Protein phosphorylation in a dopamine-sensitive homogenate of rat caudate: Effect of adenosine 3′,5′-cyclic monophosphate and putative neurotransmitters

Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of ³²P into proteins endogenous to a homogenate of rat caudate nucleus when 4 μM [γ?³²P] ATP is usedas substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 μM no significant increase in protein phosphorylation was seen.Incubation of the homogenate with 400 μM ATP and 100 μM dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study.     

Investigation of the dark metabolism of acetate in photoheterotrophically grown cells ofRhodospirillum rubrum

The mechanism of the aerobic dark assimilation of acetate in the photoheterotrophically grown purple nonsulfur bacteriumRhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation inRsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C₄-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle inRsp. rubrum cells can function as an anaplerotic pathway under aerobic dark conditions. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO₂ in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicated that citramalate and mesaconate were intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts ofRsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function inRsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Effect of organophosphorus insecticides on the oxidative processes in rat brain synaptosomes

Abstract: This paper describes the effect of four organophosphorus insecticides: Dipterex, DDVP, Ronnel and its oxygen analogue on the respiration of rat brain synaptosomes. Dipterex and DDVP in the concentrations used, 5, 50, or 500 μM, did not change the rate of oxygen uptake and oxidative phosphorylation in rat brain synaptosomes. Ronnel in the highest concentration (500 μM) inhibited respiration in state 3 conditions and abolished respiratory control by ADP. This inhibition was correlated with a change of cytochrome c oxidase activity. The oxygen analogue of Ronnel (OAR) in micromolar concentrations (50 μM) increased the rate of respiration of synaptosomes utilizing glutamate plus malate as substrate. Higher concentrations of OAR produced inhibition of respiration, cytochrome c oxidase and NADH: cytochrome c reductase activities. These observations are typical for uncouplers of oxidative phosphorylation. Noteworthy is the fact that the uncoupling activity of OAR was observed at concentrations which did not inhibit acetylcholinesterase activity. These findings seem to suggest that disturbances in oxidative processes could play an important role in the toxicity of organophosphorus insecticides. The relation between chemical structure and the ability of insecticides to affect oxidative phosphorylation is discussed.     

The tricarboxylic acid cycle and glycolysis in relation to ion transport by the ciliary body

1. The respiration and aerobic glycolysis of pig ciliary processes in oxygenated phosphate and bicarbonate buffers have been investigated. 2. Significant amounts of lactic acid are produced only in the presence of added glucose, but this does not change the endogenous respiration rate. 3. Succinate and citrate increase the oxygen uptake considerably, but pyruvate has almost no effect; oxaloacetate and fumarate stimulate slightly in the presence of glucose. Aspartate and fumarate together stimulate pyruvate utilization and are oxidized as fast as citrate. 4. Ouabain inhibits the oxidation of glucose and other substrates by limiting the ADP supply from the sodium transport system. Cyanide and azide inhibit respiration and stimulate glycolysis. 5. The transport mechanism depends largely on ATP from oxidative phosphorylation and regulates the rate of respiration and glycolysis by controlling ADP production from the Na(+)-K(+)-activated adenosine triphosphatase.     

Transmembrane ferricyanide reduction in tobacco callus cells

Transmembrane ferricyanide reduction in whole cells of normal and of transformed tobacco (Nicotiana tabacum) callus tissue was compared. It was found that low concentrations of indoleacetic acid (IAA, 0.1 μM), gibberellic acid (GA, 0.3 μM), and benzyl adenine (BA, 0.03 μM) stimulate external ferricyanide reduction in normal tobacco callus cells, but inhibit this reaction up to 67% in transformed cells when hormones are applied to cells 10 min prior to assay. Higher concentrations of these growth regulators (1 μM or greater) inhibit transmembrane ferricyanide reduction in both types of cells, with the exception of IAA, giving an initial stimulation of the rate (12%), followed by 24% inhibition after 2 min. The observed external ferricyanide reduction by whole tobacco callus cells may be explained on the basis of a transplasmalemma redox system, which may be associated with the iron metabolism of these cells.     

Active cell aggregation by immature rat Sertoli cells in primary culture: a role for cell surface glycoproteins.

Follicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10-day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH-stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0- to 18-hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose-specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of ³H-aminoacid incorporation as well as cell aggregation in 24-hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease ¹²⁵I-FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.     

Interaction of a synthetic polyanion with rat liver mitochondria

The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied.The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state.The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration).The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria.The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2-³H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria.It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c, and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.     

