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.     

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.     

The proton stoichiometry of electron transport in Ehrlich ascites tumor mitochondria.

Initial rate measurements of the stoichiometric relationships between H+ ejection, K+ and Ca2+ uptake, and electron transport were carried out on mitochondria from Ehrlich ascites tumor cells grown in mice. With succinate as substrate and N-ethylmaleimide to prevent interfering H+ reuptake via the phosphate carrier, close to 8 H+ were ejected per oxygen atom reduced (H+/O ejection ratio = 8.0); with the NAD-linked substrates pyruvate or pyruvate + malate, the H+/O ejection ratio was close to 12. The average H+/site ratio (H+ ejected/2e-/energy-conserving site) was thus close to 4. The simultaneous uptake of charge-compensating cations, either K+ (in the presence of valinomycin) or Ca2+, was also measured, yielding average K+/site uptake ratios of very close to 4 and Ca2+/site ratios close to 2. It was also demonstrated that each calcium ion enters the respiring tumor mitochondria carrying two positive electric charges. These stoichiometric data observed in mitochondria from Ehrlich ascites tumor cells thus are in complete agreement with similar data on normal rat liver and rat heart mitochondria and suggest that the H+/site ratio of mitochondrial electron transport may be 4 generally. It was also observed that the rate of deltaH+ back-decay in anaerobic tumor mitochondria following oxygen pulses is some 6- to 8-fold greater than in rat liver mitochondria tested at equal amounts of mitochondrial protein.     

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.     

Nutritional effects on mitochondrial bioenergetics. Alterations in calcium uptake by rat liver mitochondria

The uptake of Ca2+ by energized liver mitochondria was compared in normal fed as well as in protein-energy malnourished rats. In the presence of phosphate, mitochondria obtained from both groups were able to accumulate Ca2+ from the suspending medium and eject H+ during oxidation of common substrates which activate different segments of the respiratory chain. The rate of Ca2+ uptake was significantly lower in mitochondria from protein-energy malnourished rats. The rates of oxygen consumption and H+ ejection were decreased by 20-30% during oxidation of substrates at the three coupling sites. Similarly, mitochondria from protein-energy malnourished rats exhibit a 34% decrease in the maximal rate of Ca2+ uptake and a 25% lower capacity for Ca2+ load. The stoichiometric relationship of Ca2+/2e- remained unaffected. In steady state, with succinate as a substrate in the presence of rotenone and N-ethylmaleimide, mitochondria from normal fed and protein-energy malnourished rats showed a similar rate of Ca2+ uptake. Furthermore in both groups the stoichiometry of the H+/O ratio was close to 8.0 (H+/site ratio close to 4.0), and of Ca2+/site was close to 2.0. The diminished rate of Ca2+ uptake observed in mitochondria from protein-energy malnourished rats could be explained on the basis of a depressed rate of electron transport in the respiratory chain rather than by an effect at the level of the Ca2+ or H+ transport mechanism per se.     

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.     

A theoretical analysis of the effect of phosphate on apparent H+/O stoichiometries in oxygen-pulse experiments with rat liver mitochondria

It is now generally accepted that, in oxygen-pulse experiments on rat-liver mitochondria suspended in KCl-based media, the rapid import of H+ with phosphate leads to an approx. 33% lowering of apparent H+/O stoichiometry. However, in low-K+ media, N-ethylmaleimide has no effect on stoichiometry, and there appears to be no import of H+ with phosphate. In this paper the quantitative effect of extramitochondrial phosphate on apparent H+/O stoichiometry is calculated theoretically, on the basis of internal and external buffering powers. The lack of appreciable phosphate uptake in low-K+ media is quantitatively explained in terms of several factors, including the initial pH gradient and initial phosphate distribution.     

Reconstitution of the mitochondrial non-selective Na+/H+ (K+/H+) antiporter into proteoliposomes

Mitochondria contain two Na+/H+ antiporters, one of which transports K+ as well as Na+. The physiological role of this non-selective Na+/H+ (K+/H+) antiporter is to provide mitochondrial volume homeostasis. The properties of this carrier have been well documented in intact mitochondria, and it has been identified as an 82,000-dalton inner membrane protein. The present studies were designed to solubilize and reconstitute this antiporter in order to permit its isolation and molecular characterization. Proteins from mitoplasts made from rat liver mitochondria were extracted with Triton X-100 in the presence of cardiolipin and reconstituted into phospholipid vesicles. The reconstituted proteoliposomes exhibited electroneutral 86Rb+ transport which was reversibly inhibited by Mg2+ and quinine with K0.5 values of approximately 150 and 300 microM, respectively. Incubation of reconstituted vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of 86Rb+ uptake into proteoliposomes. Incubation of vesicles with [14C]dicyclohexylcarbodiimide resulted in labeling of an 82,000-dalton protein. These properties, which are also characteristic of the native Na+/H+ (K+/H+) antiporter, lead us to conclude that this mitochondrial carrier has been reconstituted into proteoliposomes with its known native properties intact.     

