ATP synthesis during exogenous NADH oxidation. A reappraisal

This paper reports a reinvestigation on the pathway for mitochondrial oxidation of exogenous NADH and on the related ATP synthesis, first reported 30 years ago (Lehninger, A.L. (1951) J. Biol. Chem. 190, 345-359). NADH oxidation, both in intact and in water-treated mitochondria, is 90% inhibited by mersalyl, an inhibitor of the outer membrane NADH-cytochrome b5 reductase, and 10% inhibited by rotenone. The mersalyl-sensitive, but not the rotenone-sensitive, portion of NADH oxidation is stimulated by exogenous cytochrome c. Part of ATP synthesis is independent of exogenous NADH and cytochrome c, and is inhibited by rotenone and antimycin A, and is therefore due to oxidation of endogenous substrates. Another part of ATP synthesis is dependent on exogenous NADH and cytochrome c, is insensitive to rotenone and antimycin A, and is due to operation of cytochrome oxidase. It is concluded that (i) oxidation of exogenous NADH in the presence of cytochrome c proceeds mostly through NADH-cytochrome b5 reductase and cytochrome b5 on the outer membrane and then through cytochrome oxidase via the cytochrome c shuttle, and (ii) ATP synthesis during oxidation of exogenous NADH is partly due to oxidation of endogenous substrates and partly to operation of cytochrome oxidase receiving electrons from the outer membrane via cytochrome c.     

The oxidative activities of membrane vesicles from Bacillus caldolyticus. Energy-dependence of succinate oxidation

1. The properties of membrane vesicles from the extreme thermophile Bacillus caldolyticus were investigated. 2. Vesicles prepared by exposure of spheroplasts to ultrasound contained cytochromes a, b and c, and at 50 degrees C they rapidly oxidized NADH and ascorbate in the presence of tetramethyl-p-phenylenediamine. Succinate and l-malate were oxidized more slowly, and dl-lactate, l-alanine and glycerol 1-phosphate were not oxidized. 3. In the absence of proton-conducting uncouplers the oxidation of NADH was accompanied by a net translocation of H(+) into the vesicles. Hydrolysis of ATP by a dicyclohexylcarbodi-imide-sensitive adenosine triphosphatase was accompanied by a similarly directed net translocation of H(+). 4. Uncouplers (carbonyl cyanide p-trifluoromethoxyphenylhydrazone or valinomycin plus NH(4) (+)) prevented net H(+) translocation but stimulated ATP hydrolysis, NADH oxidation and ascorbate oxidation. The last result suggested an energy-conserving site in the respiratory chain between cytochrome c and oxygen. 5. Under anaerobic conditions the reduction of cytochrome b by ascorbate (with tetramethyl-p-phenylenediamine) was stimulated by ATP hydrolysis, indicating an energy-conserving site between cytochrome b and cytochrome c. However, no reduction of NAD(+) supported by oxidation of succinate, malate or ascorbate occurred, neither did it with these substrates in the presence of ATP under anaerobic conditions, suggesting that there was no energy-conserving site between NADH and cytochrome b. 6. Succinate oxidation, in contrast with that of NADH and ascorbate, was strongly inhibited by uncouplers and stimulated by ATP hydrolysis. These effects were not observed when phenazine methosulphate, which transfers electrons from succinate dehydrogenase directly to oxygen, was present. It was concluded that in these vesicles the oxidation of succinate was energy-dependent and that the reoxidation of reduced succinate dehydrogenase was dependent on the outward movement of H(+) by the protonmotive force. 7. In support of the foregoing conclusion it was shown that the reduction of fumarate by NADH was an energy-conserving process. 8. If the activities of vesicles accurately represent those of the intact organism it appears that in B. caldolyticus the reduction of fumarate to succinate at the expense of reducing equivalents from NADH is energetically favoured over succinate oxidation even under aerobic conditions. This may be related to the need for an ample supply of succinate for haem synthesis in order to provide cytochromes for the organism.     

