The Respiratory Chain of Plant Mitochondria. II. Oxidative Phosphorylation in Skunk Cabbage Mitochondria

Mitochondria were prepared from the spadices of skunk cabbage (Symplocarpus foetidus) whose respiratory rate with succinate and malate showed 15% to 30% sensitivity to cyanide inhibition, and which showed respiratory control by added ADP. The observed respiratory control ratios ranged from 1.1 to 1.4. The change in pH of the mitochondrial suspension was recorded simultaneously with oxygen uptake: alkalinization of the medium, expected for phosphorylation of ADP, coincided with the period of acceleration in oxygen uptake caused by addition of an ADP aliquot. The ADP/O ratios obtained were 1.3 for succinate and 1.9 for malate. In the presence of 0.3 mm cyanide, the ADP/O ratio for succinate was zero, while that for malate was 0.7. These results are consistent with the existence of an alternate oxidase which interacts with the flavoprotein and pyridine nucleotide components of the respiratory chain and which, in the presence of cyanide, allows the first phosphorylation site to function with an efficiency of about 70%. In the absence of respiratory inhibitors, the efficiency of each phosphorylation site is also about 70%. This result implies that diversion of reducing equivalents through the alternate oxidase, thereby bypassing the 2 phosphorylation sites associated with the cytochrome components of these mitochondria, occurs to a negligible extent during the oxidative phosphorylation of ADP or State 3.Addition of ADP or uncoupler to skunk cabbage mitochondria respiring in the controlled state or State 4, results in reduction of cytochrome c and the oxidation of the cytochromes b, ubiquinone and pyridine nucleotide. A site of interaction of ADP with the respiratory chain between cytochromes b and cytochrome c is thereby identified by means of the crossover theorem. Flavoprotein measured by fluorescence is also oxidized upon addition of ADP or uncoupler, but flavoprotein measured by optical absorbance changes becomes more reduced under these conditions. Depletion of the mitochondria by pretreatment with ADP and uncoupler prevents reduction of most of the fluorescent flavoprotein by succinate. These results indicate that skunk cabbage mitochondria contain both high and low potential flavo-proteins characterized by different fluorescence/absorbance ratios similar to those demonstrated to be part of the respiratory chain in mitochondria from animal tissues.     

Interrelationships of copper, cytochrome oxidase, phospholipid synthesis and adenine nucleotide binding.

Studies are reported on the interrelationships in liver mitochondria of copper status, cytochrome oxidase activity, adenine nucleotide binding capacity and phospholipid synthesis. Direct exposure of mitochondria to cyanide or diethyldithiocarbamate depressed cytochrome oxidase activity; ADP-binding and phospholipid synthesis. Fractionation of mitochondria to increase the specific activity of cytochrome oxidase about 10-fold did not increase the affinity to bind ADP. Ageing of mitochondria or dialysis of mitochondria or mitochondrial membrane preparations against water or diethyldithiocarbamate at 0--2 degrees for 18 h did not decrease cytochrome oxidase activity or copper content of reisolated and resuspended mitochondria or mitochondrial membrane preparations, but considerably reduced the affinity to bind ADP. The respiratory inhibitors, fluoride and azide, at concentrations inhibitory to cytochrome oxidase did not reduce ADP-binding or phospholipid synthesis. Atractyloside did not inhibit cytochrome oxidase activity but did inhibit ADP-binding and phospholipid synthesis. Pre-incubation of mitochondrial membrane preparations with Cu++ increased the copper content and ADP-binding affinity. The results indicate that cytochrome oxidase is not the ADP-binding site of the mitochondrial membrane system and that reduced cytochrome oxidase activity per se does not depress binding affinity. Copper appears to be a component of the adenine nucleotide binding sites of mitochondrial membranes because the copper-complexing agents, cyanide and diethyldithiocarbamate, depressed ADP-binding, while increased mitochondrial membrane copper content increased ADP-binding.     

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 these 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 cyanide 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 obtain-red 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.     

