Effect of O2 limitation on growth and respiration of the wild type and an ascorbate-tetramethyl-p-phenylenediamine-oxidase-negative mutant strain ofAzotobacter vinelandii

Azotobacter vinelandii strain AVOP (wild type) and an ascorbate-N,N,N,N-tetramethylene-p-phenylenediamine oxidase-negative mutant (AV11) were each grown in O₂-limited chemostat cultures. The results showed that the mutant strain grew and used O₂ less efficiently than the wild-type strain. Respiration rates of membrane particles with NADH or malate as the substrate were similar for each strain. Succinate oxidase activity was about fourfold lower in membrane particles prepared from mutant than from wild-type strain. Cyanide at a concentration that completely inhibited ascorbate-TMPD oxidase activity resulted in a 50% inhibition of NADH oxidase activity in membrane particles of AVOP. These data suggest that the cytochromeo,a ₁, oxidase branch of the respiratory chain may be important in the physiology ofA. vinelandii under O₂-limiting growth conditions.     

Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles

1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle.2. The activity of cytochrome c oxidase in the reconstituted vesicles is not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c.3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH^? inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externally added ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions.     

Use of 3,3′-diaminobenzidine as a biochemical electron donor for studies on terminal cytochrome oxidase activity inAzotobacter vinelandii

Azotobacter vinelandii cells readily oxidize the dye 3,3′-diaminobenzidine (DAB), which has been previously used as an electron donor for studies on the mitochondrial cytochromec oxidase reaction. The DAB oxidase activity inA. vinelandii cells was 10-fold lower than that noted for theN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase reaction, which is commonly used to measure terminal oxidase activity both in bacteria and mitochondria. Analyses of cell-free extracts show that DAB oxidase activity is concentrated almost exclusively in theA. vinelandii membrane fractions, most notably in the “R₃” electron transport particle (ETP). Oxidation studies, which employed both whole cells and the ETP fraction, show DAB oxidase activity to be markedly sensitive to KCN, NaN₃, and NH₂OH. A manometric assay system was developed which readily measured DAB oxidase activity in bacteria. Preliminary studies indicate that ascorbate-DAB oxidation inAzotobacter vinelandii measures terminal cytochrome oxidase activity in a manner similar to the TMPD oxidase reaction.     

Cyanide-resistant electron transport in sporulating Bacillus megaterium KM

The NADH oxidase activity of stage V mother-cell membranes, isolated from sporulating Bacillus megaterium KM, shows a greater inhibition by cyanide and displays this response at lower concentrations of cyanide than the stage V forespore inner membrane. Comparison of the effects of various respiratory inhibitors reveals that the difference in cyanide sensitivity between these membranes is located on the oxidase side of the 2-heptyl-4-hydroxyquinoline N-oxide-sensitive step. Both membranes contain cytochromes a+a₃, b-562, b-555, c and d, with three potential oxidases: cytochromes a+a₃, o and d. Cyanide difference spectra suggest that cytochromes b-562 and d may be the components involved in the cyanide-resistant electron transport pathway. Membrane ascorbate-N,N,N′,N′-tetramethylphenylenediamine and ascorbate 2,6-dichlorophenolindophenol oxidase activities are highly sensitive to cyanide. Evidence is presented for terminal branching of the respiratory chain with branches differing in cyanide sensitivity. The cyanide sensitivity of the NADH oxidase of membranes prepared from various stages of sporulation is compared. Morphogenesis of the mother-cell plasma membrane to a cyanide-sensitive form during stages II and III of sporulation is postulated.     

Regulation of respiration in paracoccus denitrificans: the dependence on redox state of cytochrome c and [ATP]/[ADP][Pi].

The respiratory rates of Paracoccus denitrificans cells, membrane fragments, and detergent-solubilized, ammonium sulfate-precipitated membrane fractions were measured with NADH and ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine as the substrates. It was found that the turnover numbers for cytochrome c were the same in the three preparations giving maximum values of approximately 300 s^?1 at 100% reduction of cytochrome c. NADH was rapidly oxidized in the detergent-solubilized, ammonium sulfate-precipitated membrane fraction which contained tightly bound cytochrome c. It is suggested that tight binding of cytochrome c in P. denitrificans does not impair its electron transport activity. The respiration of intact cells was dependent on the redox state of cytochrome c over a wide range of cytochrome c reduction, on the intracellular [ATP]/[ADP][P_i] and on the pH of the suspending medium. A conclusion is drawn that the basic principle(s) underlying regulation of cellular respiration is the same in the prokaryotic P. denitrificans and in mitochondria-containing eukaryotic organisms.     