Catalytic and regulatory site composition of yeast AMP deaminase by comparative binding and rate studies. Resolution of the cooperative mechanism

Yeast AMP deaminase is allosterically activated by ATP and MgATP and inhibited by GTP and PO4. The tetrameric enzyme binds 2 mol each of ATP, GTP, and PO4/subunit with Kd values of 8.4 +/- 4.0, 4.1 +/- 0.6, and 169 +/- 12 microM, respectively. At 0.7 M KCl, ATP binds to the enzyme, but no longer activates. Titration with coformycin 5'-monophosphate, a slow, tight-binding inhibitor, indicates a single catalytic site/subunit. ATP and GTP bind at regulatory sites distinct from the catalytic site and their binding is mutually exclusive. Inorganic phosphate competes poorly with ATP for the ATP sites (Kd = 20.1 +/- 4.1 mM). However, near-saturating ATP reduces the moles of phosphate bound per subunit to 1 PO4, which binds with a Kd = 275 +/- 22 microM. In the presence of ATP, PO4 cannot effectively compete with ATP for the nucleotide triphosphate sites. The PO4 which binds in the presence of ATP is competitive with AMP at the catalytic site since the Kd equals the kinetic inhibition constant for PO4. Initial reaction rate curves are a cooperative function of AMP concentration and activation by ATP is also cooperative. However, no cooperativity is observed in the binding of any of the regulator ligands and ATP binding and kinetic activation by ATP is independent of substrate analog concentration. Cooperativity in initial rate curves results, therefore, from altered rate constants for product formation from each (enzyme.substrate)n species and not from cooperative substrate binding. The traditional cooperative binding models of allosteric regulation do not apply to yeast AMP deaminase, which regulates catalytic activity by kinetic control of product formation. The data are used to estimate the rates of AMP hydrolysis under reported metabolite concentrations in yeast.     

Kinetics of inhibition and binding of dicyclohexylcarbodiimide to the 82,000-dalton mitochondrial K+/H+ antiporter

Inhibition of K+/H+ antiport by N,N'-dicyclohexylcarbodiimide in Mg2+ depleted mitochondria follows first order kinetics, exhibiting a half-time of 13 min when mitochondria are incubated with 50 nmol/mg inhibitor at 0 degrees C. 14C radiolabeled N,N'-dicyclohexylcarbodiimide binds to the 82,000-dalton protein, and the second order rate constant for binding is found to be approximately the same as the second order rate constant for inhibition. These findings provide additional confirmation of the identification of this porter with the 82,000-dalton protein and permit us to estimate that rat liver mitochondria contain about 8 pmol/mg of K+/H+ antiporter with a turnover number of 700 s-1. The K+/H+ antiporter of rat liver mitochondria is protected from N,N'-dicyclohexylcarbodiimide inhibition and binding by quinine and by endogenous Mg2+. An 82,000-dalton, [14C]N,N'-dicyclohexylcarbodiimide-binding protein is also observed in rat liver submitochondrial particles, establishing this as an integral protein of the inner membrane. Submitochondrial particles, presumed to be inverted in membrane orientation, are protected from radiolabeling by external Mg2+, supporting the contention that the Mg2+ binding site is localized to the matrix side of the K+/H+ antiporter.     

Blocking of Na+/K+ transport by the MgPO4 complex analogue Co(NH3)4PO4 leaves the Na+/Na(+)-exchange reaction of the sodium pump unaltered and shifts its high-affinity ATP-binding site to a Na(+)-like form.

