Mechanism of respiration-driven proton translocation in the inner mitochondrial membrane. Kinetics of proton translocation and role of cations

The kinetics and mechanism of passive and active proton translocation in submitochondrial vesicles, obtained by sonication of beef heart mitochondria, have been studied.Analysis of the anaerobic release of the protons taken up by submitochondrial particles in the respiring steady state shows that proton diffusion consists of two parallel, apparent first-order processes: a fast reaction which, on the basis of its kinetic properties and response to cations and various effectors, is considered to consist of a proton/monovalent cation exchange; and a slow process which, on analogous grounds, is considered as a single electrogenic flux.The study of the various parameters of the respiration-linked active proton translocation and of the accompanying migration of permeant anions and K⁺ led to the following conclusions: (i) The oxidoreduction-linked proton translocation is electrogenic. (ii) Cation counterflow is not a necessary factor in the respiration-driven proton translocation. (iii) The membrane potential developed by active proton translocation exerts a coupling with respect to permeant cations and anions. (iv) The respiration-driven proton translocation is secondarily coupled, through the Δμ_H component of the electrochemical proton gradient and at the level of a proton-cation exchange system of the membrane, to the flow of K⁺ and Na⁺.     

Ion transport processes in corn mitochondria : I. Effect of the local anesthetic dibucaine

The local anesthetic dibucaine inhibited respiration-dependent contraction mediated by the K⁺/H⁺ antiport system of isolated corn mitochondria. Respiration declined concurrently. Nigericin, an exogenous K⁺/H⁺ exchanger, restored ion efflux in dibucaine-blocked corn mitochondria. It was concluded that dibucaine inhibited ion efflux via blockage of the K⁺/H⁺ antiport. Further experiments determined that dibucaine also inhibited proton influx facilitated by protonophores and by the ATPase complex during state III respiration. These results are discussed in relation to the mechanism by which dibucaine inhibits proton translocation across the inner mitochondrial membrane.     

Respiration-driven proton translocation in micrococcus denitrificans

The polarity and stoichiometry of respiration-driven proton translocation was studied by electrometric and spectrophotometric techniques inMicrococcus denitrificans in the context of the energy transduction mechanism in bacterial oxidative phosphorylation.

1. Protons are ejected through the plasma membrane during respiratory pulses and thereafter diffuse slowly back.
2. In presence of ionic species mobile across the membrane (K⁺-valinomycin, K⁺-gramicidin, or SCN^?), limiting→H⁺/O quotients of 8 were obtained with endogenous respiratory substrates, and the rate of translocation (14·3 μg ions of H⁺/sec g cell dry weight) was commensurate with that of respiration optimally stimulated by FCCP at an →H⁺/O quotient of 8.
3. The rate of decay of the proton pulses was greatly increased by FCCP, but there was little or no effect on the →H⁺/O quotient characteristic of the respiratory system.
4. Various interpretations of the observations are discussed, and it is concluded that respiration is probably coupled directly or indirectly to electrogenic proton translocation. The observations are compatible with the chemiosmotic hypothesis of coupling between respiration and phosphorylation.

     

On the mechanism of action of oligomycin and acidic uncouplers on proton translocation and energy transfer in “sonic” submitochondrial particles

A study is presented of the effect of acidic uncouplers and oligomycin on energy-linked and passive proton translocation, oxidative phosphorylation, and energy-linked nicotinamide-adenine-nucleotide transhydrogenase in EDTA submitochondrial particles from beef-heart. A flow potentiometric technique has been applied to resolve the kinetics of the initial rapid phase of the redox proton pump. Rapid kinetics analysis shows that carbonyl-cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) does not exert any direct effect on redox-linked active proton transport. The uncoupling action of FCCP on oxidative phosphorylation and energy-linked transhydrogenase is shown to be quantitatively accounted for by its promoting effect of passive proton-diffusion across the mitochondrial membrane. Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase. In fact, rapid kinetic analysis demonstrates that oligomycin does not directly affect the redox-linked proton pump. The present results show that there does not exist any labile intermediate in the redox-linked proton pump which is sensitive to acidic uncouplers.     