Cyanide-insensitive oxidation of ascorbate + NNN''N''-tetramethyl-p-phenylenediamine mixture by mung-bean (Phaseolus aureus) mitochondria. An energy-linked function.

Freshly prepared washed or purified mung-bean (Phaseolus aureus) mitochondria utilize oxygen with ascorbate/tetramethyl-p-phenylenediamine mixture as electron donor in the presence of KCN. ATP control of the oxygen uptake can be observed with very fresh mitochondria. The electron flow, which is inhibited by antimycin A, salicylhydroxamic acid or octylguanidine, takes place by reversed electron transport through phosphorylation site II and thence to oxygen through the cyanide-insensitive pathway. Oligomycin and low concentrations of uncoupler partially inhibit the oxygen uptake in a manner similar to that observed for other energy-linked functions of plant mitochondria. An antimycin A-insensitive oxygen uptake occurs if high concentrations of uncoupler are used, indicating that the pathway of electron flow has been altered. The process of cyanide-insensitive ascorbate oxidation is self-starting, and, since it occurs in the presence of oligomycin, it is concluded that the reaction can be energized by a single energy-conservation site associated with the cyanide-insensitive oxidase pathway.     

The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

The phosphorylation potential generated by respiring mitochondria

1. 1. The phosphorylation potential, ΔG_P = ΔG₀′ + 1.36 log ([ATP]/[ADP][P_i]), where ΔG_O′ is the standard free energy of hydrolysis of ATP at a given pH, and [ATP], [ADP] and [P_i] refer to concentrations in the suspending medium, has been determined in rat-liver mitochondria under various conditions.

2. 2. The ATP/ADP ratio is relatively constant, over a 10-fold range of phosphate concentration. Thus, the phosphate potential is higher at low phosphate concentration. State-4 rat-liver mitochondria in the presence of succinate, oxygen and low concentrations of phosphate in State 4 maintain a phosphorylation potential of 16.1 kcal (67.3 kJ) per mole ATP.

3. 3. High concentrations of ATP inhibit ADP uptake, and it is suggested that this is the reason for the independence of the ATP/ADP ratio on the phosphate concentration. A steady-state ratio is set up dependent upon two processes that are relatively slow compared with State-3 respiration, namely ADP transport and ATP hydrolysis.

4. 4. The phosphorylation potential calculated from the concentrations of total ADP, ATP and P_i within State-4 mitochondria is 4.5 kcal/mole less than that in the suspending medium.

5. 5. It was shown experimentally that the phosphorylation potential cannot be calculated from the ΔG of the redox couple, the respiratory-control ratio and the P:O ratio, as has been suggested in the literature.

6. 6. The measured phosphorylation potential is 83% of that calculated from the span succinate to oxygen, assuming thermodynamic equilibrium, and 95% of that calculated from the span NADH to oxygen.

7. 7. Based on the measurements of the phosphorylation potential and of the redox potentials and redox states of redox components in mitochondria, ubiquinone and cytochrome b are found at their expected position at the junction of the phosphorylations at Sites 1 and 2. The iron-sulphur centres 2 and 5 and the iron-sulphur centre of succinate dehydrogenase also probably lie at this junction. Cytochrome a₃ lies at its expected junction between phosphorylation Sites 2 and 3. A number of electron carriers (cytochromes c, c₁, and a, the iron-sulphur centre of Complex III and the EPR-detectable copper), however, lie in the ‘no-man's land’ within Site 2.

8. 8. A phosphorylation potential of 16.1 kcal/mole corresponds to a membrane potential of 350 mV in State 4, on the basis of the chemiosmotic hypothesis.

An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation.

Rat liver mitochondria were incubated in the presence of varying concentrations of ATP, followed by ADP to initiate phosphorylation. Analysis of phosphorylation to oxygen ratios (P/O) was carried out with varied initial phosphorylation potentials (or ATP/ADP ratios). Rates of phosphorylation and respiration and magnitude of membrane potential (delta psi) were measured. The results are discussed in the framework of P/total O and P/"extra" O ratios in determination of the mechanistic P/O ratio. It is concluded that the former underestimates, and the latter overestimates the mechanistic P/O ratio.     