The oxidation of external NADH by an intermembrane electron transfer in mitochondria from the ubiquinone-deficient mutant E3-24 of Saccharomyces cerevisiae

Cells of the E3-24 mutant of the strain D273-10B of Saccharomyces cerevisiae, grown in a fermentable substrate not showing catabolite repression of respiration (2% galactose), are able to respire, in spite of their ubiquinone deficiency in mitochondrial membranes. Mitochondria isolated from these mutant cells oxidize exogenous NADH through a pathway insensitive to antimycin A but inhibited by cyanide. Addition of methanolic solutions of ubiquinone homologs stimulates the oxidation rate and restores antimycin A sensitivity in both isolated mitochondria and whole cells. Mersalyl preincubation of isolated mitochondria inhibits both NADH oxidation and NADH-cytochrome c oxido-reductase activity (assayed in the presence of cyanide) with the same pattern. Electrons resulting from the oxidation of exogenous NADH reduce both cytochrome b5 and endogenous cytochrome c. The increase in ionic strength stimulates NADH oxidation, which is also coupled to the ATP synthesis with an ATP/O ratio similar to that obtained with ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) as substrate. The effect of cyanide on these activities and on NADH-induced endogenous cytochrome c reduction is also comparable. These results support the existence in vivo and in isolated mitochondria of a energy-conserving pathway for the oxidation of cytoplasmatic NADH not related to the dehydrogenases of the inner membrane, the ubiquinone, and the b-c1 complex, but involving a cytochrome c shuttle between the NADH-cytochrome c reductase of the outer membrane and cytochrome oxidase in the inner membrane.     

The Effect of Respiratory Inhibitors on NADH, Succinate and Malate Oxidation in Corn Mitochondria

The effect of a series of respiratory inhibitors on the oxidation of NADH in state 4 and state 3 conditions was studied with corn shoot mitochondria. Comparisons were made using malate and succinate as substrates. The inhibitors, rotenone, amytal, antimycin A and cyanide, inhibited oxidation of NADH in state 3 but rotenone and amytal did not inhibit oxidation in state 4. The inhibition by antimycin A was partially overcome by the presence of cytochrome c. The results indicate the presence of alternative pathways available for NADH oxidation depending on the metabolic condition of the mitochondria. Under state 4 conditions, NADH oxidation bypasses the amytal and rotenone sensitive sites but under state 3 conditions a component of the NADH respiration appears to be oxidized by an internal pathway which is sensitive to these inhibitors. Still a third pathway for NADH oxidation is dependent on the addition of cytochrome c and is insensitive to antimycin A. Succinate oxidation was sensitive to cyanide and antimycin A under both state 4 and state 3 conditions as well as amytal and rotenone under state 3 conditions but was not inhibited by amytal and rotenone under state 4 conditions. Malate oxidation was inhibited by cyanide, rotenone and amytal under both state 4 and state 3 conditions. Antimycin A inhibited state 3 but did not appreciably alter state 4 rates of malate oxidation. With all substrates tested inhibition by antimycin A was greatly facilitated by preswelling the mitochondria for 10 min. This was interpreted to indicate that swelling increases the accessibility of antimycin A to the site of inhibition.     

Nutritional effects on mitochondrial bioenergetics. Alterations in oxidative phosphorylation by rat liver mitochondria.

Rats malnourished since birth and fed on a protein-free diet for 2 weeks showed a 23-27% decrease in the State-3 oxidation of glutamate, succinate and ascorbate + NNN' N'-tetramethyl-p-phenylenediamine by liver mitochondria compared with control fed animals. ATP synthesis and the respiratory control index were diminished at the three coupling sites, but significant alterations were not observed in ADP/O ratios. Vmax. for NADH oxidation in electron-transport particles was 40% lower. Mitochondrial cytochromes b and c1 remained unchanged, but cytochrome c was increased by 26%. Cytochromes a + a3 were diminished by 22%. Vmax. for mitochondrial ATPase was 23% lower. These results suggest that the lower content of cytochrome a + a3 at the rate-controlling step of oxidative phosphorylation in malnourished rats might be mainly responsible for the decrease in substrate oxidations as well as ATP synthesis at the three coupling sites. The decreased synthesis and hydrolysis of ATP suggests that other energy-dependent mitochondrial processes could be decreased during malnutrition.     

Mitochondria from human term placenta. II. Characterization of respiratory pathways and coupling mechanisms.