The effect of carbonyl cyanidep-trifluoromethoxy-phenylhydrazone on yeast cells

Carbonyl cyanidep-trifluoromethoxy-phenylhydrazone (FCCP) induced anaerobic fermentation of endogenous reserves and stimulated endogenous respiration in yeast. Under the given experimental conditions it stimulated the oxidation of glucose in concentrations of 0.4–3 μM; higher concentrations inhibited the oxidation and fermentation of glucose. The oxidation of pyruvate, ethanol andd(?) and L(+)-lactate was already inhibited by very low FCCP concentrations. Phosphorylation coupled to oxidation of succinate in subcellular particles was uncoupled by 0.1–1 μM FCCP. The effect on the intact cell depended on the ratio of the FCCP and cell concentration, since FCCP was absorbed from the medium by the cells. Absorption of FCCP by the cells was completely prevented by cysteine, which also blocked the inhibitory effect of FCCP on the metabolism of whole cells.     

Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Microaerobic respiration and oxidative phosphorylation by soybean nodule mitochondria: implications for nitrogen fixation

The infected cells of soybean (Glycine max) root nodules require ATP production for ammonia assimilation and purine synthesis under microaerobic conditions. It is likely that the bulk of this demand is supplied through mitochondrial oxidative phosphorylation. Mitochondria purified from root nodules respired and synthesized ATP in sub-micromolar oxygen concentrations as measured by leghaemoglobin spectroscopy and luciferase luminescence. Both oxygen uptake and the apparent ATP/O ratio declined significantly as the oxygen concentration fell below 100 μmol m^?3. Cytochrome-pathway respiration by root nodule mitochondria had a higher apparent affinity for oxygen (Km 50 μmol m^?3) than did mitochondria isolated from roots (Km 125 μmol m^?3). Electron micrographs showed that mitochondria predominated at the periphery of infected cells adjacent to gas-filled intercellular spaces, where the oxygen concentration is predicted to be highest. Calculations of oxygen concentration and nitrogen fixation rates on an infected cell basis suggest that the measured rates of ATP production by isolated mitochondria are sufficient for the quantifiable in vivo requirements of ammonia assimilation and purine synthesis. The possible roles of mitochondrial respiration in the control of infected cell metabolism are also discussed.     

The inhibition of hexose transport and metabolism by small amounts of adenosine 5′-triphosphate in isolated rat adipocytes

The effects of ATP on glucose transport and metabolism were studied in rat adipocytes. Over a concentration range of 10–250 μm, ATP was found to inhibit several aspects of adipocyte glucose metabolism, particularly when stimulated by insulin. Much of the effect of ATP on glucose metabolism appeared related to impairment of glucose transport, reflected by inhibition of both basal and insulin-stimulated rates of 3-O-methylglucose transport. ATP inhibited the V of insulin-stimulated 3-O-methylglucose transport, but had no effect on the K_m. The inhibitory effects of ATP were much less apparent when cells were preincubated with insulin, suggesting that ATP inhibited only the components of hexose transport not yet activated by the hormone. At very high medium glucose concentrations, where transport was no longer rate limiting for metabolism, there was no inhibition of glucose oxidation by 250 μm ATP. However, when hexose transport was blocked with cytochalasin B (50 μm), a small inhibitory effect of ATP persisted on basal and insulin-stimulated glucose and fructose oxidation, suggesting that intracellular metabolism was impaired. The mechanism of the intracellular effect did not appear to be caused by uptake of exogenous ATP. These studies provide further evidence that energy metabolism may play an important role in the regulation of facilitated glucose transport.     

Modification of swelling–contraction–aggregation processes in rat muscle mitochondria by the 1,4-dihydropyridines,cerebrocrast and glutapyrone,themselves and in the presence of azidothymidine