Inactivation of Na+/K(+)-ATPase activity by the MgPO4 complex analogue Co(NH3)4PO4 leads, in everted red blood cell vesicles, to the parallel inactivation of 22Na+/K+ flux and 86Rb/Rb+ exchange, but leaves the 22Na+/Na(+)-exchange activity and the uncoupled ATP-supported 22Na+ transport unaffected. Furthermore, inactivation of purified Na+/K(+)-ATPase by Co(NH3)4PO4 leads to a parallel decrease of the capacity of the [3H]ouabain receptor site, when binding was studied by the Mg2+/Pi-supported pathway (ouabain-enzyme complex II) but the capacity of the ouabain receptor site was unaltered, when the Na+/Mg2+/ATP-supported pathway (ouabain-enzyme complex I) was used. No change in the dissociation constants of either ouabain receptor complex was observed following inactivation of Na+/K(+)-ATPase. When eosin was used as a marker for the high-affinity ATP-binding site of the E1 conformation, formation of stable E'2.Co(NH3)4PO4 complex led to a shift in the high-affinity ATP-binding site towards the sodium form. This led to an increase in the dissociation constant of the enzyme complex with K+, from 1.4 mM with the unmodified enzyme to 280 mM with the Co(NH3)4PO4-inactivated enzyme. It was concluded, that the effects of Co(NH3)4PO4 on the partial activities of the sodium pump are difficult to reconcile with an alpha, beta-protomeric enzyme working according the Albers-Post scheme. The data are consistent with an alpha 2, beta 2 diprotomeric enzyme of interacting catalytic subunits working with a modified version of the Albers-Post model.     

Energy transduction by the reconstituted b-c1 complex from yeast mitochondria. Inhibitory effects of dicyclohexylcarbodiimide

A purified cytochrome b-c1 complex isolated from yeast mitochondria has been reconstituted into proteoliposomes. The reconstituted comp]lex catalyzed antimycin A-sensitive electron transfer from different analogues of coenzyme Q to cytochrome c. The reconstituted complex was also capable of energy conservation as indicated by uncoupler-stimulated rates of electron transfer, electrogenic proton ejection, and reversed electron flow from cytochrome b to coenzyme Q2 in the presence of antimycin A driven by a valinomycin-induced K+-diffusion potential (negative inside). Close to four protons were ejected per two electrons transported through the reconstituted b-c1 complex with ferricyanide as an artificial and impermeable electron acceptor.l The H+/2e- ratio decreased to two in the presence of the proton-conducting agent, carbonyl cyanide m-chlorophenylhydrazone. The same processes were studied in parallel in energy-conserving site 2 of rat liver mitochondria with similar results. In the reconstituted b-c1 complex, dicyclohexylcarbodiimide (DCCD) blocked the function of the electrogenic proton translocating device in the forward direction of proton ejection as well as in the backwards direction, measured as reversed electron flow from cytochrome b to coenzyme Q2 driven by a K+-diffusion potential. The primary effect of DCCD is localized on the proton ejection process, as the low proton conductance of the proteoliposome membrane was totally preserved after DCCD treatment.     

The effect of endogenous phosphate on the H+/Mn2+ ratio and the state of Mn2+ in the mitochondrial matrix.

1. Kinetics and stoichiometry of H+ extrusion and reuptake and of Mn2+ uptake and release have been measured in respiring liver mitochondria in the absence of external added Pi. H+ and Mn2+ fluxes are parallel during aerobic cation uptake but not during uncoupler induced cation release. The H+/Mn2+ is 1.24. Addition of SH reagents, in concentrations inhibiting the Pi carrier, modifies the kinetics of H+ extrusion and of Mn2+ uptake and release. The slow phase of uncoupler induced Mn2+ release is diminished. The H+/Mn2+ is increased to 1.72. Addition of SH reagents, after the phase of aerobic uptake is completed, results in a significant reduction of the extent of uncoupler-induced Mn2+ release. The extent of reuptake of endogenous Pi during aerobic uptake of Mn2+ is about 8 nmol x mg protein-1. 2. Aerobic uptake of Mn2+ in the absence of external Pi results in an electron spin resonance spectrum which is the sum of two components. One, denoted as S, corresponds to Mn(H2O)2+(6). Another denoted as E, reflects spin exchange narrowing. In contrast to previous claims the following evidence suggests that the spin exchange component is due to Mn3(PO4)2 precipitate: (a) the dimension of the spin exchange spectrum is markedly reduced by abolition of Pi transport; (b) the spin exchange spectrum is released very slowly by addition of uncouplers under conditions where uncouplers cause a rapid deenergization of mitochondria, reuptake of H+ and release of cations; (c) the free matrix Mn2+ is released slowly after addition of uncoupler if there is a large spin exchange signal; howeover the free matrix Mn2+ is abolished rapidly by uncoupler when formation of the spin exchange signal is prevented by pretreatment with Ca2+; (d) the band width of the spin exchange fraction is independent of the Mn2+/protein ratio either under kinetic or steady state conditions; (e) the experimental spectrum recalls closely that obtained by computer simulation by assuming it as a combination of Mn(H2O)2+(6) and Mn3(PO4)2. 3. It is concluded that endogenous Pi affects the process of aerobic divalent cation uptake. A part of Mn2+ uptake in the absence of externally added anions, consists of a Mn3(PO4)2 precipitate. This accounts for a H+/Mn2+ ratio lower than 2.     