Pathways of electron transport utilized for respiration in human term placental mitochondrial preparations were differentiated and characterized through the use of classical respiratory chain inhibitors and multiple sources of reducing equivalents. Mechanisms of associated energy conservation and utilization were examined in the preparations with uncouplers and inhibitors of phosphorylation. Inhibition by rotenone, antimycin A and cyanide established the classical electron transport chain as the major pathway of respiration with glutamate and succinate as substrates. Approximately 20% of glutamate-supported respiration was insensitive to inhibitors and may proceed by the cytochrome P-450 linked pathway of electron transport. Approximately 50% of ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine supported respiration was insensitive to 10-3 M cycanide and must utilize an undefined by-pass of cytochrome oxidase. A rotenone- and antimycin-insensitive, exterior pathway for NADH oxidation was demonstrated which could be artificially linked by exogenous cytochrome c to the cytochrome oxidase region of the classical electron transport system. Glycerol 3-phosphate also supported oxidative phosphorylation yielding ADP/O ratios of 2. Respiration of placental mitochondria was stimulated by 2,4-dinitrophenol and gramicidin. With succinate, dinitrophenol-stimulated respiration exceeded that obtained in the presence of ADP. Oligomycin and atractyloside prevented the stimulation of respiration by ADP. Thus, respiration appeared coupled through normal mechanisms to ATP formation and ion transport. A preferential coupling of respiration to the energy-utilizing processes of steroid hormone biosynthesis may exist.     

The activation of non-phosphorylating electron transport by adenine nucleotides in Jerusalem-artichoke (Helianthus tuberosus) mitochondria.

In isolated plant mitochondria the oxidation of both succinate and exogenous NADH responded in the expected manner to the addition of ADP or uncoupling agents, and the uncoupled rate of respiration was often in excess of the rate obtained in the presence of ADP. However, the oxidation of NAD+-linked substrates responded in a much more complex manner to the addition of ADP or uncoupling agents such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone to mitochondria oxidizing pyruvate plus malate failed to result in a reliable stimulation; this uncoupled rate could be stimulated by adding AMP or ADP in the presence of oligomycin or bongkrekic acid. Spectrophometric measurements showed that the addition of AMP or ADP resulted in the simultaneous oxidation of endogenous nicotinamide nucleotide and the reduction of cytochrome b. ADP was only effective in bringing about these changes in redox state in the presence of Mg2+ whereas AMP did not require Mg2+. It was concluded that AMP activated the flow of electrons from endogenous nicotinamide nucleotide to cytochrome b, possible at the level of the internal NADH dehydrogenase.     

Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola

Beffa, T., Pezet, R. and Turian, G. 1987. Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola. Mitochondria from young dormant α spores of Phomopsis viticola Sacc. (ATCC 44940) were isolated by grinding and differential centrifugation. They presented a good integrity of their inner and outer membranes as measured by biochemical assays. Electron microscopic analysis revealed an homogenous population. The highest respiratory activities were observed with NADH and ascorbate + tetra-methyl-p-phenylenediamine (TMPD). Malate stimulated the oxidation of pyruvate, citrate or α-ketoglutarate. The coupling of respiration to oxidative phosphorylation appeared at the time of spore germination. The respiratory activities of mitochondria isolated from young dormant α spores of P. viticola were strongly inhibited by S°. The sensitivity of mitochondrial oxidation of different substrates (NADH, pyruvate + malate, succinate and ascorbate + TMPD) to S° was heterogenous and indicated multiple-site action. Thus preincubation of mitochondria with 30 μM S° before addition of substrates fully prevented NADH oxidation (>98%), and strongly inhibited oxidation of pyruvate + malate (85%), succinate (60%) and ascorbate + TMPD (74%). S° inhibited more rapidly the oxidation of succinate than that of other substrates. In the presence of dithiothreitol (DTT), S°-inhibited oxidation of all substrates (except ascorbate + TMPD) could only be transiently and weakly reestablished. The inhibitory action of S° on the oxidation of NADH, pyruvate + malate and succinate was higher than that observed with sulfhydryl group reagents such as mersalyl, Hg-acetate or p - chloromercuribenzoate. In contrast to S° these SH-group reagents could not inhibit oxidation of ascorbate + TMPD. S°, by its dual capacity to oxidize the SH-groups and to self-reduce, probably at the level of cytochrome c oxidase, could produce a modification of the oxidation state of the respiratory complexes thereby disturbing the electron flux.     

