate H(2)O(2) production during the preconditioning phase and a strong decrease in oxidant generation during the subsequent ischemic and reperfusion phases.     

The microbial metabolism of C1 compounds. The stoicheiometry of respiration-driven proton translocation in Pseudomonas AM1 and in a mutant lacking cytochrome c

This paper clarifies the role of cytochrome c in Pseudomonas AM1 by measuring the stoicheiometry of proton translocation driven by respiration of endogenous or added substrates in wild-type bacteria and in a mutant lacking cytochrome c (mutant PCT76). The maximum -->H(+)/O ratio (protons translocated out of the bacteria per atom of oxygen consumed during respiration) was about 4 and, except when respiration was markedly affected, this ratio was similar in mutant and wild-type bacteria. The -->H(+)/O ratios were unaltered when the usual oxidase (cytochrome a(3)) was inhibited by 300mum-KCN and respiration involved the single cytochrome b functioning as an alternative oxidase. Ratios measured in cells respiring endogenous substrate and in cells loaded with malate or 3-hydroxybutyrate suggest that there are two proton-translocating segments operating during the oxidation of NADH. By contrast, during oxidation of formaldehyde or methylamine only one pair of protons is translocated. Proton translocation could not be measured with methanol as substrate, because its oxidation was inhibited (90-95%) by 5mm-KSCN. It is tentatively proposed that the electron-transport chain for NADH oxidation in Pseudomonas AM1 is arranged such that the NADH-ubiquinone oxidoreductase forms one proton-translocating segment and the second segment consists of ubiquinone and cytochromes b and a/a(3). The cytochrome c appears to be essential only for respiration and proton translocation from methanol (and possibly from methylamine); there is no conclusive evidence that cytochrome c ever mediates between cytochromes b and a/a(3) in Pseudomonas AM1.     

Malate valves to balance cellular energy supply

In green parts of the plant, during illumination ATP and NAD(P)H act as energy sources that are generated mainly in photosynthesis and respiration, whereas in darkness, glycolysis, respiration and the oxidative pentose-phosphate pathway (OPP) generate the required energy forms. In non-green parts, sugar oxidation in glycolysis, respiration and OPP are the only means of producing energy. For energy-consuming reactions, the delivery of NADPH, NADH, reduced ferredoxin and ATP has to take place at the required rates and in the specific compartments, since the pool sizes of these energy carriers are rather limited and, in general, they are not directly transported across biomembranes. Indirect transport of reducing equivalents can be achieved by malateoxaloacetate shuttles, involving malate dehydrogenase (MDH) for the interconversion. Isoenzymes of MDH are present in each cellular compartment. Chloroplasts contain the redox-controlled NADP-MDH that is only active in the light. In addition, a plastid NAD-MDH that is permanently active and is present in all plastid types has been found. Export of excess NAD(P)H through the malate valves will allow for the continued production of ATP (1) in photosynthesis, and (2) in oxidative phosphorylation. In the latter case, the coupled production of NADH is catalysed by the bispecific NAD(P)-GAPDH (GapAB) in chloroplasts that is active with NAD even in darkness, or by the specific plastid NAD-GAPDH (GapCp) in non-green tissues. When plants are subjected to conditions such as high light, high CO(2), NH(4) (+) nutrition, cold stress, which require changed activities of the enzymes of the malate valves, changed expression levels of the MDH isoforms can be observed. In nodules, the induction of a nodule-specific plastid NAD-MDH indicates the changed requirements for energy supply during N(2) fixation. Furthermore, the induction of glucose 6-phosphate dehydrogenase isoforms by ammonium and of ferredoxin and ferredoxin-NADP reductase by nitrate has been described. All these findings are in line with the assumption that a changed redox state caused by metabolic variability leads to the induction of enzymes involved in redox poise.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1).The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.     

NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.     

The F420H2 dehydrogenase from Methanosarcina mazei is a Redox-driven proton pump closely related to NADH dehydrogenases

The F(420)H(2) dehydrogenase is part of the energy conserving electron transport system of the methanogenic archaeon Methanosarcina mazei G?1. Here it is shown that cofactor F(420)H(2)-dependent reduction of 2-hydroxyphenazine as catalyzed by the membrane-bound enzyme is coupled to proton translocation across the cytoplasmic membrane, exhibiting a stoichiometry of 0.9 H(+) translocated per two electrons transferred. The electrochemical proton gradient thereby generated was shown to drive ATP synthesis from ADP + P(i). The gene cluster encoding the F(420)H(2) dehydrogenase of M. mazei G?1 comprises 12 genes that are referred to as fpoA, B, C, D, H, I, J, K, L, M, N, and O. Analysis of the deduced amino acid sequences revealed that the enzyme is closely related to proton translocating NADH dehydrogenases of respiratory chains from bacteria (NDH-1) and eukarya (complex I). Like the NADH-dependent enzymes, the F(420)H(2) dehydrogenase is composed of three subcomplexes. The gene products FpoA, H, J, K, L, M, and N are highly hydrophobic and are homologous to subunits that form the membrane integral module of NDH-1. FpoB, C, D, and I have their counterparts in the amphipathic membrane-associated module of NDH-1. Homologues to the hydrophilic NADH-oxidizing input module are not present in M. mazei G?1. Instead, the gene product FpoF may be responsible for F(420)H(2) oxidation and may function as the electron input part. Thus, the F(420)H(2) dehydrogenase from M. mazei G?1 resembles eukaryotic and bacterial proton translocating NADH dehydrogenases in many ways. The enzyme from the methanogenic archaeon functions as a NDH-1/complex I homologue and is equipped with an alternative electron input unit for the oxidation of reduced cofactor F(420) and a modified output module adopted to the reduction of methanophenazine.