Effect of crosslinking cytochrome c oxidase

Bovine heart cytochrome c oxidase and rat liver mitochondria were crosslinked in the presence and absence of cytochrome c. Biimidate treatment of purified cytochrome oxidase, which results in the crosslinkage of all of the oxidase protomers except subunit I when ? 20% of the free amines are modified, inhibits ascorbate-N,N,N′,N′-tetramethyl-p-phenylene diamine oxidase activity. Intermolecular crosslinking of cytochrome oxidase molecules, which results in the formation of large enzyme aggregates displaying rotational correlation times ? 1 ms, does not affect oxidase activity. Crosslinking of mitochondria covalently binds the cytochrome bc₁ and aa₃ complexes to cytochrome c, and inhibits steady-state oxidase activity. Addition of cytochrome c to purified cytochrome oxidase or to cytochrome c-depleted mitoplasts increases this inhibition slightly. Cytochrome c oligomers act as competitive inhibitors of native cytochrome c; however, crosslinking of cytochrome c to cytochrome c-depleted mitoplasts or purified cytochrome oxidase results in a catalytically inactive complex. These experiments indicate that cytochrome c oxidase subunit interactions are required for activity, and that cytochrome c mobility may be essential for electron transport between cytochrome c reductase and oxidase.     

Cytochrome c interactions with membranes. A photoaffinity-labeled cytochrome c.

The aryl azide, 2,4-dinitro-5-fluorophenylazide, was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c, with one to two dinitroazidophenyl groups per mole of the enzyme, has a half-reduction potential the same (± 10 mV) as native cytochrome c. The dissociation constant for the modified cytochrome c from cytochrome c-depleted mitochondrial membranes and the apparent K_m for the reaction with cytochrome c oxidase were each five to six times greater than the values for native cytochrome c. Irradiation of cytochrome c-depleted mitochondrial membranes supplemented with an excess of photoaffinity-labeled cytochrome c resulted in covalent binding of the derivative to the mitochondrial membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on agarose, Bio-Gel A, showed that labeled cytochrome c was bound covalently to cytochrome c oxidase in a 1:1 molar complex. The covalently linked cytochrome c-cytochrome c oxidase complex was active in mediating the electron transfer between N,N,N′,N′-tetramethyl-p-phenylenediamine/ascorbate and the oxidase.     

Respiratory physiology and cytochrome content ofLegionella pneumophila

The cytochrome composition of membrane vesicles ofLegionella pneumophila has been examined by low temperature (77°K) and room temperature difference spectroscopy, and cytochromes of thec, b, a, andd types have been detected. The presence ofc-type cytochrome was verified by formation of the pyridine ferrohemochromogen. A carbon monoxide-bindingc-type cytochrome was detected in CO-reduced minus reduced difference spectra and may also function in cytochromec reductase activity. Respiratory activities were determined for membrane vesicles, and reduced nicotinamide adenine dinucleotide (NADH) was the most rapidly oxidized substrate (199 nmol per min per mg protein), followed by succinate and malate. Cytochrome oxidase activity was demonstrated using ascorbate andN,N,N,N-tetramethyl-p-phenylenediamine (TMPD) (39 nmol per min per mg of protein). High levels of cyanide (K _i =10 mM) inhibited NADH oxidation, while low levels of 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO, 18 and 37 M) inhibited NADH oxidation by nearly 90%. The respiratory chain appeared to be complex and terminated by at least three terminal oxidases. Superoxide dismutase activity, but not catalase activity, was detected in cellular extracts.     

The terminal oxidases of Azotobacter vinelandii

The terminal respiratory chain of the aerobic nitrogen-fixing bacterium Azotobacter vinelandii was investigated by photochemical action spectra. Terminal cytochrome oxidases a₂, a₁ and o were confirmed as being the terminal oxidases for the physiological substrates NADH and L-malate. TMPD/ascorbate, not giving coupled phosphorylation uses cytochrome a₁ and possibly an o type. DCPIP/ascorbate, giving coupled phosphorylation uses neither cytochrome a₁ nor a₂.     

