The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scaler H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods. A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3- and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 h+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems.

Adenine Nucleotides↗

PubMed1976

Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements.

The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0.

Animals↗

PubMed1976

Re-evaluation of the H+/site ratio of mitochondrial electron transport with the oxygen pulse technique.

The number of protons ejected per pair of electrons passing each energy-conserving site in the electron transport chain (the H+/site ratio) has been investigated in rat liver mitochondria by means of the oxygen pulse technique introduced by Mitchell and Moyle (1967) (Biochem. J. 105, 1147-1162). The usual H+/site values of 2.0 observed by this method were found to be substantially underestimated as a result of the influx of phosphate into the mitochondria. This was shown by three different kinds of experiments. 1. Addition of N-ethylmaleimide or mersalyl, inhibitors of mitochondrial phosphate transport, increased the H+/site ratio from 2.0 to 3.0. The dependence of this effect on the concentration of either inhibitor was identical with that for inhibition of phosphate transport. Added phosphate diminished the H+/site ratio to values below 2.0 in the absence of N-ethylmaleimide. N-Ethylmaleimide protected the elevated H+/site ratio of 3.0 against the deleterious effect of added phosphate, but did not prevent a lowering effect of weak acid anions such as 3-hydroxybutyrate. 2. Prior washing of mitochondria to remove the endogenous phosphate that leaks out during the anaerobic preincubation led to H+/site ratios near 3.0, which were not increased by N-ethylmaleimide. Addition of low concentrations of phosphate to such phosphate-depleted mitochondria decreased the H+/site ratio to 2.0; addition of N-ethylmaleimide returned the ratio to 3.0. 3. Lowering the temperature to 5 degrees, which slows down phosphate transport, led to H+/site values of 3.0 even in the absence of N-ethylmaleimide. The H+/site ratio of 3.0 observed in the absence of phosphate movements was not dependent on any narrowly limited set of experimental conditions. It occurred with either Ca2+ or K+ (in the presence of valinomycin) as mobile permeant cation. It was independent of the concentration of succinate, oxygen, mitochondria, or rotenone, additions of Ca2+, Li+, or Na+ and was independent of medium pH between 6.5 and 7.5. Inhibitors of the transport of ions or acids other than phosphate did not affect the H+/site ratio. These results indicate that re-uptake of endogenous phosphate, lost from mitochondria during anaerobic preincubation, reduces the observed H+ ejection and leads to underestimated H+/site ratios of 2.0 in the oxygen pulse method. When phosphate movements are eliminated by the procedures described above, the observed H+/site ratio is about 3.0. This value appears to be closer to the true H+/site ratio for the primary H+ ejection process during electron transport.

Animals↗

PubMed1976

Stoichiometry of H+ ejection during respiration-dependent accumulation of Ca2+ by rat liver mitochondria.

We have investigated the energy-dependent uptake of Ca2+ by rat liver mitochondria with succinate as respiratory substrate with rotenone added to block NAD-linked electron transport. In the presence of 3-hydroxybutyric or other permeant monocarboxylic acids Ca2+ was taken up to extents approaching those seen in the presence of phosphate. The quantitative relationship between cation and anion uptake was determined from the slope of a plot of 3-hydroxybutyrate uptake against Ca2+ uptake, a method which allowed determination of the stoichiometry without requiring ambiguous corrections for early nonenergized or nonstoichiometric binding events. This procedure showed that 2 molecules of 3-hydroxtbutyrate were accumulated with each Ca2+ ion. Under these conditions close to 2 Ca2+ ions and 4 molecules of 3-hydroxybutyrate were accumulated per pair of electrons per energy-conserving site of the respiratory chain. Since 3-hydroxybutyrate must be protonated to pass the membrane as the undissociated free acid, it is concluded that 4 protons were ejected (and subsequently reabsorbed) per pair of electrons per energy-conserving site, in contrast to the value 2.0 postulated by the chemiosmotic hypothesis.

Animals↗

PubMed1976

The H+/site ratio of mitochondrial electron transport.

The number of H+ ejected during passage of 2e- through each energy-conserving site of the mitochondrial respiratory chain (the H+/site ratio) was measured in three ways. In each case transmembrane movements of endogenous phosphate were minimized. (1) Measurement of the uptake of weak acids during loading of mitochondria with Ca2+ demonstrated that 2.0 weak acid anions were accumulated per Ca2+ ion. Since 1.7 to 2.0 Ca2+ ions were were taken up per site, these data correspond to an H+/site ratio of 3.5 to 4.0. (2) More direct measurement of H+ ejection using the oxygen pulse technique demonstrated that the H+/site ratio was 3.0. In these experiments phosphate movements were prevented by addition of N-ethylmaleimide to inhibit phosphate-hydroxide antiport, by washing the mitochondria to remove endogenous phosphate, or by working at 5 degrees C to reduce the rate of phosphate transport. When phosphate movements were allowed, H+/site ratios of 2.0 were observed. (3) Measurement of the initial steady rates of oxygen consumption and H+ ejection following addition of substrate to aerobic, substrate-limited mitochondria yielded H+/site ratios of 2.0, which were elevated to 4.0 when phosphate transport was prevented as described above. Previous determinations of the H+/site ratio were thus underestimates due to the unrecognized movements of endogenous phosphate; our results show that the H+/site ratio is at least 3.0 andmay be as high as 4.0.

Acids↗

PubMed1976

Stoichiometric relationship between energy-dependent proton ejection and electron transport in mitochondria.

The number of protons ejected during electron transport per pair of electrons per energy-conserving site (the H+/site ratio) was measured in rat liver mitochondria by three different methods under conditions in which transmembrane movements of endogenous phosphate were minized or eliminated. (1) In the Ca2+ pulse method, between 3.5 and 4.0 molecules of 3-hydroxybutyrate and 1.75 to 2.0 Ca2+ ions were accumulated per 2 e- per site during Ca2+ induced electron transport in the presence of rotenone, when measured under conditions in which movements of endogenous phosphate were negligible. Since entry of 3-hydroxybutyrate requires its protonation to the free acid these data correspond to an H+/site ratio of 3.5-4.0 (2) In the oxygen pulse method addition of known amounts of oxygen to anaerobic mitochondria in the presence of substrate yielded H+/site ratios of 3.0 when phosphate transport was eliminated by addition of N-ethylmaleimide or by anaerobic washing to remove endogenous phosphate. In the absence of such measures the observed H+/site ratio was 2.0. (3) In the reductant pulse method measurement of the initial steady rates of H+ ejection and oxygen consumption by mitochondria in an aerobic medium after addition of substrate gave H+/site near 4.0 in the presence of N-ethylmaleimide; in the absence of the inhibitor the observed ratio was only 2.0. These and other experiments reported indicate that the values of 2.0 earlier obtained for the H+/site ratio by Mitchell and Moyle [Biochem J. (1967) 105, 1147-1162] and others were underestimates due to the unrecognized masking of H+ ejection by movements of endogenous phosphate. The results presented here show that the H+/site ratio of mitochondrial electron transport is at least 3.0 and may be as high as 4.0.

Animals↗

PubMed1975

Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria.

The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP.

Adenosine Triphosphate↗