Estimation of H+-translocation stoicheiometry of mitochondrial ATPase by comparison of proton-motive forces with clamped phosphorylation potentials in submitochondrial particles

The proton-motive forces generated in submitochondrial particles by both hydrolysis of ATP and oxidation of succinate have been measured by flow dialysis and compared with the ambient phosphorylation potentials. It is concluded that three H⁺ are translocated for each ATP molecule hydrolysed or synthesised. By utilising rat liver mitochondria respiring with β-hydroxybutyrate as a new system for regeneration of ATP from ADP and P_i, phosphorylation potentials were clamped at a range of values by using mixtures of particles and mitochondria in various ratios. As the rate of ATP hydrolysis by the particles was lowered, the proton-motive force decreased only slightly except at the very lowest rates, these results paralleling earlier studies on the relation between rate of respiration-driven proton translocation and proton-motive force.     

Proton translocation in membranes of submitochondrial particles]

Effect of an electrophilous inhibitor, chlorophenacyl, on energy-dependent functions of submitochondrial particles is studied. Chlorophenacyl at concentrations up to 1 mM is found practically not to affect the generation of membrane potential under NADH and succinate oxidation and ATP hydrolysis and to be a strong inhibitor of oxidative phosphorylation and reverse electron transport. The mechanism of the inhibition of energy-dependent functions of submitochondrial particles with chlorophenacyl is different from that of electron transport inhibitor, energy transport inhibitors and classical uncoupling agents--protonophors. The data obtained are suggested to be due to the existence of two ways of proton translocation in submitochondrial particle membrane, phosphorylating and non-phosphorylating, the effect of chlorophenacyl being directed on phosphorylating way only.     

On the mechanism of H+ translocation by mitochondrial H+-ATPase. Studies with chemical modifier of tyrosine residues

In this paper a detailed study of the effect of nitration of tyrosine residues by tetranitromethane on H⁺ conduction and other reactions catalyzed by the H⁺-ATPase complex in phosphorylating submitochondrial particles, uncoupled particles, and the purified complex is presented. Tetranitromethane treatment of submitochondrial particles results in marked inhibition of ATP hydrolysis, ATP-³³P_i exchange, and proton conduction by the H⁺-ATPase complex. These effects are caused by nitration of tyrosine residues of H⁺-ATPase complex as shown by the appearance of the absorption peak at 360 nm (specific for nitrotyrosine formation) and inhibition of ATP hydrolysis and ATP-³³P_i exchange in the complex purified from tetranitromethane-treated particles. H⁺ conduction in phospholipid vesicles inlaid with F₀ is also inhibited by tetranitromethane treatment. These observations indicate that tyrosine residue(s) of F₀ are critically involved in energy-linked proton translocation in the ATP-ase complex.     

Energy-dependent accumulation of the uncoupler picrate and proton flux in submitochondrial particles

In the presence of ATP and oxidizable substrate, submitochondrial particles accumulate up to 7 nmol of picrate/mg of protein. Half of this value is reached at 5 μM picrate in the medium, and maximal energy-dependent accumulation occurs at 25 μM picrate. Mitochondrial proton fluxes calculated under such conditions are 0.80 and 1.08 pmol H⁺/cm²·sec at 10 μM and 25 μM picrate, respectively. These values are similar to those reported for state 4, and are therefore not large enough for uncoupling by picrate through proton translocation. The energy-dependent spectral response of oxonol VI is reversed to 50 % by 40 μM picrate, suggesting that abolishment of membrane potential is responsible for uncoupling of submitochondrial particles by picrate.     

Anilinonaphthalenesulfonate fluorescence changes induced by non-enzymatic generation of membrane potential in mitochondria and submitochondrial particles

Changes in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS⁻) accompanying non-enzymatic generation of the membrane potential in mitochondria and sonicated submitochondrial particles have been demonstrated. Generation of the membrane potential was induced by addition of an ionophore (valinomycin for K⁺, or tetrachlorotri-fluoromethylbenzimidazole for H⁺) under conditions where there existed K⁺ (or H⁺) gradients across the mitochondrial membrane. The ANS⁻ fluorescence decreased when the mitochondrial (or particle) interior became more negative, and increased when it became more positive. Collapse of the membrane potential reversed the ANS⁻ responses. A hypothesis is put forward to explain the energy-dependent ANS⁻ responses in mitochondria and particles by the membrane potential-induced redistribution of ANS⁻ between the membrane and water phases.     