Pyruvate metabolism in castor-bean mitochondria.

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation.     

Control of oxidative phosphorylation in rat heart mitochondria. The role of the adenine nucleotide carrier

Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F1-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). Concomitantly with the inhibition of O2 consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.     

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.     

Oxidative phosphorylation in yeast IX. Modification of the mitochondrial adenine nucleotide translocation system in the oxidative phosphorylation-deficient mutant op1

The properties of intact cells and isolated mitochondria of the op₁ mutant of Saccharomyces cerevisiae, which had been shown previously to be deficient in oxidative phosphorylation, have been studied further.

1. 1. When isolated mutant mitochondria were preincubated with substrate and phosphorylation was started by the addition of external ADP, low P/O ratios were obtained under standard conditions. The P/O ratios could be raised to normal values approaching 2 with citrate and succinate in the presence of unusually high concentrations of ADP. Under these conditions the Michaelis constant for ADP of respiration and phosphorylation was found to be 2.9 mM. When isolated mitochondria were added to a medium containing substrate and adenine nucleotide, the Michaelis constant for ADP was found to be lower, about 0.5 mM and maximal P/O ratios of only 0.8 were achieved.

2. 2. Adenine nucleotide translocation across the membrane of the mutant mitochondria was found to be different from that in wild-type mitochondria and dependent on the energy level in mitochondria. When the intramitochondrial nucleotide pool consisted mostly of ADP and AMP, the rate of adenine nucleotide translocation was approx. 30 times lower than in wild-type mitochondria and the Michaelis constants for ADP of the translocation process were similar in the two types of mitochondria, being lower than 10 μM. When the nucleotide pool was enriched in ATP, the translocation rate in mutant mitochondria was as high as in wild-type mitochondria but the Michaelis constant for external adenine nucleotide was more than 100 times higher in the former than in the latter.

3. 3. An examination of the effects of the uncoupler, oligomycin, valinomycin and nigericin on the translocation process in the mutant mitochondria provided additional evidence that varying energization of mutant mitochondria was responsible for the variations of the translocation rates and the Michaelis constants under different experimental conditions.

4. 4. The properties of the adenine nucleotide carrier of mutant mitochondria were studied and found to be different from those of wild-type mitochondria.

5. 5. It has been concluded that the modification of adenine nucleotide translocation across the mitochondrial membrane is responsible for oxidative phosphorylation deficiency in the op₁ mutant. The implications of these findings for the understanding of the adenine nucleotide translocation mechanism and the role of the translocation system in the control of cellular syntheses and growth are discussed.

Mechanism of citrinin-induced dysfunction of mitochondria. II. Effect on respiration, enzyme activities, and membrane potential of liver mitochondria.

The mycotoxin citrinin, depressed the phosphorylation efficiency of liver mitochondria as deduced from a decrease of respiratory coefficient and of the ADP/O ratio. Citrinin (1.0 mM) inhibited some enzymes linked to the respiratory chain, namely NADH oxidase and NADH cytochrome c reductase involved with complex I. The activities of enzymes related with other enzymatic complexes of the respiratory chain were either unaffected or enhanced. ATPase activity was inhibited by the mycotoxin. Malate, glutamate, and 2-oxoglutarate dehydrogenases were also inhibited. The transmembrane potential (delta psi), developed by energized mitochondria and depolarization on the addition of ADP, was decreased. The results suggest that citrinin promotes a partial dissipation of the transmembrane potential, different from that resulting from a classical uncoupler such as 2,4-dinitrophenol.     

Differential effect of temperature on mitrochondrial activity in shoots from freshly harvested and moderately aged kernels of maize (Zea mays L.)