Reconstitution of the beef heart and rat liver mitochondrial K+/H+ (Na+/H+) antiporter. Quantitation of K+ transport with the novel fluorescent probe, PBFI

New indicators for fluorescent measurement of Na+ and K+ ions should prove particularly useful for studies of reconstituted carriers of these ions. We show that PBFI, a K(+)-specific probe, provides a convenient and sensitive assay for the study of K+ uptake mediated by the reconstituted mitochondrial K+/H+ (Na+/H+) antiporter. Fluorescent measurements have enabled us for the first time to establish reconstitution of the K+/H+ (Na+/H+) antiporter from beef heart as well as from rat liver mitochondria. This technique has also enabled us to establish that dicyclohexylcarbodiimide is capable of complete inhibition of K+/H+ antiport in the reconstituted system, in accord with findings in intact mitochondria. PBFI fluorescence, which measures net K+ uptake, was essential for this corroboration, since dicyclohexylcarbodiimide is not capable of complete inhibition of 42K+/K+ or 86Rb+/Rb+ exchange, presumably because it acts selectively on proton transport within the carrier.     

The H+/ATP transport ratio of the (K+ + H+)-ATPase of pig gastric membrane vesicles

Various values have been reported for the H+/ATP transport ratio of the (K+ + H+)-ATPase of the gastric parietal cell: 4, 2 and 1. We have, therefore, reinvestigated this matter with a vesicle preparation isolated from pig gastric mucosa. The vesicles are suspended in glycylglycine buffer (pH 6.11) at 22 degrees C, and incubated until equalization of the K+ concentration inside and outside (75 mM). After addition of ATP, the initial rates of H+ uptake and ATP hydrolysis are then measured. Proton uptake is inhibited in the absence of K+ or in the presence of nigericin. The K0.5 value for proton transport is 154 microM and the Km value for ATP hydrolysis is 61 microM. The Lineweaver-Burk plot for ATP hydrolysis vs. ATP concentration is linear with a Vmax of 5.5 nmol/mg protein per s, but that for H+ uptake is not. Thus with increasing ATP concentration (6.7 to 1670 microM) the transport ratio increases from 0.3 to 1.8. Extrapolation to infinite ATP concentration gives a value of 1.89. (S.E. 0.13, N = 5) and a Hill coefficient of n = 1.21 (S.E. 0.06, N = 5) implying that the true transport ratio is 2 H+/ATP with positive cooperativity between the protons.     

Characterization of the Na+/H+ exchanger in human epidermoid carcinoma A431 vesicles

A previous report from this laboratory (Rothenberg et al., 1983a) demonstrated the presence of an Na+/H+ exchanger in human epidermoid carcinoma A431 cells. We now characterize surface-derived membrane vesicles from this cell line which contain a functional Na+/H+ exchanger. The Na+/H+ exchanger in A431 vesicles shares a number of characteristics in common with previously described Na+/H+ exchangers including the following: (1) Na+ uptake is stimulated by an outward-directed pH gradient and inhibited by an inward-directed pH gradient. (2) Na+ uptake is inhibited by amiloride and its analogs and their relative effectiveness is similar in vesicles and A431 cells. (3) The Na+/H+ exchanger uses Na+ or Li+ as a substrate but not K+ or Cs+. (4) H+ efflux is stimulated by an inward-directed Na+ gradient and inhibited by the amiloride analog 5-N-dimethylamiloride. The Na+/H+ exchanger in these membrane vesicles is activated allosterically by low intravesicular pH. The apparent pKa of the activating site is 6.4-6.6, characteristic of the NA+/H+ exchanger before activation by mitogens.     

Steady-state H+/O stoichiometry of liver mitochondria.

