Localized energization of the mitochondrial inner membrane by ATP.

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane. The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F1 . F0) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211-222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F1), reflecting the localized energization of the membrane by ATP.     

Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.     

Conformational coupling in H+-pumps and ATP synthesis — its analysis with anisotropic inhibitors of energy transduction in oxidative phosphorylation

Summary The analysis of anisotropic inhibitor-induced phenomena in mitochondria revealed that two kinds of negative charges are generated near surface of the C-side of mitochondrial inner membranes in the energized state, on the redox complexes (I, III & IV) and F₀, respectively, and that positively charged anisotropic inhibitors (AI⁺) inhibit energy transduction in oxidative phosphorylation by binding to these negative charges. Thus, AI⁺ have two different inhibition sites in oxidative phosphorylation, the redox complexes and F₀. The membrane components generating the negative charges in energized mitochondria were examined by the technique of photoaffinity labeling with monoazide ethidium, which is an AI⁺. Results showed that monoazide ethidium specifically binds to two kinds of hydrophobic protein (of 8 K and 13 K daltons) of mitochondria energized with succinate, and these proteins were named chargerin I and II, respectively. Chargerin I and II, which may be components of the redox complexes and F₀, seem to generate the negative charges described above, and these may be essential for H⁺-pumps in the redox complexes and F₁ · F₀. AI⁺ seem to inhibit ATP synthesis by binding to negatively charged sites of chargerin I and II.Based on these findings and the salient results on energy-transducing membranes obtained recently in other laboratories, a conformational model of H⁺-pumps and ATP synthesis in mitochondria is proposed, which is also applicable to ATP synthesis in other energy-transducing membranes and ATP-linked active transport of ions.     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The precursor of cytochrome b₂ (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b₂ precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.     

The effect of mitochondrial energization on cytochrome c oxidase kinetics as measured at low temperatures. I. The reaction with carbon monoxide

The kinetics of CO binding by the cytochrome c oxidase of pigeon heart mitochondria were studied as a function of membrane energization at temperatures from 180 to 280°K in an ethylene glycol/water medium. Samples energized by ATP showed acceleration of CO binding compared to those untreated or uncoupled by carbonylcyanide p-trifluoromethoxyphenylhydrazone but only at relatively low temperatures and high CO concentrations. Experiments using samples in a “mixed valency” (partially oxidized) state showed that the acceleration of ligand binding is not due to the formation of a partially oxidized state via reverse electron transport.It is concluded that in the deenergized state one CO molecule can be closely associated with the cytochrome a₃ heme site without actually being bound to the heme iron; in the energized state, two or more ligand molecules can occupy this intermediate position.The change in the apparent ligand capacity of a region near the heme iron in response to energization is evidence for an interaction between cytochrome oxidase and the ATPase system. Furthermore, these results suggest a control mechanism for O₂ binding.     

Both ATP and an energized inner membrane are required to import a purified precursor protein into mitochondria.

We have investigated the energy requirement of mitochondrial protein import with a simplified system containing only isolated yeast mitochondria, energy sources and a purified precursor protein. This precursor was a fusion protein composed of 22 residues of the cytochrome oxidase subunit IV pre-sequence fused to mouse dihydrofolate reductase. Import of this protein required not only an energized inner membrane, but also ATP. ATP could be replaced by GTP, but not by CTP, TTP or non-hydrolyzable ATP analogs. Added ATP did not increase the membrane potential of respiring mitochondria; it supported import even if the proton-translocating mitochondrial ATPase and the entry of ATP into the matrix were blocked. We conclude that ATP exerts its effect on mitochondrial protein import outside the inner membrane.     

The metabolic effects and uptake of ethidium bromide by rat liver mitochondria.

The uptake of ethidium bromide by rat liver mitochondria and its effect on mitochondria, submitochondrial particles, and F₁ were studied. Ethidium bromide inhibited the State 4-State 3 transition with glutamate or succinate as substrates. With glutamate, ethidium bromide did not affect State 4 respiration, but with succinate it induced maximal release of respiration. These effects appear to depend on the uptake and concentration of the dye within the mitochondrion. In submitochondrial particles, the aerobic oxidation of NADH is much more sensitive to ethidium bromide than that of succinate. Ethidium bromide partially inhibited the ATPase activity of submitochondrial particles and of a soluble F₁ preparation. Ethidium bromide behaves as a lipophilic cation which is concentrated through an energy-dependent process within the mitochondria, producing its effects at different levels of mitochondrial function. The ability of mitochondria to concentrate ethidium bromide may be involved in the selectivity of the dye as a mitochondrial mutagen.     

ADP-Inhibition of H<Superscript>+</Superscript>-F<Subscript>O</Subscript>F<Subscript>1</Subscript>-ATP Synthase

H⁺-F_OF₁-ATP synthase (F-ATPase, F-type ATPase, F_OF₁ complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.     