This paper reports on a study of mitochondrial activity in etiolated shoots of freshly harvested and moderately aged kernels of maize. Activity was investigated after incubation at a favourable temperature (25°C), sub-optimal temperature (13°C) and after a heat shock (46°C for 2h). Although impaired mitochondrial activity in shoots from moderately aged maize kernels was not detected at 25°C, deficiencies became evident under low temperature stress (13°C). State 3 oxygen uptake, cyanide-insensitive oxygen uptake and cytochrome oxidase activity were lower in mitochondria from these shoots at 13°C than in mitochondria from shoots of freshly harvested kernels at this temperature. After a heat shock, cyanide-insensitive oxygen uptake was higher, and cytochrome oxidase activity lower, in shoots of aged kernels than in shoots of fresh kernels. No significant differences in ADP: O ratio or succinate dehydrogenase activity occurred between mitochondria from shoots of the two seed lots in any of the temperature treatments.     

Uncoupling proteins in mitochondria of plants and some microorganisms.

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.     

Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation in Rhizobium.

Cellular ATP level, ATP/ADP ratio and nitrogenase activity rise when oxyleghaemoglobin is added to respiring suspensions of Rhizobium japonicum bacteroids from soybean root nodules. Increased gaseous O2 tension is much less efficient than oxyleghaemoglobin in stimulation of bacteroid ATP production. Studies with the inhibitor carbonyl cyanide m-chlorophenylhydrazone show this ATP to be generated as a consequence of oxidative phosphorylation. N-Phenylimidazole, a specific cytochrome P-450 inhibitor, also lowers the efficiency of bacteroid oxidative phosphorylation. An approximately linear relationship is observed between ATP/ADP ratio and nitrogenase activity as N-phenylimidazole concentration is lowered. It is suggested that cytochrome P-450 is a component of the leghaemoglobin-facilitated respiration pathway and that it may act as intracellular O2 carrier rather than terminal oxidase. A less efficient oxidase appears to function when cytochrome P-450 is inhibited.     

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.     

Electron Transport and Oxidative Phosphorylation in Arum Spadix Mitochondria

Arum spadix mitochondria exhibited a rapid cyanide-resistantoxygen uptake when oxidizing malate, NADH₂ or succinate, anda slower, cyanide-sensitive oxygen uptake when oxidizing ascorbate+tetramethylphenylenediamine(TMPD). Cytochrome oxidase does not therefore appear to functionas the terminal oxidase in the presence of cyanide, and therather low cytochrome c oxidase activity obtained using ascorbate+TMPDmay exclude it from possessing a major role even in the absenceof cyanide. ATP synthesis has been shown to accompany substrateoxidation. In the presence of antimycin A the P: O ratio accompanyingmalate oxidation was reduced by half, while phosphorylationaccompanying NADH₂ or succinate oxidation was almost completelyabolished. It is proposed that electrons from exogenous NADH₂enter the electron transport chain at a site after that whereendogenous NADH2 donates electrons and that electrons from exogenousNADH₂ are not coupled to ATP synthesis at site 1. The cyanide-resistant,non-phosphorylating electron-transport pathway may functionin the absence of cyanide and account for the low efficiencyof energy conservation observed in this tissue.     

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with α-ketoglutarate and malate as substrates. Whereas α-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.

Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD⁺, glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

     

Evidence for a structural interacti on betwen ATP synthetase and cytochrome c oxidase in mitochondria

The reaction of cyanide with the oxidized form of cytochrome c oxidase in mitochondria is strongly inhibited by adenosine triphosphate (ATP). This inhibition is strictly dependent on the ATP concentration and is insensitive to changes in the concentrations of adenosine diphosphate (ADP) and orthophosphate. It is completely prevented by oligomycin or uncouplers of oxidative phosphorylation. The ATP is kinetically competitive with respect to cyanide and has a measured inhibitor constant of less than 2 μm The stoichiometry is one ATP/cyanide. This ATP effect is proposed to result from a structural interaction of ATP synthetase with cytochrome c oxidase, such that the formation of an ATP complex of the synthetase results in a decrease in the affinity of the oxidized form of cytochrome c oxidase for cyanide in the formation of an intermediate in the overall measured cyanide reaction.     

Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.