Studies on yeast mitochondria: Some properties of mitochondria isolated from Saccharomyces Carlsbergensis grown in normal glucose medium

1. Mitochondria isolated from Saccharomyces Carlsbergensis are found to have three phosphorylation sites in the respiratory chain for the oxidation of NADH and NAD⁺-linked substrates and two for succinate oxidation. Freshly isolated mitochondria exist in an inhibited state with no respiratory control, but on ageing for 2–3 h a good coupled state is obtained. -Ketogultarate and -glycerophosphate are poorly oxidized in these mitochondria.

2. Exogenous NADH is a very good substrate for yeast mitochondrial respiration and apparently has a very low K_m. However, one-third of the added NADH is not available for oxidation probably due to some form of compartmentation. Studies of both oxygen uptake and the redox changes of cytochrome b show complete oxidation of two-third of the added NADH.

3. Difference spectra of yeast mitochondria at liquid-nitrogen temperatures show all the characteristic peaks of cytochromes a (600 nm), b (558, 525 and 428 nm), c₁ (552 nm) and c (545 and 516 nm).

4. The reduction of cytochrome b by dicumarol in antimycin A inhibited mitochondria provides evidence for an energy conservation site on the substrate side of cytochrome b.

5. In the absence of added ADP, the oxidation of malate and pyruvate occurs in the yeast mitochondria in a new respiratory state (State X) where the oxygen uptake occurs at State 4 rate but the redox level of the flavins, cytochrome b and c are similar to State 3. State X respiration is believed to be due to depletion of the high energy intermediate C I caused by the substrate anions accumulation.

6. The responses of yeast mitochondria to Ca²⁺ are qualitatively similar to those in rat liver mitochondria, particularly with respect to respiratory stimulation, membrane alkalinization and its accumulation in the mitochondria with succinate as the substrate in the presence and absence of acetate.     

Acetyl-coenzyme A deacylase activity in liver is not an artifact. Subcellular distribution and substrate specificity of acetyl-coenzyme A deacylase activities in rat liver

The stoicheiometry of proton translocation, the amounts of cytochromes firmly bound to membranes, and cell yields with respect to succinate and O(2) have been measured in the facultative methylotroph Pseudomonas AM1 and in the mutant lacking cytochrome c (mutant PCT76) during carbon-limited growth and carbon-excess growth. -->H(+)/O ratios during endogenous respiration of about 4 were measured in wild-type bacteria grown in carbon-excess conditions, and in the mutant in all growth conditions. During methanol- or succinate-limited growth of wild-type bacteria the -->H(+)/O ratio increased to about 6. Cell yields with respect to succinate and O(2) were higher in wild-type than in the mutant lacking cytochrome c by an amount suggesting loss in the mutant of 30% of the ATP-generating capacity of wild-type bacteria. During carbon-limited growth on methanol or succinate some cytochrome c was tightly bound to bacterial membranes, whereas none was tightly bound in bacteria grown in batch-culture or in NH(4) (+)-limited conditions. It is proposed that the role of cytochrome c in Pseudomonas AM1 depends on growth conditions and hence on the ;needs' of the bacteria. During growth in carbon-excess conditions it is only required for methanol oxidation, mediating between methanol dehydrogenase and cytochrome a/a(3). In these conditions oxidation of NADH and succinate by way of cytochrome b and cytochrome a/a(3) occurs without the mediation of cytochrome c. This is the only route for oxidation of NADH and succinate in the cytochrome c-deficient mutant in all growth conditions. During carbon-limited growth the cytochrome c becomes bound to the membrane in such a way that it can mediate between cytochromes b and a/a(3), hence becoming involved in proton translocation and ATP synthesis during NADH and succinate oxidation. An alternative possibility is that in wild-type bacteria the cytochrome c is always involved in electron transport, but that its involvement in measurable proton translocation only occurs in carbon-limited conditions.     

Effect of perchlorate on mitochondrial function

Administration of sodium perchlorate to rats for 45 days leads to decreased activities of citric acid cycle enzymes. The oxidation of succinate both in state 3 and state 4 conditions and endogenous ATP content of mitochondria decreased during perchlorate toxicity. The significant decrease in cytochrome aa3 in perchlorate-treated rats may be one of the prime factors involved in the decreased rate of respiration. The permeability of mitochondria of perchlorate-treated rats is altered as indicated by increased oxidation of NADH and low respiratory control ratio (RCR).     