Regulation of lipogenesis in adipose tissue: the significance of the activation of pyruvate dehydrogenase by insulin

Simultaneous measurements were made of lipogenesis and pyruvate dehydrogenase activity in segments of rat epididymal adipose tissue incubated with saturating amounts of [U-¹⁴C]glucose and insulin. Glucose was converted to fatty acids at a rate only 64–79% of that permitted by the tissue's content of the active form of pyruvate dehydrogenase (PDH_a). Addition of either of the electron acceptors, phenazine methosulfate (10 μm) or N,N,N′,N′-tetramethyl-p-phenylenediamine (50 μm), increased lipogenesis until it equalled the PDH_a activity of the tissue. Pyruvate release was increased 2-fold or more by the electron acceptors, suggesting that the increase in lipogenesis might have resulted from an increase in the intracellular pyruvate levels such that PDH_a became saturated with substrate. Higher levels of the electron acceptors decreased PDH_a activity, and reduced lipogenesis correspondingly. The data suggest that the maximal rate of lipogenesis in the presence of glucose and insulin is limited by the inability of the tissue to elevate pyruvate levels sufficiently to saturate PDH_a. Although glycerol release was increased by either electron acceptor and insulin partially overcame this effect, the effects of the electron acceptors on PDH_a activity could not be attributed to an increase in lipolysis.     

The respiratory system of Pseudomonas putida: participation of cytochromes in electron transport

Rates of oxygen utilization by Pseudomonas putida respiratory particles were measured using the electron donors, reduced nicotinamide adenine dinucleotide (NADH) and succinate, and the oxidation-reduction dyes, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethyl-p-phenylenediamine. The maximal rates produced by NADH and succinate were similar for particles from either log- or stationary-phase cells, but rates measured using the dyes were much higher in stationary-phase particles. Cyanide and azide were very effective inhibitors of dye oxidation in both cases, but they produced only partial inhibition of NADH and succinate oxidation in log-phase particles and had no effect in the stationary phase. Spectral examination of the cytochromes at several levels of reduction produced by the various electron donors and inhibitors indicated that most of the cytochromes that were reduced by the dyes lie on a cyanide sensitive pathway of electron transport. These findings support the hypothesis that P. putida produces an electron transport system in the stationary phase which involves branching at the level of the cytochromes.Inhibition of oxygen utilization by CO was nearly complete for all four substrates in logphase particles. Inhibition was also reasonably effective for dye oxidation in the stationary phase, but there was no effect on NADH or succinate oxidation. Photochemical action spectra of the relief of CO inhibition revealed that NADH and succinate oxidation in log-phase particles probably involves cytochrome o. Oxidation of the dyes by either type of particles also appeared to involve cytochrome o, and the possibility of the participation of an a- or d-type cytochrome was also indicated.     

Direct Spectrophotometric Measurement of Photosystem I and Photosystem II Activities of Photosynthetic Membrane Preparations from Cyanophora paradoxa, Phormidium laminosum, and Spinach

Vesicles prepared with the French press from membranes of cyanelles of Cyanophora paradoxa retain O₂ evolution activity with rates up to 500 micromoles 2,6-dichlorophenolindophenol reduced per hour per milligram chlorophyll. This activity is immediately lost when the vesicles are transferred from the sucrose-phosphate-citrate preparation buffer into dilute phosphate buffer. Similar preparations from Phormidium laminosum, a thermophilic cyanobacterium retain activity under such conditions. Photosystem I activities of both cyanobacterial vesicle preparations were determined by direct spectrophotometric measurement of N,N,N′,N′-tetramethyl-p-phenylenediamine photooxidation in the presence of anthraquinone-2-sulfonate. The rates so determined were compared with rates of O₂ taken up in the presence of methyl viologen or anthraquinone-2-sulfonate as electron acceptors. The predicted stoichiometry of two was observed for moles of N,N,N′,N′-tetramethyl-p-phenylenediamine oxidized per mole of oxygen taken up. Anthraquinone-2-sulfonate was the better electron acceptor, and maximal rates of 943 micromoles per hour per milligram chlorophyll for O₂ uptake were observed for Phormidium laminosum preparations in the presence of superoxide dismutase. For purposes of comparison, spinach chloroplasts were assayed for similar activities. All preparations were readily assayed for photosystem I activity by the direct spectrophotometric method, which has advantages of simplicity and freedom from errors introduced by photoxidation of other substrates by photosystem I when O₂ uptake is measured.     

Rhizobium phaseoli cytochrome c-deficient mutant induces empty nodules on Phaseolus vulgaris L.

