Mechanism of uncoupling of oxidative phosphorylation by gramicidin

The mechanism of the uncoupling of oxidative phosphorylation in rat liver mitochondria by gramicidin and truncated gramicidin derivatives was investigated. The derivatives desformylgramicidin and des(formylvalyl)gramicidin are not expected to form head to head, dimeric, ion-conducting channels, and thus allow an evaluation of the relevance of the stimulation of transmembrane cation conductance (and the resulting collapse of the proton electrochemical gradient) to the uncoupling of oxidative phosphorylation. When assayed for the enhancement of the passive diffusion of KSCN, gramicidin was 100-fold more potent than desformylgramicidin and 50-fold more potent than des(formylvalyl)gramicidin. Yet, in a medium devoid of alkalai cations, all three compounds were nearly equally potent uncouplers at low concentrations. Moreover, this uncoupling was not associated with stimulation of cation transport or a reduction of the magnitude of the proton electrochemical potential. In the same medium, gramicidin stimulated 86Rb uptake 50-fold more than desformylgramicidin and 10 times more than des(formylvalyl)gramicidin. At higher concentrations, gramicidin induced further uncoupling, which was associated with reduction of membrane potential (and presumably with transport of alkali cations), while the truncated derivatives were considerably less effective than gramicidin in this range. Thus, with the truncated derivatives, a better separation between decoupling (i.e., uncoupling not associated with reduction of delta mu H) and uncoupling is observed. In the same medium, gramicidin, but not the truncated derivatives, strongly inhibits the formation of both the membrane potential and delta pH by the H+-ATPase. This finding suggests direct interaction of gramicidin with the H+-ATPase. The truncated derivatives stimulated the ATPase without collapsing the membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the relationship between rate of ATP synthesis and H+ electrochemical gradient in rat-liver mitochondria

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, delta mu H, has been analyzed in intact mitochondria. Onset of phosphorylation causes a depression of delta mu H of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of delta mu H to its state-4 value during titrations with oligomycin or atractyloside. Titrations with ionophores display the following features: (a) delta mu H can be depressed by 3-4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing delta mu H by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of delta mu H of only 1 kJ/mol. The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of delta mu H per se. The consistence of the present data with either a chemiosmotic model where delta mu H is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.     

Nature of proton cycling during gramicidin uncoupling of oxidative phosphorylation

Addition of gramicidin D to liver mitochondria, incubated in low- or high-salt media, results in stimulation of respiration in the absence or presence of depression of delta muH, respectively. Gramicidin D concentrations 2 orders of magnitude higher are required in the low-salt media with full uncoupling at 1 nmol of gramicidin.mg-1. The stimulation of respiration is not accompanied by increased passive proton influx in low-salt media. In high-salt media, the extent of respiratory stimulation and the extent of delta muH depression differ according to the nature and concentration of cation. The flow-force relationship is very steep when gramicidin D induced uncoupling occurs in low-salt media and much less steep in high-salt media. A multiplicity of flow-force relationship, respiratory rate vs delta muH, is obtained, the slope of which depends on the nature and concentration of cation, and which can be reproduced by computer simulation by introducing a variable extent of proton cycling either in the membrane or in the pump. The apparent proton conductance, as analyzed in the relationship of Je/delta muH vs delta muH, increases in the so-called ohmic and nonohmic regions according to whether gramicidin D is added in high-salt or low-salt media, respectively. Titration with antimycin of the respiratory control ratio (RCR) in gramicidin D treated mitochondria leads to a depression of the RCR in high-salt but not in low-salt media. The view is discussed that in low-salt media the gramicidin D induced uncoupling is due to a cycling of protons within a proton domain operationally located at or near the proton pump.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free fatty acids decouple oxidative phosphorylation by dissipating intramembranal protons without inhibiting ATP synthesis driven by the proton electrochemical gradient

Free fatty acids (FFA) uncouple oxidative phosphorylation and reverse electron transport and inhibit ATP-Pi exchange in beef heart submitochondrial particles. In this, they resemble classical uncouplers and ionophores. However, in contrast to the latter agents, FFA do not collapse the substrate generated proton electrochemical potential and do not inhibit ATP synthesis when the latter is driven by artificially imposed delta microH. These results lend further support to the suggestion that oxidative phosphorylation depends, in part, on direct intramembranal proton transfer - a process which is specifically uncoupled by FFA and other membrane perturbing agents (e.g. general anesthetics).     