Influence of increased environmental temperature on oxidation processes in rat liver mitochondria

Exposure of rats to elevated temperature of 28 degrees C or 35 degrees C for 3 days six hours daily resulted in a decreased rate of oxidation with succinate or glutamate + malate as substrates, by the mitochondria of liver. The higher decrease was observed in environment temperature of 35 degrees C. There was no change in ADP/O ratio. The activities of NADH: cytochrome c reductase and cytochrome oxidase were stimulated but activities of succinate dehydrogenase and succinate cytochrome reductase were decreased.     

The Respiratory Chain of Chlorella protothecoides: II. Isolation and Characterization of Mitochondria

Mitochondria were isolated from glucose-bleached Chlorella protothecoides Krüger, Indiana strain 25. These mitochondria oxidized succinate, NADH, and l-malate at high rates. Oxygen uptake with these substrates was partially inhibited by 1 mmm-chlorobenzhydroxamic acid (mCLAM). Respiratory control was seen with succinate as substrate in the presence of mCLAM. The apparent Km for succinate was determined to be 0.83 mm. Chlorella mitochondria catalyzed the oxidation of reduced horse heart cytochrome c.     

Regulation of the heart mitochondrial respiration rate. Comparison of oxidation of succinate and NAD-dependent substrates]

Regulation of respiration at all rates between State 4 and State 3 was studied in heart mitochondria oxidizing FAD- and NAD-dependent substrates (succinate, pyruvate + + malate and palmitoylcarnitine). The creatine phosphokinase ADP-regenerating system was used which allows to fix the concentrations of extramitochondrial adenine nucleotides in such a way that the rate of respiration is controlled by mitochondrial processes alone. It was shown that respiration is controlled by delta mu(H+)-utilizing system within the respiration rate interval from State 4 till 70-80% of the maximal rate in State 3 (corresponding to physiological rates) both for NAD- and FAD-dependent substrates. The main step in the control of respiration near State 4 is proton leakage through the inner mitochondrial membrane, whereas in all the other parts of the mentioned interval this role is assigned to the adenine nucleotide translocator (ANT). The control coefficient for ANT is higher, while that of proton leakage is lower at the same relative rates of respiration with NAD-dependent substrates compared with succinate. These differences were found to be related to much higher values of the membrane potential generated at the same relative rates of succinate oxidation in comparison with the case with pyruvate + + malate. The contribution of delta mu(H+)-utilizing system to respiration control sharply decreases, whereas that of the delta mu(H+)-generating system increases at maximal rates of respiration near State 3. This phenomenon in more characteristic of succinate. In this case the control coefficient of ANT drops to zero, while that of succinate dehydrogenase rises to 0.7.     

Suppression of the mitochondrial oxidation of (-)-palmitylcarnitine by the malate-aspartate and alpha-glycerophosphate shuttles.

Palmitylcarnitine oxidation by isolated liver mitochondria has been used to investigate the interaction of fatty acid oxidation with malate, glutamate, succinate, and the malate-aspartate shuttle. Mitochondria preincubated with fluorocitrate were added to a medium containing 2mM ATP and ATPase. This system, characterized by a high energy change, allowed titration of respiration to any desired rate between States 4 and 3 (Chance, B., and Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134). When respiration (reference, with palmitylcarnitine and malate as substrates) was set at 75% of State 3, the oxidation of palmitylcarnitine was limited by acetoacetate formation. The addition of malate or glutamate approximately doubled the rate of beta oxidation. Malate circumvented this limitation by citrate formation, but the effect of glutamate apparently was due to enhancement of the capacity for ketogenesis. The rate of beta oxidation was curtailed when malate and glutamate were both present. This curtailment was more pronounced when the malate-aspartate shuttle was fully reconstituted. Among the oxidizable substrates examined, succinate was most effective in inhibiting palmitylcarnitine oxidation. Mitochondrial NADH/NAD+ ratios were correlated positively with suppression of beta oxidation. The degree of suppression of beta oxidation by the malate-aspartate shuttle (NADH oxidation) or by succinate oxidation was dependent on the respiratory state. Both substrates extensively reduced mitochondrial NAD+ and markedly suppressed beta oxidation as respiration approached State 4. Calculations of the rates of flux of hydrogen equivalents through beta oxidation show that the suppression of beta oxidation by glutamate or by the malate-aspartate shuttle is accounted for by increased flux of reducing equivalents through mitochondrial malic dehydrogenase. This increased Flux is accompanied by an increase in the steady state NADH/NAD+ ratio and a marked decrease in the synthesis of citrate. The alpha-glycerophosphate shuttle was reconstituted with mitochondria isolated from rats treated with L-thyroxine. This shuttle was about equal to the reconstructed malate-aspartate shuttle in supression of palmitylcarnitine oxidation. This interaction could not be demonstrated in euthyroid animals owing to the low activity of the mitochondrial alpha-glycerol phosphate dehydrogenase. It is concluded that beta oxidation can be regulated by the NADH/NAD+ ratio. The observed stimulation of flux through malate dehydrogenase both by glutamate and by the malate-aspartate shuttle results in an increased steady state NADH/NAD+ ratio, and is linked to a stoichiometric outward transport of aspartate. We suggest, therefore, that some of the reducing pressure exerted by the malate-aspartate shuttle and by glutamate plus malate is provided through the energy-linked, electrogenic transport of aspartate out of the mitochondria. These results are discussed with respect to the mechanism of the genesis of ethanol-induced fatty liver.     

