Effects of oxygen on the dark recombination between photoreduced secondary quinone and oxidized bacteriochlorophyll in Rhodobacter sphaeroides reaction centers

The influence of duration of exposure to actinic light (from 1 sec to 10 min) and temperature (from 3 to 35^°C) on the temporary stabilization of the photomobilized electron in the secondary quinone acceptor (Q_B) locus of Rhodobacter sphaeroides reaction centers (RC) was studied under aerobic or anaerobic conditions. Optical spectrophotometry and ESR methods were used. The stabilization time increased significantly upon increasing the exposure duration under aerobic conditions. The stabilization time decreased under anaerobic conditions, its dependence on light exposure duration being significantly less pronounced. Generation of superoxide radical in photoactivated aerobic samples was revealed by the ESR method. Possible interpretation of the effects is suggested in terms of interaction between the semiquinone Q_B with oxygen, the interaction efficiency being determined by the conformational transitions in the structure of RC triggered by actinic light on and off.     

Some properties and occurrence of cytochrome c-552 in the aerobic photosynthetic bacterium Roseobacter denitrificans

Characteristics and occurrence of cytochrome c-552 from an aerobic photosynthetic bacterium, Roseobacter denitrificans, were described.Relative molecular mass of the cytrochrome was 13.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 15,000 by gel filtration. This cytochrome was a acidic protein having a pI of 5.6 and E_m was +215 mV at pH 7.0. Absorption peaks were at 278, 408 and 524 nm in the oxidized form and 416, 523 and 552 nm in the reduced form.Amino acid composition and N-terminal amino acid sequence of cytochrome c-552 determined for 24 residues had low similarities to those of cytochrome c-551 of this bacterium, which is homologous to cytochrome c ₂, although the physico-chemical properties of these two cytochromes were similar to each other.Cytochrome c-552 was maximally synthesized in the light under aerobic conditions but not in the dark. The synthesis also occurred in the presence of alternative acceptors such as trimethylamine N-oxide (TMAO) and nitrate under anaerobic conditions. Our results suggest that cytochrome c-552 is involved in TMAO respiration and denitrification in R. denitrificans, although the effect of light remains to be solved.Abbreviations E_m Midpoint redox potential - PAGE Polyacrylamide ge electrophoresis - SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - TMAO Trimethylamine N-oxide     

Effect of CuSO4 and Cu(II)(Gly)2 on some indirect assays for superoxide dismutase activity

The effect of CuSO₄ and Cu(II)(Gly)₂ has been compared with that of superoxide dismutase on the ferricytochrome c reduction and on the nitroblue tetrazolium reduction by an enzymic or chemical flux of superoxide anion radicals as well as on o-dianisidine photooxidation. Both CuSO₄ and Cu(II)(Gly)₂ have been found to inhibit ferricytochrome c reduction as well as the aerobic and anaerobic nitroblue tetrazolium reduction with approximately equal efficiency. Unlike superoxide dismutase they proved capable of inhibiting o-dianisdine photooxidation. The effect of copper either as CuSO₄ or as Cu(II)(Cly)₂ has been established as being due to its interference with the indirect assays for superoxide dismutase activity used. The reasons for this interference have been examined and it is concluded that copper can react with a component of the indirect assay system and depending on the method used it either mimics SOD or acts contrary to the enzyme.     

Effect of oxygen on the cyanobacterium Oscillatoria limnetica

Oscillatoria limnetica grown photoautotrophically under aerobic or anaerobic conditions contained a single superoxide dismutase (SOD) of identical electrophoretic mobility in both cases. Its activity was cyanide resistant and H₂O₂ sensitive, implicating Fe-SOD. The enzyme level was high in aerobically and low in anaerobically growing cells. Anaerobically grown cells were more sensitive than aerobic to photooxidation, as expressed by bleaching of phycocyanin and disintegration of the trichomes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - SOD superoxide dismutase     

Oxygen and temperature-dependent structural and redox changes in a novel cytochrome c(4) from the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina

A novel cytochrome c₄, the first of this type in purple phototrophic bacteria has been discovered in Thiocapsa roseopersicina. The fact that cytochrome c₄ has been found in an anaerobic organism puts in question the up hereto suggested role of cytochromes c₄ in the aerobic respiratory metabolism. The structure of cytochrome c₄ was studied under both aerobic and anaerobic conditions, using differential scanning calorimetry and a combination of redox potentiostatic measurements with CD and UV-Vis absorption techniques. Cytochrome c₄ maintained its functional capability at high temperature (60 °C) if it was kept under anaerobic conditions. With increasing temperature under aerobic conditions, however, there are dramatic conformational changes in the protein and coordination changes on the iron side. Presumably oxygen binds to the iron at the position left vacant by the methionine and facilitates conformational changes with low reversibility.     