Effects of quercetin and F1 inhibitor on mitochondrial ATPase and energy-linked reactions in submitochondrial particles

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) shares certain properties with the mitochondrial ATPase inhibitor protein. At low concentrations it inhibits both soluble and particulate mitochondrial ATPase and has no effect on oxidative phosphorylation in submitochondrial particles. Unlike the mitochondrial inhibitor protein quercetin inhibits the ATP-dependent reduction of NAD⁺ by succinate in fully reconstituted submitochondrial particles. A comparison of various flavones indicates that the hydroxyl groups at the 3′ and perhaps 3 position are important for the inhibition of ATPase activity.     

Estimation of H+-translation stoicheiometry of mitochondrial ATPase by comparison of proton-motive forces with clamped phosphorylation potentials in submitochondrial particles

The proton-motive forces generated in submitochondrial particles by both hydrolysis of ATP and oxidation of succinate have been measured by flow dialysis and compared with the ambient phosphorylation potentials. It is concluded that three H+ are translocated for each ATP molecule hydrolysed or synthesised. By utilising rat liver mitochondria respiring with beta-hydroxybutyrate as a new system for regeneration of ATP from ADP and Pi, phosphorylation potentials were clamped at a range of values by using mixtures of particles and mitochondria in various ratios. As the rate of ATP hydrolysis by the particles was lowered, the proton-motive force decreased only slightly except at the very lowest rates, these results paralleling earlier studies on the relation between rate of respiration-driven proton translocation and proton-motive force.     

Proton translation in submitochondrial vesicles

An investigation of proton translocation in submitochondrial vesicles from rat liver has been made under simple experimental conditions. Choline chloride was used both as the oxidizable substrate and the ionic medium for the measurement of activity during oxygen pulse experiments:

1. The passive permeability measured from the decay of proton efflux after an oxygen pulse could be described by a first-order equation. An H⁺/O ratio of 2·5 was obtained for choline oxidation in the presence of oligomycin and/or MgCl₂. Oligomycin decreased the passive proton permeability and respiration, concomitant with an increase in proton uptake. Respiratory control was directly related to the passive proton permeability and inversely related to the magnitude of the proton gradient. The decreased respiration and passive permeability reflecting respiratory control is most evident in the pH rang 5·8–7·5.
2. Preparation of submitochondrial vesicles in the presence of EDTA resulted in proton production during an oxygen pulse given at alkaline pH. Cytochromec enhanced proton uptake by approximately 1 H⁺/cytochromec, but only in the presence of Triton X-100. These results are indicative of the asymmetric behavior of the coupling membrane and provide direct evidence of the participation of electron transport components in proton translocation.

     

Acid-base titration across the plasma membrane ofMicrococcus denitrificans: Factors affecting the effective proton conductance and the respiratory rate

The object of this work was to measure the effective proton conductance of the plasma membrane ofMicrococcus denitrificans under various conditions and to investigate possible connections between respiration and proton translocation.

1. Pulsed acid-base titrations of suspensions ofM. denitrificans in a medium containing the permeant thiocyanate ion, or when K⁺ ion permeability was induced by valinomycin in a KCl medium, showed that the normal effective proton conductance of the membrane system was less than 1 μmho/cm².
2. A pH-overshoot artefact was suppressed by adding carbonic anhydrase.
3. The effective proton conductance was increased by the uncoupler FCCP in the same concentration range as was required to stimulate respiration. Concentrations of FCCP above 1·5 μM inhibited respiration after an initial stimulation.
4. The effective proton conductance in presence of 2 μM FCCP was at least 17 μmho/cm².
5. The quantitative relationships between the respiratory rate, the stoichiometry of respiration-driven proton translocation, and the effective proton conductance of the membrane of the cells are compatible with the suggestion that stimulation of respiration by FCCP is due to a release of back-pressure exerted by a protonmotive potential on the respiratory chain system in the membrane. Only one amongst other possible explanations of the stimulation of respiration by FCCP is, however, excluded.