Stimulation of dopamine oxidation in liver mitochondria by palmitic acid in the presence of ATP and <Emphasis Type="Italic">tert</Emphasis>-butylhydroperoxide

The effect of palmitic acid on the oxidation of dopamine, i.e., on the monoamine oxidase (MA-oxidase) activity, was investigated on deenergized liver mitochondria, upon energization by ATP and also in the presence of an oxidizing agent tert-butylhydroperoxide (TBH). It was found that palmitic acid reduces the value of the apparent K _m for dopamine without alteration of the apparent V _max. This points to stimulation of the mitochondrial MA-oxidase activity by palmitic acid at low concentrations of dopamine. Stimulatory effect of palmitic acid may be related to the ability of amphiphilic compounds to increase the negative charge density on the outer mitochondrial membrane. This leads to an increase in the local concentration of positively charged ions of dopamine in the layer adjacent to the membrane near the active site of monoamine oxidase. ATP eliminates the ability of palmitic acid to stimulate the MA-oxidase activity of mitochondria. This effect of ATP is not observed in the presence of the F _O F ₁-ATP-synthase inhibitor oligomycin. Apparently, in the case of vector transport of H⁺ from the matrix induced by ATP-hydrolysis, protonation of palmitic acid anions occurs on the outer mitochondrial membrane, followed by the movement of the neutral molecules to the outer and then to the inner monolayer of the inner membrane. It was found that TBH at a concentration of 300 μM has no significant effect on the ATPase activity of mitochondria and in the presence of ATP and palmitic acid reduces the value of the apparent K _m for dopamine without alteration of the apparent V _max. Antioxidant thiourea eliminates this effect of TBH. We propose that the TBH-induced oxidative stress in the case of ATP-energized mitochondria results in the movement of palmitic acid molecules from the inner to the outer membrane. This leads to an increase in the density of negative charges on the surface of this membrane and, therefore, to the stimulation of the dopamine oxidation.     

Characterization of a highly diverged mitochondrial ATP synthase Fo subunit in Trypanosoma brucei

The mitochondrial F₁F_o ATP synthase of the parasite Trypanosoma brucei has been previously studied in detail. This unusual enzyme switches direction in functionality during the life cycle of the parasite, acting as an ATP synthase in the insect stages, and as an ATPase to generate mitochondrial membrane potential in the mammalian bloodstream stages. Whereas the trypanosome F₁ moiety is relatively highly conserved in structure and composition, the F_o subcomplex and the peripheral stalk have been shown to be more variable. Interestingly, a core subunit of the latter, the normally conserved subunit b, has been resistant to identification by sequence alignment or biochemical methods. Here, we identified a 17 kDa mitochondrial protein of the inner membrane, Tb927.8.3070, that is essential for normal growth, efficient oxidative phosphorylation, and membrane potential maintenance. Pull-down experiments and native PAGE analysis indicated that the protein is both associated with the F₁F_o ATP synthase and integral to its assembly. In addition, its knockdown reduced the levels of F_o subunits, but not those of F₁, and disturbed the cell cycle. Finally, analysis of structural homology using the HHpred algorithm showed that this protein has structural similarities to F_o subunit b of other species, indicating that this subunit may be a highly diverged form of the elusive subunit b.     

Relation between the initial kinetics of ATP synthesis and of conformational changes in the chloroplast ATPase studied by external field pulses

ATP formation and the energy-dependent release of tightly bound [14C]-adenine nucleotides from the chloroplast coupling factor CF1 has been studied as a function of the time of energization of the membrane in the range of 500 mus up to 60 ms. The high time resolution was achieved because the energization was generated artificially by external electric field pulses. Applying external electric field pulses to a chloroplast suspension induces an electric potential difference across the thylakoid membrane. The following results were obtained: (1) The amount of ATP generated increases linearly with the time of energization. The steady-state rate of ATP formation is reached in less than 500 mus. (2) A fraction of the adenine nucleotides tightly bound to CF1 is released on energization with a half-rise-time of about 2 ms. The size of the fraction, i.e., the amplitude of the fast phase of the release, increases with the magnitude of the induced transmembrane electric potential difference. A further slow release is superimposed. (3) The initial rate of the release of adenine nucleotides is practically identical with the rate of ATP formation. It is concluded that the release of tightly bound nucleotides monitors an initial conformational change by which the ATPase turns from an inactive into an activated state. For the explanation of the results a reaction scheme is proposed which takes into account a preceding activation of the ATPase.     

Membrane studies with polarity-dependant and excimer-forming fluorescent probes

1. The interaction of electron-transporting particles from heavy mitochondria of ox heart with several fluorescent probes was examined. 2. 1-Anilinonaphthalene-8-sulphonate and 2-(N-methylanilino)naphthalene-6-sulphonate both show an energy-dependent response. 3. Energy transfer between the electron-transporting particles and the dyes and the kinetics of the dye-particle interaction were studied in order to locate the binding regions in the membrane. 4. The energy-dependent probe responses were shown to be a result of changes in the quantum yield of fluorescence of the bound dyes together with increased binding of the dyes to the energized membrane. 5. Fluorescence lifetime measurements were also used to observe changes on energization. 6. A new type of probe was found in pyrene-3-sulphonate, which may be regarded as a ;volume indicator' for the internal membrane binding region, since it shows a concentration-dependent excimer fluorescence. 7. By comparing the responses of all these dyes when energized particles are uncoupled, a membrane transition with a time-constant of 2-3s is inferred.     