Involvement of the alternative oxidase in respiration of Yarrowia lipolytica mitochondria is controlled by the activity of the cytochrome pathway

The degree of involvement of cyanide-resistant alternative oxidase in the respiration of Yarrowia lipolytica mitochondria was evaluated by comparing the rate of oxygen consumption in the presence of cyanide, which shows the activity of the cyanide-resistant alternative oxidase, and the oxidation rate of cytochrome c by ferricyanide, which shows the activity of the main cytochrome pathway. The oxidation of succinate by mitochondria in the presence of ferricyanide and cyanide was associated with oxygen consumption due to the functioning of the alternative oxidase. The subsequent addition of ADP or FCCP (an uncoupler of oxidative phosphorylation) completely inhibited oxygen consumption by the mitochondria. Under these conditions, the inhibition of the alternative oxidase by benzohydroxamic acid (BHA) failed to affect the reduction of ferricyanide at the level of cytochrome c. BHA did not influence the rate of ferricyanide reduction by the cytochrome pathway occurring in controlled state 4, nor could it change the phosphorylation quotient ATP/O upon the oxidation of various substrates. These data indicate that the alternative system is unable to compete with the cytochrome respiratory chain for electrons. The alternative oxidase only transfers the electrons that are superfluous for the cytochrome respiratory chain.     

Uncoupled oxidation in rat heart mitochondria

Isolated heart mitochondria possessing a high phosphorylation efficiency with pyruvate and malate as substrates oxidize NADH and ascorbate unassociated with ADP phosphorylation. This uncoupled pathway is expressed partially when succinate or NAD-linked substrates are oxidized. The uncoupled oxidation is likely to be the result of the presence of a mitochondrial population with the high-permeable inner membrane in intact tissues. The nature and origin of a uncoupled respiratory system and its role in the thermoproduction of endotherms are discussed.     

Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart mitochondria.

The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re-investigated in pentane-extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent 'Km' for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The Km value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.     

The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.     

Cyanide-and rotenone-resistant respiration in mitochondria of sugar beet taproots during plant growth and development

Effects of cyanide and rotenone were examined on respiration (oxygen uptake) in mitochondria isolated from sugar beet (Beta vulgaris L.) taproots at various stages of plant growth and development. In mitochondria from growing and cool-stored taproots, the ability of cyanide-resistant, salicylhydroxamic acid-sensitive alternative oxidase (AO) to oxidize malate, succinate, and other substrates of tricarboxylic acid cycle (TCA) was low and constituted less than 10% compared to predominant activity of the cytochrome oxidase pathway during State 3 respiration. Artificial aging of storage tissue (2-day incubation of tissue sections under high humidity at 20°C) substantially activated AO, but the highest capacity (V _alt) of this pathway of mitochondrial oxidation was only observed in the presence of pyruvate and a reducing agent dithiothreitol. At the same time, mitochondria from growing taproots exhibited high rates of rotenone-resistant respiration, and these rates gradually declined during plant growth and development. The slowest rates of this respiration were observed during oxidation of NAD-dependent TCA substrates in mitochondria from dormant storage organ. The results are discussed in relation to significance of alternative electron transport pathways during growth and storage of sugar beet taproots.