The influence of the 1,4-dihydropyridines (DHPs), water-soluble glutapyrone available as sodium, potassium and ammonium salts of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-DHP-4-carboxamide)glutaric acid, from one side, and a lipophylic cerebrocrast, 2-propoxyethyl 2,6-dimethyl-4-(2-difluoromethoxyphenyl)-1,4-DHP-3,5-dicarboxylate, from the other side, on partially damaged mitochondria of the Wistar rat hindlimb muscle was also studied. The following tests were made: (1) rates of endogenous respiration and substrate (succinate) oxidation and oxidative phosphorylation; (2) rates and amplitudes of high-amplitude swelling and contraction after the addition of ATP, ADP and succinate to the previously swollen mitochondria and (3) rate of reversible self-aggregation of mitochondria isolated in salt media after ATP-induced contraction without and in the presence of azidothymidine (AZT). Cerebrocrast (10–100 μM ) partially normalized the endogenous respiration rate and slightly augmented the respiration rate after the addition of succinate and to lesser extent ADP. Cerebrocrast in a concentration-dependent manner (2·5–50 μM ) increased (two-fold at 20–50 μM ) the active contraction amplitude of swollen mitochondria, induced by single or repeated additions of ATP. The influence of cerebrocrast on the ADP- and succinate-induced contractions was less obvious. Unlike cerebrocrast glutapyrone caused a reduction of the ATP-induced contraction amplitude (two-fold at 0·5–5·0 mM ), not impairing the mitochondrial contraction ability in response to ATP or succinate. Pre-exposure to 2·5 mM glutapyrone resulted in at least a 10-fold inhibition of the reversible aggregation rate in the presence of 99 and 198 μM AZT. The results suggest the usefulness of further study of cerebrocrast and glutapyrone in preventing AZT-induced and some other mitochondrial myopathies. © 1997 John Wiley & Sons, Ltd.     

Adenine nucleotide pool variations in intact Nitrobacter winogradskyi cells

1. The ATP pool in Nitrobacter winogradskyi cells was determined by means of the luciferin-luciferase enzyme system and the ADP and AMP pools were measured after enzymatic conversion into ATP.
2. In the fist 10 min after addition of nitrite to endogenously respiring cells, which had stood for 5–16 days after completion of the nitrite oxidation, the ATP pool dropped about 60%.
3. During the log phase the ATP pool was approx. 20–40 pmoles/5 μg cell-N. During growth it increased exponentially by 3–4 times the amount until the nitrite had been used up. Subsequently the ATP pool decreased at first rapidly and then more slowly without sinking to 0 in the first 2 months after nitrification.
4. Nitrite oxidizing cells had an energy charge of 0.37 during the log-phase. After approx. 90% of the substrate had been used up the energy charge had reached 0.57.
5. If the CO₂ assimilation was inhibited in growing cultures by increased oxygen partial pressure, nitrite oxidation continued but the ATP pool increased.
6. The ATP pool and the activity of the endogenous respiration decreased by more than 50% during the first hours after the substrate had been used up.

     

Comparison of oxygen consumption rates in minimally transformed BALB/3T3 and virus-transformed 3T3B-SV40 cells

In the recent years, bioenergetics of tumor cells and particularly cell respiration have been attracting great attention because of the involvement of mitochondria in apoptosis and growing evidence of the possibility to diagnose and treat cancer by affecting the system of oxidative phosphorylation in mitochondria. In the present work, a comparative study of oxygen consumption in 3T3B-SV40 cells transformed with oncovirus SV40 and parental BALB/3T3 cells was conducted. Such fractions of oxygen consumption as “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration observed in the presence of protonophore were determined. Maximal respiration was shown to be only slightly decreased in 3T3B-SV40 cells as compared to BALB/3T3. However, in the case of certain fractions of cellular respiration, the changes were significant. “Phosphorylating” respiration was found to be reduced to 54% and “reserve” respiration, on the contrary, increased up to 160% in virus-transformed 3T3B-SV40 cells. The low rate of “phosphorylating” respiration and high “reserve” respiration indicate that under normal incubation conditions the larger part of mitochondrial respiratory chains of the virus-transformed cells is in the resting state (i.e. there is no electron transfer to oxygen). The high “reserve” respiration is suggested to play an important role in preventing apoptosis of 3T3B-SV40 cells.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, K_D = 1–2 μM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (K_I = 240–300 μM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This “tightly bound” ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetic studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, P_i can maintain the active form of the enzyme.     

Energy coupling for membrane hyperpolarization in Lemna: respiration rate,ATP level and membrane potential at low oxygen concentrations

Respiration rate, ATP content and membrane potential of Lemna have been measured as a function of the concentration of dissolved oxygen. Kinetic analysis showed that within the range from 1 μM to 20 μM O₂, the respiration rate of isolated mitochondria and intact plants was a hyperbolic function of the oxygen concentration. The apparent Michaelis constant (K _m ) for the oxygen of respiration of intact plants (1.15±0.08 μM) is close to that for isolated mitochondria (1.07±0.06 μM), so that diffusion of oxygen within the tissue was obviously not rate-limiting under the applied experimental conditions. The ATP level decreased in parallel with the respiration rate when the oxygen concentration was reduced. In contrast, the hyperpolarization of the membrane potential above the diffusion potential had already decreased at oxygen concentrations where the respiration rate and ATP level remained practically unchanged and was completely abolished at oxygen concentrations above the K _m of respiration. This result is discussed according to the current models for electrogenic pumps. It is concluded that ATP cannot be the fuel for the electrogenic process under investigation.     