Biosynthesis of superoxide dismutase and catalase inSaccharomyces cerevisiae: effects of oxygen and cytochromec deficiency

Summary Two strains ofSaccharomyces cerevisiae were used to study the synthesis of superoxide dismutase. One strain (cytochromec-deficient) contained 5–10% of the normal amounts of total cytochromec, while the other strain was a wild type. The cytochromec-deficient mutant had lower specific growth rate, growth yield, and oxygen uptake than the wild type. The superoxide dismutase and catalase activities, in both strains, were significantly lower under anaerobic than under aerobic conditions. Furthermore, under aerobic conditions the mutant contained higher levels of superoxide dismutase than the wild type which may be attributed to the higher intracellular flux of superoxide radicals caused by the cytochromec deficiency. The mutant also showed a lower level of catalase which was due to glucose repression.Paper Number 10007 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, U.S.A. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned.     

Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate.

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic condition the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O2-.; this reaction is strongly inhibited by superoxide dismutase and by a number of O2-. scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O2-. is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.     

Cytochrome c reduction by semiquinone radicals can be indirectly inhibited by superoxide dismutase

The inhibition by superoxide dismutase of cytochrome c reduction by a range of semiquinone radicals has been studied. The semiquinones were produced from the parent quinones by reduction with xanthine and xanthine oxidase. Most of the quinones studied were favored over O₂ as the enzyme substrate, and in air as well as N₂, semiquinone radicals rather than superoxide were produced and they caused the cytochrome c reduction. With all but one of the quinones (benzoquinone), cytochrome c reduction in air was inhibited by superoxide dismutase, but the amount of enzyme required for inhibition was up to 100 times greater than that required to inhibit reduction by superoxide. It was highest for the quinones with the highest redox potential. These results demonstrate how superoxide dismutase can inhibit cytochrome c reduction by species other than superoxide. They can be explained by the dismutase displacing the equilibrium: semiquinone + O₂ ? quinone + O₂^? to the right, thereby allowing the forward reaction to out-compete other reactions of the semiquinone. The implication from these findings that superoxide dismutase-inhibitable reduction of cytochrome c may not be a specific test for superoxide production is discussed.     

Occurrence of cytochromes in R and S yeast mutants of the genusSaccharomyces

Visible region of an absorption spectrum was followed in cells of original strains and of rough mutants ofSaccharomyces cerevisiae andS. cerevisiae var.ellipsoideus. It was found that there are no substantial differences in relative content of cytochromesb andc in aerobically grown rough and smooth yeast forms, in spite of the fact that both forms differ substantially in the metabolic oxygen quotient. If the cytochromes present were not reduced in washed cells by dithionite or by substrate addition, the rough forms exhibited a lower cytochrome b:c ratio than the smooth forms. Under anaerobic conditions of cultivation, the rough forms retained a typical aerobic spectrum, lacking, however, the cytochromea and a₃ band; the ratio of cytochromesb andc was changed in favour of cytochromeb (from the original 1.7: 1 up to 3.4: 1). The inability of the rough mutants to produce anaerobic cytochrome spectrum represented by cytochrome b₁ was connected with their inability to reproduce under anaerobic conditions.     

Superoxide and hydrogen peroxide production and NADPH oxidation stimulated by nitrofurantoin in lung microsomes: possible implications for toxicity.

The addition of nitrofurantoin to aerobic incubation mixtures containing rat lung microsomes strongly enhanced the generation of adrenochrome from epinephrine. Adrenochrome formation in this system was blocked by superoxide dismutase, but not by catalase. Hydrogen peroxide production was also strongly enhanced by nitrofurantoin in these preparations; superoxide dismutase did not significantly alter the amount of H₂O₂ measured, but no H₂O₂ was detected in incubation mixtures in the presence of catalase. Nitrofurantoin enhanced the oxidation of NADPH in lung microsomal suspensions under aerobic conditions; the enhancement was unaffected by catalase but was partially prevented by superoxide dismutase. Neither adrenochrome formation nor H₂O₂ production were enhanced by nitrofurantoin under anaerobic (N₂) conditions, but NADPH oxidation in the presence of nitrofurantoin was greater under anaerobic conditions than under aerobic conditions. These results are consistent with the view that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen species, emphasizing some $\text{in vitro}$ metabolic similarities with the lung-toxic herbicide, paraquat.     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c ₂ from mutant H41 demonstrated at the same time the participation of the c ₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation

The oxidation of NADH and accompanying reduction of oxygen to H₂O₂ stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H₂O₂ all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H₂0₂-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.     