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uncoupler-inhibitor titrations of ATP-driven reverse electron transfer in bovine submitochondrial particles provide evidence for direct interaction between ATPase and NADH:Q oxidoreductase

From the chemiosmotic hypothesis it follows that no change is expected in potency of an uncoupler to inhibit an energy-driven reaction in an energy-transducing membrane if the energy-requiring part of the reaction, the so-called secondary proton pump, is partially inhibited by a specific, tightly bound inhibitor. An increase in potency upon inhibition of the primary pump may be expected, due to a lower rate of the total proton flow that can be used by the secondary pump and dissipated by the uncoupler. Contrary to this prediction several uncouplers (S13, SF6847, 2,4-dinitrophenol, valinomycin + nigericin) show an increase in uncoupling efficiency in ATP-driven reverse electron transfer (reversal) upon inhibition of the secondary pump in this reaction, the NADH:Q oxidoreductase, by rotenone. The increase in uncoupling efficiency is proportional to the decrease in the rate of reversal, that is to the decrease in concentration of active secondary pump. Similarly, upon inhibition of the primary pump, the ATPase, with oligomycin, an increase in uncoupling efficiency was found, also proportional to the decrease in the rate of reversal. When the pore-forming uncoupler gramicidin was used, no change in uncoupling potency was found upon inhibition of NADH:Q oxidoreductase. Inhibition of the ATPase, however, resulted in a proportionally lower uncoupling titre for gramicidin, just as was found for S13 in the presence of oligomycin. A difference was also found in the relative concentrations of S13 and gramicidin required to stimulate ATP hydrolysis or to inhibit reversal. The amount of S13 needed to stimulate ATP hydrolysis was clearly higher than the amount needed to inhibit reversal. On the contrary, the titre of gramicidin for both actions was about the same. To explain these results we propose that gramicidin uncouples via dissipation of the bulk delta mu H+, whereas the carrier-type uncouplers preferentially interfere with the direct energy transduction between the ATPase and redox enzymes. This is in accordance with the recently developed collision hypothesis.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (delta mu H+) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and delta mu H+ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of delta mu H+, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on delta mu H+; for example, the excess of oligomycin produced a 90% inhibition of the respiration while delta mu H+ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing delta mu H+ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of delta mu H+, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and delta mu H+.     

Temperature jump as a new technique to study the kinetics of fast transport of protons across membranes

Application of a temperature jump (2.5 degrees C) to a suspension of liposomes, having phosphate (delta pK/delta T approximately 0.005) as the internal buffer and tris(hydroxymethyl)aminomethane (delta pK/delta T approximately 0.031) as the external buffer, created a delta pH (pHin - pHout) of positive sign in ca. 5 microseconds. Decay of this delta pH was monitored by using the fluorescent pH indicator 8-hydroxy-1,3,6-pyrenetrisulfonic acid entrapped inside the liposome. This technique is useful to study transmembrane proton movement in the time range 5 microseconds-10 s at physiological pH values. The kinetics of proton transport aided by ion carriers such as nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and valinomycin were studied by our method. The electrogenic nature of transport by CCCP and valinomycin and electroneutral ion transport by nigericin and monensin were shown. From the kinetics of proton transport aided by gramicidin, the time-averaged single-channel conductance of gramicidin channels was estimated to be (2.1 +/- 0.5) X 10(-16) S for H+ at pH 7.5.     