The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo‐ATP synthase

Mitochondrial F₁F_o‐ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear‐ and mitochondria‐encoded subunits. Whereas chaperones for formation of the matrix‐exposed hexameric F₁‐ATPase core domain have been identified, insight into how the nuclear‐encoded F₁‐domain assembles with the membrane‐embedded F_o‐region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F₁‐module and peripheral stalk, but not with the assembled F₁F_o‐ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F₁‐module to the membrane embedded F_o‐domain. We conclude that INAC represents a matrix‐exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F₁F_o‐ATP synthase.     

The Vacuolar ATPase of Red Beet Storage Tissue: Electron Microscopic Demonstration of the “Head-and-Stalk” Structure*

Tonoplast membranes were prepared from tissue homogenates and from vacuoles isolated from beetroot storage tissue (Beta vulgaris L., ssp. conditiva) for transmission electron microscopic analysis of the structure of the beetroot vacuolar ATPase using the negative staining technique. By comparison of the specific inhibitor sensitivities of the ATPase activity, i.e. ATP hydrolysis and H⁺-pumping, the purity of the tonoplast preparations with respect to contamination with mitochondrial inner membranes was assessed to avoid confusion with mitochondrial F₁F₀-ATPase. Membranes prepared in Hepes/Tris or BTP/Mes-containing media rarely showed typical head-and-stalk structures although characteristic nitrate- and bafilomycin A₁-sensitive ATP-hydrolysis and H⁺-pumping could be measured. However, typical head-and-stalk structures were observed regularly when these buffers were replaced by K-phosphate buffer. Under these conditions, the beetroot vacuolar ATPase is characterized by a large head group with a central cleft, a thin stalk, connecting it to the membrane and by basal components projecting from the base of the stalks near the vacuolar membrane and forming a distinct layer of electron-light particles between the vacuolar membrane and the layer of non-stained head groups.     

The role of the adenine nucleotide translocator in oxidative phosphorylation. A theoretical investigation on the basis of a comprehensive rate law of the translocator

A minimum model of adenine nucleotide exchange through the inner membrane of mitochondria is presented. The model is based on a sequential mechanism, which presumes ternary complexes formed by binding of metabolites from both sides of the membrane. The model explains the asymmetric kinetics of ADP-ATP exchange as a consequence of its electrogenic character. In energized mitochondria, a part of the membrane potential suppresses the binding of extramitochondrial ATP in competition with ADP. The remaining part of the potential difference inhibits the back exchange of internal ADP for external ATP. The assumption of particular energy-dependent conformational states of the translocator is not necessary. The model is not only compatible with the kinetic properties reported in the literature about the adenine nucleotide exchange, but it also correctly describes the response of mitochondrial respiration to the extramitochondrial ATP/ADP ratio under different conditions. The model computations reveal that the translocation step requires some loss of free energy as driving force. The size of the driving force depends on the flux rate as well as on the extra- and intramitochondrial ATP/ADP quotients. By both quotients the translocator controls the export of ATP formed by oxidative phosphorylation in mitochondria.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

Noncatalytic nucleotide binding sites: Properties and mechanism of involvement in ATP synthase activity regulation

ATP synthases (F_oF₁-ATPases) of chloroplasts, mitochondria, and bacteria catalyze ATP synthesis or hydrolysis coupled with the transmembrane transfer of protons or sodium ions. Their activity is regulated through their reversible inactivation resulting from a decreased transmembrane potential difference. The inactivation is believed to conserve ATP previously synthesized under conditions of sufficient energy supply against unproductive hydrolysis. This review is focused on the mechanism of nucleotide-dependent regulation of the ATP synthase activity where the so-called noncatalytic nucleotide binding sites are involved. Properties of these sites varying upon free enzyme transition to its membrane-bound form, their dependence on membrane energization, and putative mechanisms of noncatalytic site-mediated regulation of the ATP synthase activity are discussed.     

An analysis of the binding of fluorescence probes in mitochondrial systems

Summary Measurements of the binding of the fluorescent probes 8-anilinonaphthalene-1-sulfonate (ANS) and ethidium ions to whole and disrupted mitochondria and submitochondrial particles suggest that the inner mitochondrial membrane is freely permeable to the two probes. Equations relating the binding of permeant probes to the electro-chemical balance across the membrane of vesicular systems are derived and these equations used to analyze Scatchard plots of the binding of the two probes to energized and nonenergized mitochondria and EDTA particles.     

Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties

The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to ΔpH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (K_i = 12 μM) and ATPase activity of inverted inner membrane vesicles (K_i = 126 μM) and partially purified F₁-ATPase (K_i = 177 μM). The smaller K_i for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 μM), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. Δψ-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of Δψ in isolated mitochondria.