Superoxide anion generation by the cytochrome bc1 complex

We have measured the rates of superoxide anion generation by cytochrome bc₁ complexes isolated from bovine heart and yeast mitochondria and by cytochrome bc₁ complexes from yeast mutants in which the midpoint potentials of the cytochrome b hemes and the Rieske iron-sulfur cluster were altered by mutations in those proteins. With all of the bc₁ complexes the rate of superoxide anion production was greatest in the absence of bc₁ inhibitor and ranged from 3% to 5% of the rate of cytochrome c reduction. Stigmatellin, an inhibitor that binds to the ubiquinol oxidation site in the bc₁ complex, eliminated superoxide anion formation, while myxothiazol, another inhibitor of ubiquinol oxidation, allowed superoxide anion formation at a low rate. Antimycin, an inhibitor that binds to the ubiquinone reduction site in the bc₁ complex, also allowed superoxide anion formation and at a slightly greater rate than myxothiazol. Changes in the midpoint potentials of the cytochrome b hemes had no significant effect on the rate of cytochrome c reduction and only a small effect on the rate of superoxide anion formation. A mutation in the Rieske iron-sulfur protein that lowers its midpoint potential from +285 to +220 mV caused the rate of superoxide anion to decline in parallel with a decline in cytochrome c reductase activity. These results indicate that superoxide anion is formed by similar mechanisms in mammalian and yeast bc₁ complexes. The results also show that changes in the midpoint potentials of the redox components that accept electrons during ubiquinol oxidation have only small effects on the formation of superoxide anion, except to the extent that they affect the activity of the enzyme.     

Electrochemical Sensors for Direct Reagentless Measurement of Superoxide Production by Human Neutrophils

Electrochemical sensors based on immobilised cytochrome c or superoxide dismutase for the measurement of superoxide radical production by stimulated neutrophils are described. Cytochrome c was immobilised covalently at a surface-modified gold electrode and by passive adsorption to novel platinised activated carbon electrodes (PACE). The reoxidation of cytochrome c at the electrode surface upon reduction by superoxide was monitored using both xanthine/xanthine oxidase and stimulated neutrophils as sources of the free radical. In addition, bovine Cu/Zn superoxide dismutase was immobilised to PACE by passive adsorption and superoxide, generated by xanthine/xanthine oxidase, detected by oxidation of hydrogen peroxide produced by the enzymic dismutation of the superoxide radical. A biopsy needle probe electrode based on cytochrome c immobilised at PACE and suitable for continuous monitoring of free radical production was constructed and characterised.     

Superoxide dismutase and hydrogen peroxide formation in Campylobacter sputorum subspecies bubulus

Cell-free extracts of Campylobacter sputorum subspecies bubulus contained superoxide dismutase. The enzyme was located in the cytoplasmic fraction and insensitive to cyanide. After centrifuging a cell-free extract at 144000 x g for 1.5 h the total activity in the supernatant fraction was threefold higher than in the crude cell-free extract. The pellet fraction thus obtained was shown to have a lowering effect on superoxide dismutase activities from different sources in the assay method used here. C. sputorum responded to a raised oxygen tension in the culture by an increase in the superoxide dismutase activity. The ability to produce superoxide anion radicals (O₂ ^-·) during oxidation of formate and lactate was demonstrated. Furthermore C. sputorum was found to produce H₂O₂ while oxidizing formate. In experiments in which the reduction of cytochrome c by formate was followed, step-wise kinetics were observed. One of the steady states then obtained was attributed to the oxidizing action of H₂O₂, because it was abolished by the addition of catalase and lengthened by H₂O₂ added in addition to H₂O₂ formed as a product of formate oxidation. An overall reaction for formate oxidation by C. sputorum is discussed.Abbreviations O₂ ^-· superoxide anion radical - NBT p-nitro blue tetrazolium chloride - ABTS 2,2-azino-di-[3-ethylbenzthiazoline sulfonate (6)] - TL-medium tryptose-lactate medium     