     

A rapid screening method for streptococcal plasmids yielding plasmid DNA suitable for restriction enzyme analysis

Abstract Anaerobically and aerobically grown cultures of Propionibacterium acnes have been used to demonstrate the presence of respiration, respiration-driven proton translocation and stimulation of membrane potential formation by O₂ in such cultures. The results obtained indicate that P. acnes has the capacity to carry out oxidative phosphorylation.     

Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane

Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD⁺) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP⁺, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH → AcPyAD⁺ reaction of reconstituted enzyme. NAD⁺ had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H⁺ translocation that was comparable to the decrease in the rate of NADPH → AcPyAD⁺ transhydrogenation. It is concluded that tetranitromethane modification controls the H⁺ translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H⁺-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5′-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue.     

Spegazzinine,a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I₅₀ was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca²⁺ and the proton ejection associated with the ATP-dependent Ca²⁺ uptake were not affected by the alkaloid.Oxidative phosphorylation and the P_i-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I₅₀, 50 μM) while the ATP-dependent reactions, reduction of NAD⁺ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I₅₀, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.     

Oxidative phosphorylation and respiratory control in lysolecithin treated electron transport particles

Lysolecithin treatment of electron transport particles (ETP) generated non-vesicular fragments of membrane that can catalyze oxidative phosphorylation. Electron micrographs of ultrathin sections of lysolecithin treated ETP were devoid of circular patterns characteristic of closed vesicular structures. No synergistic uncoupling of oxidative phosphorylation by valinomycin plus nigericin in the presence of K⁺ was observed in such fragments of membrane, which remained sensitive to classical uncouplers and to oligomycin. Preceding total destruction of closed vesicular structure, lysolecithin caused a drastic alteration in the membrane as evidenced by a greatly diminished effect of the ionophores in releasing respiratory control.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1,N⁶-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [³H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[³H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F₁-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg²⁺-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F₁-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F₁-ATPase displays similar K_m values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F₁-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F₁-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Measurement of mitochondrial oxidative phosphorylation: Selective inhibition of adenylate kinase activity by P1,P5-di-(adenosine-5′)-pentaphosphate

A biochemical assay for the measurement of ATP synthesis coupled to electron transport in the presence of adenylate kinase was developed as an alternative to using the conventional Clark-type oxygen electrode. The assay utilizes P¹,P⁵-di-(adenosine-5′)-pentaphosphate which is shown to be a competitive inhibitor with MgADP for rat liver mitochondrial adenylate kinase (K_i = 7.04 × 10^?8m) and was found to have no effect on oxidative phosphorylation of either intact mitochondria or submitochondrial particles.     

Kinetic properties of aspartate transport in rat heart mitochondrial inner membranes.

The mitochondrial glutamate-aspartate exchange carrier catalyzes the electrogenic exchange of intramitochondrial aspartate for extramitochondrial glutamate. Protons are cotransported with glutamate in a 1:1 ratio. In the present study, the effects of pH and glutamate concentration on glutamate entry into intact mitochondria were determined. Hydrogen ions were found to decrease the K_m for glutamate entry. In addition, using glutamate-loaded submitochondrial particles, aspartate transport into the particles was measured as a function of internal and external glutamate concentrations, pH, and electrical potential across the membrane. Glutamate, was a competitive inhibitor of aspartate transport when both amino acids were present on the same side of the membrane, while H⁺ was a noncompetitive inhibitor of aspartate entry into the particles. A decrease in glutamate concentration on the inside of the particles brought about a parallel decrease in V and K_m for aspartate outside of the particles, thus suggesting a ping-pong mechanism for the carrier. The uncoupling agent, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), lowered both the K_m and V of aspartate transport, while the effect on V was somewhat larger. Data obtained in the presence of KSCN was similar to that obtained with FCCP, and therefore it is concluded that both K_m and V changes are dependent on a change of electrical potential across the membrane. A model for the carrier is proposed, which is consistent with the data presented. The model includes a single binding site specific for either glutamate or aspartate, and a separate binding site for the cotransported proton. The affinity of the binding site for protons is increased by simultaneous glutamate binding, but decreased by aspartate binding. The data suggest that an increase in the membrane potential increases the mobility of the charged carrier-aspartate complex, but also facilitates some additional step in the exchange cycle involving subsequent return of the carrier to the matrix side of the membrane. The additional membrane-potential-dependent step could be proton binding on the cytosolic side of the carrier.