Control of proteoliposomal cytochrome c oxidase: the overall reaction

The control of cytochrome c oxidase incorporated into proteoliposomes has been investigated as a function of membrane potential (delta psi) and pH gradient (delta pH). The oxidase generates a pH gradient (alkaline inside) and a membrane potential (negative inside) when respiring on external cytochrome c. Low levels of valinomycin collapse delta psi and increase delta pH; the respiration rate decreases. High levels of valinomycin, however, decrease delta pH as valinomycin can also act as a protonophore. Nigericin (in the absence of valinomycin) increases delta psi and collapses delta pH; the respiration rate increases. On a millivolt equivalent basis delta pH is a more effective inhibitor of activity than is delta psi. In the absence of any ionophores the cytochrome oxidase proteoliposomes enter a steady state, in which there are both delta pH and delta psi components of control. Present and previous data suggest that the respiration rate responds in a linear way ("ohmically") to increasing delta pH but in a nonlinear way to delta psi ("non-ohmically"). High levels of both delta psi and delta pH do not completely inhibit turnover (maximal respiratory control values lie between 6 and 10). The controlled steady state involves the electrophoretic entry and electroneutral exit of K+ from the vesicles. A model is presented in which the enzyme responds to both delta pH and delta psi components of the proton-motive force, but is more sensitive to delta pH than to delta psi at an equivalent delta mu H+. The steady state of the proteoliposome system can be represented for any set of permeabilities and enzyme activity levels using the computer simulation programme Stella.     

Stimulation of menaquinone-dependent electron transfer in the respiratory chain of Bacillus subtilis by membrane energization

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Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.     

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.     

Flow-force relationships in mitochondrial oxidative phosphorylation

The rates of oxidation and phosphorylation in isolated rat-liver mitochondria have a steep dependence on the protonmotive force (delta mu H+) across the membrane. These experimentally observed relationships proved to be independent of the way in which delta mu H+ was varied. These results were obtained when the membrane potential (delta psi) was calculated from the distribution of K+ (in the presence of valinomycin). When triphenylmethylphosphonium (TPMP+) was used as a probe for delta psi, slightly different flow-force relationships were obtained. We conclude that unique relationships exist between delta mu H+ and the rates of oxidation and phosphorylation, and that under some conditions the behaviour of the probe TPMP+ is anomalous.     

The anti-cancer agent nemorosone is a new potent protonophoric mitochondrial uncoupler

Nemorosone, a natural-occurring polycyclic polyprenylated acylphloroglucinol, has received increasing attention due to its strong in vitro anti-cancer action. Here, we have demonstrated the toxic effect of nemorosone (1-25 μM) on HepG2 cells by means of the MTT assay, as well as early mitochondrial membrane potential dissipation and ATP depletion in this cancer cell line. In mitochondria isolated from rat liver, nemorosone (50-500 nM) displayed a protonophoric uncoupling activity, showing potency comparable to the classic protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Nemorosone enhanced the succinate-supported state 4 respiration rate, dissipated mitochondrial membrane potential, released Ca(2+) from Ca(2+)-loaded mitochondria, decreased Ca(2+) uptake and depleted ATP. The protonophoric property of nemorosone was attested by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium in the presence of valinomycin. In addition, uncoupling concentrations of nemorosone in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. Therefore, nemorosone is a new potent protonophoric mitochondrial uncoupler and this property is potentially involved in its toxicity on cancer cells.     

Effect of inductors of alkali cation permeability on cytochrome c bound to mitochondrial membrane]

The effect of inductors of alkali cation permeability--valinomycin, gramicidin A, gramicidin S and its N,N'-diacetyl derivative--on rat liver mitochondria during respiration has been studied. It is shown that valinomycin, gramycidin A and diacetylgramicidin S at optimal concentration for uncoupling cause two-phase activation of mitochondrial respiration and that this effect results from cytochrome c solubilization. Gramicidin S at optimal concentration cannot remove cytochrome c from the respirating mitochondria. It is suggested that this property of gramicidin S is owned to cytochrome c immobilisation in membrane, due to the effect of this compound.     

Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization.

This paper reports an investigation on the relationship between the proton electrochemical gradient (delta mu H+) and the cyclosporin A-sensitive permeability transition pore (PTP) in rat liver mitochondria. Using the SH group cross-linker phenylarsine oxide as the inducer, we show that both matrix pH and the membrane potential can modulate the process of PTP induction independently of Ca2+. We find that membrane depolarization induces the PTP per se when pHi is above 7.0, while at acidic matrix pH values PTP induction is effectively prevented. Since Ca2+ uptake leads to major modifications of the delta mu H+ (i.e. matrix alkalinization and membrane depolarization), we have explored the possibility that the Ca(2+)-induced changes of the delta mu H+ may contribute to PTP induction by Ca2+. Our data in mitochondria treated with Ca2+ plus N-ethylmaleimide and Ca2+ plus phosphate show that membrane depolarization is a powerful inducer of the PTP. Taken together, our observations indicate that the PTP can be controlled directly by the delta mu H+ both in the absence and presence of Ca2+, and suggest that a collapse of the membrane potential may be the cause rather than the consequence of PTP induction under many experimental conditions. Thus, many inducers may converge on dissipation of the membrane potential component of the delta mu H+ by a variety of mechanisms.     