A Rhizobium phaseoli cytochrome mutant, unable to oxidize N,N,N′,N′ -tetramethyl-p-phenylend(amine (TMPD), was isolated after Mu-dl (Kan lac) mutagenesis of the wild-type strain CE-3. Mutant strain CFN4202 had sixfold less haem-c but similar levels of b type, o and aa₃ cytochromes than the wild-type strain. CFN402 strain also showed reduced NADH- and TMPD-oxidase activity than the wild-type strain. Succinate-oxidase activities were very similar. Western blot experiments, using antiserum against bovine c₁ and c cytochromes, revealed that both proteins were present in CFN4202 membranes, suggesting a defect of haem binding to cytochrome c. Nodules formed by this strain in Phaseolus vulgaris did not contain bacteroids. These data suggest that the cytochrome c-aa₃ chain or some other respiratory chain, containing c-type cytochromes in R. phaseoli, is essential for bacterial division during the early steps of the symbiotic interaction with the legume-host.     

Exogenous cytochrome c-dependent light-induced membrane potential generation in Rhodospirillum rubrum chromatophores

Rhodospirillum rubrum chromatophores associated with a planar phospholipid macromembrane by bivalent cations in the presence of quinone, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) and ascorbate generate a transmembrane electrical potential difference in the light. Photoelectrical activity is also observed if chromatophores are preincubated with cytochrome c; the maximum values of responses are reached upon subsequent addition of ascorbate and menadion in the absence of bivalent cations and TMPD. The cytochrome c-dependent responses of the illuminated chromatophores are inhibited by Ca²⁺ and prevented by quinones. The possibility of cytochrome c (c₂) translocation across the chromatophore membrane and the mechanism of charge transfer across the planar phospholipid membrane are discussed.     

Orientation and reactivity of cytochrome aa3 heme groups in proteoliposomes

Reduction of cytochrome aa₃ in proteoliposomes with ascorbate plus cytochrome c confirms that not more than 55% of the molecules are externally accessible and that the remainder are reduced only on the addition of membrane-permeable N,N,N′,N′tetramethyl-p-henylenediamine. Reduction in the presence of terminal inhibitors such as cyanide, azide, and carbon monoxide shows that likewise 50% of the cytochrome a is accessible and 50% inaccessible. Dithionite reduces part of the cytochrome a₃ in the presence of azide, and none in the presence of cyanide. Methyl viologen, which is somewhat membrane permeable, can reduce part of the cyanide-complexed cytochrome a₃ at low concentrations and all of it at high concentrations. Cytochrome a₃ is therefore also distributed randomly inside and outside the vesicles. Cytochrome c oxidase with externally facing cytochrome a is stimulated to high activity by its membrane association. Its turnover is dependent on the external pH and it is inhibited by external azide; trapping of azide cannot be used to demonstrate the orientation of the cytochrome a₃ hemes associated with externally facing cytochrome a. Cytochrome c oxidase with internally facing cytochrome a is rather sluggishly reactive. Its low activity accounts for the apparent failure of detergents to release extra activity on lysing proteoliposomes. Double reciprocal plots of the reaction of added cytochrome c with proteoliposomes indicate apparent biphasic binding in the energized state, which is abolished upon the addition of uncouplers and valinomycin. But no transmembraneous effect upon the oxidase reaction other than energization has been identified.     

Effect of artificial redox mediators on the photoinduced oxygen reduction by photosystem I complexes

The peculiarities of interaction of cyanobacterial photosystem I with redox mediators 2,6-dichlorophenolindophenol (DCPIP) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) were investigated. The higher donor efficiency of the reduced DCPIP form was demonstrated. The oxidized form of DCPIP was shown to be an efficient electron acceptor for terminal iron–sulfur cluster of photosystem I. Likewise methyl viologen, after one-electron reduction, DCPIP transfers an electron to the molecular oxygen. These results were discussed in terms of influence of these interactions on photosystem I reactions with the molecular oxygen and natural electron acceptors.     

Selection and organisation of denitrifying electron-transfer pathways in Paracoccus denitrificans