Some effects of chloramphenicol on the metabolism of chlorella

Summary A chloramphenicol concentration of 3 mg per ml inhibits uptake of ¹⁴C-labelled phenylalanine, lysine, and adenine by Chlorella cells. Incorporation into both the free pool and the TCA insoluble fraction is inhibited. The inhibition is not related to inhibition of protein synthesis since cycloheximide (a specific inhibitor of protein synthesis in Chlorella) does not inhibit uptake of the ¹⁴C-labelled amino acids. Uptake of ¹⁴C-uracil is not inhibited by chloramphenicol.Both chloramphenicol and 2.4-dinitrophenol stimulate endogenous respiration of Chlorella, but whereas the latter reduces the internal concentration of ATP, the former (in concentrations of 1–3 mg/ml) stimulates it about two-fold. Similar concentrations of chloramphenicol decreases slightly the concentration of ADP, and it is therefore suggested that in Chlorella, chloramphenicol concentrations of 1–3 mg per ml inhibit some energy-linked reactions by preventing ATP utilization.     

Transport of Catecholamines by Native and Reconstituted Rat Heart Synaptic Vesicles

Highly purified “heavy” synaptic vesicles were isolated from rat heart by differential centrifugation. Because of the high intravesicular concentrations of proteins, catecholamine, and ATP, resealed vesicle ghosts were prepared and used to study the detailed kinetics of catecholamine transport. ATP stimulated the uptake of /-norepinephrine and was saturable with a K_m for l-norepinephrine at 3.3 μM and 1.8 mM for ATP. The ghosts also accumulated 5-hydroxytryptamine and l-epinephrine via an ATP-dependent mechanism. Uptake was stereospecific for the l-form. A functional catecholamine transporter could be solubilized by the detergent octyl-glucoside and incorporated into phospholipid vesicles, which, after detergent removal, generated proteoliposomes that accumulated l-norepinephrine. Reserpine- and l-propranolol-sensitive accumulation against a concentration gradient is achieved by artificially creating a pH gradient across the membrane, and lends further support to the idea that at least the initial phase of catecholamine transport is driven by the trans-membrane pH gradient created by the proton-translocating ATPase.     

Respiratory control determines respiration and nitrogenase activity of Rhizobium leguminosarum bacteroids.

The relationship between the O2 input rate into a suspension of Rhizobium leguminosarum bacteroids, the cellular ATP and ADP pools, and the whole-cell nitrogenase activity during L-malate oxidation has been studied. It was observed that inhibition of nitrogenase by excess O2 coincided with an increase of the cellular ATP/ADP ratio. When under this condition the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) was added, the cellular ATP/ADP ratio was lowered while nitrogenase regained activity. To explain these observations, the effects of nitrogenase activity and CCCP on the O2 consumption rate of R. leguminosarum bacteroids were determined. From 100 to 5 microM O2, a decline in the O2 consumption rate was observed to 50 to 70% of the maximal O2 consumption rate. A determination of the redox state of the cytochromes during an O2 consumption experiment indicated that at O2 concentrations above 5 microM, electron transport to the cytochromes was rate-limiting oxidation and not the reaction of reduced cytochromes with oxygen. The kinetic properties of the respiratory chain were determined from the deoxygenation of oxyglobins. In intact cells the maximal deoxygenation activity was stimulated by nitrogenase activity or CCCP. In isolated cytoplasmic membranes NADH oxidation was inhibited by respiratory control. The dehydrogenase activities of the respiratory chain were rate-limiting oxidation at O2 concentrations (if >300 nM. Below 300 nM the terminal oxidase system followed Michaelis-Menten kinetics (Km of 45 +/- 8 nM). We conclude that (i) respiration in R. leguminosarum bacteroids takes place via a respiratory chain terminating at a high-affinity oxidase system, (ii) the activity of the respiratory chain is inhibited by the proton motive force, and (iii) ATP hydrolysis by nitrogenase can partly relieve the inhibition of respiration by the proton motive force and thus stimulate respiration at nanomolar concentrations of O2.