Inhibitors of superoxide dismutases: A cautionary tale

An extensive search resulted in the identification of pamoic acid as an inhibitor of superoxide dismutases. Pamoic acid appeared to rapidly and reversibly inhibit all types of superoxide dismutases and did so in both the cytochrome c reduction and in the dianisidine photooxidation assays, used to measure this activity. It could nevertheless be shown that pamoic acid did not at all inhibit superoxide dismutase but rather diminished the sensitivity of the assays. The mechanism proposed to account for this effect involved oxidation of pamoate, by O₂^?, to yield a pamoate radical which can then reduce cytochrome c or oxidize pyrogallol. Pamoate thus competes with superoxide dismutase for the available O₂^?, without affecting the observable effects of that O₂^? upon cytochrome c or upon pyrogallol. It consequently makes these assays less responsive to superoxide dismutase, while appearing to be without effect in the absence of superoxide dismutase. Several of the predicted consequences of this proposal were affirmed. Other workers, interested in finding inhibitors for superoxide dismutases, are hereby forwarned of this subtle snare.     

The photosynthetic apparatus and photoinduced electron transfer in the aerobic phototrophic bacteria <Emphasis Type="Italic">Roseicyclus mahoneyensis</Emphasis> and <Emphasis Type="Italic">Porphyrobacter meromictius</Emphasis>

Photosynthetic electron transfer has been examined in whole cells, isolated membranes and in partially purified reaction centers (RCs) of Roseicyclus mahoneyensis, strain ML6 and Porphyrobacter meromictius, strain ML31, two species of obligate aerobic anoxygenic phototrophic bacteria. Photochemical activity in strain ML31 was observed aerobically, but the photosynthetic apparatus was not functional under anaerobic conditions. In strain ML6 low levels of photochemistry were measured anaerobically, possibly due to incomplete reduction of the primary electron acceptor (Q_A) prior to light excitation, however, electron transfer occurred optimally under low oxygen conditions. Photoinduced electron transfer involves a soluble cytochrome c in both strains, and an additional reaction center (RC)-bound cytochrome c in ML6. The redox properties of the primary electron donor (P) and Q_A of ML31 are similar to those previously determined for other aerobic phototrophs, with midpoint redox potentials of +463 mV and −25 mV, respectively. Strain ML6 showed a very narrow range of ambient redox potentials appropriate for photosynthesis, with midpoint redox potentials of +415 mV for P and +94 mV for Q_A. Cytoplasm soluble and photosynthetic complex bound cytochromes were characterized in terms of apparent molecular mass. Fluorescence excitation spectra revealed that abundant carotenoids not intimately associated with the RC are not involved in photosynthetic energy conservation.     

CYTOCHROME c548 IN NOSTOC SP. (CYANOPHYCEAE): AN ELECTRON ACCEPTOR FROM REDUCED NADP IN THE DARK1

A c-type, carbon monoxide-binding cytochrome was isolated and studied from an endophytic Nostoc sp. Enzymatic transfer of electrons from NADPH to this cytochrome was demonstrated in cell-free preparations under dark, anaerobic conditions, probably mediated by NADPH-cytochrome reductase or ferredoxin-NADP reductase present in the extracts. This cytochrome, with an α-peak at 548 nm, had physicochemical properties similar to the low potential c-type cytochrome, cytochrome c₅₄₉, previously described by other workers from Anacystis nidulans. The participation of such c-cytochromes in dark electron transport, possibly respiratory, of bluegreen algae is strongly indicated.     

COMPARATIVE EFFECTS OF TWO PHOTOSENSITIVE COMPOUNDS ON SUPEROXIDE DISMUTASE ACTIVITY IN SPODOPTERA LITURA LARVAE*

Abstract Superoxide dismutase activity was detected in the fourth instar larvae of cutworm Spodoptera litura and effects of the two photosensitive compounds, α-terthienyl and the synthesized compound 1-phenyl-4–(3, 4-methylene dioxy) phenyl-diacetylene (signified as compound No. 5), on superoxide dismutase activity, were compared under the specified condition. Results indicated that the near ultraviolet irradiation (300–400 nm) almost had no effect on superoxide dismutase activity in vivo in the control larvae but could decrease superoxide dismutase activity in vitro. After treatment with photosensitive compounds and near ultraviolet irradiation, superoxide dismutase activity in vitro could be enhanced by the two chemicals, with α-terthienyl more effective, while in vivo it was hardly affected by the latter compound but could be inhibited by α-terthienyl. This suggested that the larvae treated by a-terthienyl were more susceptible to light than that by the latter compound whenever in vivo or in vitro. The possible mechanisms of action of the two photo-activated compounds were discussed.