(1) Under anaerobic conditions the respiratory chain in cells of Paracoccus denitrificans, from late exponential cultures grown anaerobically with nitrate as electron acceptor and succinate as carbon source, has been shown to reduce added nitrate via nitrite and nitrous oxide to nitrogen without any accumulation of these intermediates. (2) Addition of nitrous oxide to cells reducing nitrate strongly inhibited the latter reaction. The inhibition was reversed by preventing electron flow to nitrous oxide with either antimycin or acetylene. Electron flow to nitrous oxide thus resembles electron flow to oxygen in its inhibitory effect on nitrate reduction. In contrast, addition of nitrite to an anaerobic suspension of cells reducing nitrate resulted in a stimulation of nitrate reductase activity. Usually, addition of nitrite also partially overcame the inhibitory effect of nitrous oxide on nitrate reduction. The reason why added nitrous oxide, but not nitrite, inhibits nitrate reduction is suggested to be related to the higher reductase activity of the cells for nitrous oxide compared with nitrite. Explanations for the unexpected stimulation of nitrate reduction by nitrite in the presence or absence of added nitrous oxide are considered. (3) Nitrous oxide reductase was shown to be a periplasmic protein that competed with nitrite reductase for electrons from reduced cytochrome c. Added nitrous oxide strongly inhibited the reduction of added nitrite. (4) Nitrite reductase activity of cells was strongly inhibited by oxygen in the presence of physiological reductants, but nitrite reduction did occur in the presence of oxygen when isoascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine was the reductant. It is concluded that competition for available electrons by two oxidases, cytochrome aa₃ and cytochrome o, severely restricted electron flow to the nitrite reductase (cytochrome cd). For this reason it is unlikely that the oxidase activity of this cytochrome is ever functional in cells. (5) The mechanism by which electron flow to oxygen or nitrous oxide inhibits nitrate reduction in cells has been investigated. It is argued that relatively small changes in the extent of reduction of ubiquinone, or of another component of the respiratory chain with similar redox potential, critically determine the capacity for reducing nitrate. The argument is based on: (i) the response of an anthroyloxystearic acid fluorescent probe that is sensitive to changes in the oxidation state of ubiquinone; (ii) consideration of the total rates of electron flow through ubiquinone both in the presence of oxygen and in the presence of nitrate under anaerobic conditions; (iii) use of relative extents of oxidation of b-type cytochromes as an indicator of ubiquinone redox state, especially the finding that b-type cytochrome of the antimycin-sensitive part of the respiratory chain is more oxidised in the presence of added nitrous oxide, which inhibits nitrate reduction, than in the presence of added nitrite which does not inhibit. Arguments against b- or c-type cytochromes themselves controlling nitrate reduction are given. (6) In principle, control on nitrate reduction could be exerted either upon electron flow or upon the movement of nitrate to the active site of its reductase. The observations that inverted membrane vesicles and detergent-treated cells reduced nitrate and oxygen simultaneously at a range of total rates of electron flow are taken to support the latter mechanism. The failure of an additional reductant, durohydroquinone, to activate nitrate reduction under aerobic conditions in the presence of succinate is also evidence that it is not an inadequate supply of electrons that prevents the functioning of nitrate reductase under aerobic conditions. (7) In inverted membrane vesicles the division of electron flow between nitrate and oxygen is determined by a competition mechanism, in contrast to cells. This change in behaviour upon converting cells to vesicles cannot be attributed to loss of cytochrome c, and therefore of oxidase activity, from the vesicles because a similar change in behaviour was seen with vesicles prepared from cells of a cytochrome c-deficient mutant.     

Mechanisms of peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate

Peroxidase oxidation of o-dianisidine, 3,3′,5,5′-tetramethylbenzidine, and o-phenylenediamine in the presence of sodium dodecyl sulfate (SDS), an anionic surfactant, was spectrophotometrically studied. It was found that 0.1–100 mM SDS concentrations stabilize intermediates formed in the peroxidase oxidation of these substrates. The cause of the stabilization is an electrostatic interaction between positively charged intermediates and negatively charged surfactant.     

Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrificans

Light inhibited each step of the denitrification process in whole cells of Rhodobacter sphaeroides forma sp. denitrificans. This inhibition by light was prevented in the presence of exogenous electron donors like N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) plus ascorbate or in the presence of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone). Addition of myxothiazol restored the inhibition by light in uncoupled cells. Measurements of light-induced absorbance changes under these conditions showed that this inhibition is due, for the steps of reduction of nitrite to dinitrogen, to the photooxidation of cytochromes c ₁ plus c ₂ and not due to the photoinduced membrane potential. Moreover, the presence of oxygen inhibited almost all of the reduction of nitrate and nitrous oxide but only 70% of the reduction of nitrite to nitrous oxide. These inhibitions were overcome in the presence of TMPD plus ascorbate. This implies that the inhibition in presence of oxygen was due to a diversion of the reducing power from the denitrifying chain to the respiratory chain. It was concluded from this series of experiments that the reduction of nitrate to nitrite is inhibited when the ubiquinone pool is partly oxidized and that nitrite and nitrous oxide reductions are inhibited when cytochromes c ₁ plus c ₂ are oxidized by photosynthesis or respiration.Abbreviations R Rhodobacter - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - HOQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - CCCP carbonyl cyanide m-chlorophenylhydrazone - cytochrome c ₁ cytochrome c ₂ plus cytochrome c ₁