ATP synthesis in Methanobacterium thermoautotrophicum coupled to CH4 formation from H2 and CO2 in the apparent absence of an electrochemical proton potential across the cytoplasmic membrane

Methanogenic bacteria are considered to couple methane formation with the synthesis of ATP by a chemiosmotic mechanism. This hypothesis was tested with Methanobacterium thermoautotrophicum. Methane formation from H2 and CO2 (2.5 - 3 mumol X min-1 X mg cells-1) by cell suspensions of this organism resulted in the formation of an electrochemical proton potential (delta mu H +) across the cytoplasmic membrane of 230 mV (inside negative) and in the synthesis of ATP up to an intracellular concentration of 5 - 7 nmol/mg. The addition of ionophores at concentrations which completely dissipated delta mu H + without inhibiting methane formation did not result in an inhibition of ATP synthesis. It thus appears that delta mu H + across the cytoplasmic membrane is not the driving force for the synthesis of ATP in M. thermoautotrophicum.     

Role of proton motive force in genetic transformation of Bacillus subtilis

This study explored the role of the proton motive force in the processes of DNA binding and DNA transport of genetic transformation of Bacillus subtilis 168 strain 8G-5 (trpC2). Transformation was severely inhibited by the ionophores valinomycin, nigericin, and 3,5-di-tert-4-hydroxybenzylidenemalononitrite (SF-6847) and by tetraphenylphosphonium. The ionophores valinomycin and nigericin also severely inhibited binding of transforming DNA to the cell envelope, whereas SF-6847 and carbonylcyanide-p-trifluoromethoxyphenylhydrazone hardly affected binding. The proton motive force, therefore, does not contribute to the process of DNA binding, and valinomycin and nigericin interact directly with the DNA binding sites at the cell envelope. The effects of ionophores, weak acids, and tetraphenylphosphonium on the components of the proton motive force and on the entry of transforming DNA after binding to the cell envelope was investigated. DNA entry, as measured by the amount of DNase I-resistant cell-associated [3H]DNA and by the formation of DNA breakdown products, was severely inhibited under conditions of a small proton motive force and also under conditions of a small delta pH and a high electrical potential. These results suggest that the proton motive force and especially the delta pH component functions as a driving force for DNA uptake in transformation.     

DCCD-sensitive Na+-transport in the membrane vesicles of Halobacterium halobium

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) and various ionophores on light-induced 22Na+-transport were studied in right-side-out membrane vesicles from Halobacterium halobium R1M1. The light-induced Na+ efflux was inhibited at the same DCCD concentration (greater than 40 nmol/mg protein) as required for inhibition of the Na+-dependent membrane potential (delta phi) formation. This supports our previous indication that the DCCD-sensitive, Na+-dependent transformation of pH-gradient (delta pH) into delta phi is mediated by Na+/H+-antiporter (Murakami, N. and Konishi, T. (1985) J. Biochem. 98, 897-907). FCCP or a combination of valinomycin and triphenyltin (TPT) inhibits the light-induced Na+ efflux in accordance with the notion of protonmotive force (delta mu H+)-driven antiporter. However, a marked lag in initiation of the Na+ efflux occurred in the presence of valinomycin, TPMP+, or a small amount of FCCP, suggesting that a gating step is involved in the Na+ efflux. On the other hand, the delta pH-dissipating ionophore TPT did not cause the lag. A simultaneous determination of delta phi, delta pH, and Na+ efflux rate at the initial stage of illumination revealed that the antiporter is gated by delta phi rather than delta mu H+.     

Protonophore-resistance and cytochrome expression in mutant strains of the facultative alkaliphile Bacillus firmus OF4.

Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse the protonmotive force (delta mu H+) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa3, b, c and o in strain CC2. Strain CC2 also maintained a higher delta mu H+ than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of delta mu H+ than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o. Cells of stran CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium, neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of delta mu H+. Instead, both resistant alkaliphile strains maintained a higher delta mu H+ and a correspondingly higher delta Gp than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa3-type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk delta mu H+.