We have measured the H+/O stoichiometry of rat liver mitochondria respiring in a steady-state, using a novel method. This involves measuring the initial rate of H+ back-flow into mitochondria after respiratory inhibition, with the assumption that this is equal to the steady-state H+-ejection rate. Division by the steady-state O2-consumption rate yields the H+/O ratio. The H+/O values obtained were: 8.3 +/- 1.0 (mean +/- S.E.M.) for 3-hydroxybutyrate: 8.2 +/- 0.7 for glutamate plus malate; 6.0 +/- 0.2 for succinate; 4.1 +/- 0.3 for ascorbate/tetramethylphenylenediamine and 3.0 +/- 0.1 for ascorbate/ferrocyanide. These values correspond to H+/O stoichiometries for electron flow to oxygen from NAD+-linked substrates, succinate and cytochrome c of 8, 6 and 2 (charge/O ratio = 4) respectively.     

The nature of the electron spin resonance signal during aerobic uptake of Mn2+ in mitochondria from rat liver

Rat liver mitochondria take up aerobically large amounts of divalent cations in the absence of exogenous phosphate. The electron spin resonance (ESR) spectrum of matrix Mn2+ reveals the presence of two components: one, a sextet signal, corresponding to hydrated Mn2+; another, a spin exchange signal, attributed either to Mn2+ binding to specific high-energy membrane sites or to complexes of Mn2+ with inorganic phosphate. Identification of the spin exchange signal with a Mn-Pi complex is favoured by the evidence that the spin exchange signal is observed at pH 7.5 but not at pH 6.5 in the absence of exogenous Pi, but at both pH 7.5 and 6.5 in the presence of exogenous Pi. On the other hand both in the absence or presence of exogenous Pi inhibition by N-ethylmaleimide of Pi transport, abolishes the spin exchange signal. This signal is again observed when Pi is generated in the matrix, in the presence of N-ethylmaleimide, by ATP hydrolysis, and again abolished by oligomycin. Finally, addition of uncouplers results in a very slow disappearance of the signal. The amount of Mn2+ participating in the spin exchange signal has been calculated to be in the range of 50-60 nmol X mg protein-1. This amount is compatible with the amount of endogenous Pi present or generated in average mitochondrial preparations. The ESR spectrum obtained by superimposing the spectra of Mn3(PO4)2 precipitate and hydrated Mn2+, in appropriate concentrations and ratios, resembles closely the ESR spectrum during aerobic Mn2+ uptake in mitochondria. The band width of the spin exchange signal of Mn3(PO4)2 is not constant and varies between 40 and 22 mT depending on the state of aggregation of the complex. The kinetics of aggregation can be followed in solution as a function of the concentration of Mn2+, Pi and of pH. Similar kinetics can also be followed during aerobic Mn2+ uptake by controlling the rate of Mn2+ influx. The present data support the previous proposal [Pozzan et al. (1976) Eur. J. Biochem. 71, 93-99] that the spin exchange signal is essentially due to a Mn3(PO4)2 precipitate in the mitochondrial matrix.     

Phosphate transport and proteins with SH groups in rat liver mitochondria

Phosphate transport in rat liver mitochondria was studied by following [32P] phosphate uptake within physiological concentrations. Transport inhibition due to mersalyl and protection by mersalyl against N-ethylmaleimide measured in those conditions corresponded to earlier results obtained by the swelling technique. When mitochondria were incubated with [3H] N-ethylmaleimide in the presence of mersalyl, the radioactive labeling in proteins of particles obtained after sonication was decreased in all fractions, but three proteins were both highly alkylated and also highly protected by mersalyl (M.W. 48,000 - 36,000 - 31,000). Two of these (M.W. 36,000 and 31,000) were partially purified by ultrogel chromatography in the presence of sodium dodecyl sulfate. Furthermore, it was shown that both phosphate and nigericin diminished labeling by N-ethylmaleimide in the final supernatant fraction. Two proteins (M.W. 98,000 and 31,000) were significantly alkylated by [3H] N-ethylmaleimide and protected by phosphate and nigericin.     

The mechanism of proton translocation by the cytochrome system of mitochondria. Characterization of proton-transfer reactions associated with oxidoreductions of terminal respiratory carriers.

A direct kinetic analysis is presented of rapid proton-releasing reactions at the outer or C-side of the membrane, in ox heart and rat liver mitochondria, associated with aerobic oxidation of reduced terminal respiratory carriers in the presence of antimycin. Valinomycin plus K+ enhances the rate of cytochrome c oxidation and the rate and extent of H+ release. In the presence of valinomycin the leads to H+/e- ratio, computed on the basis of total electron flow from respiratory carriers to oxygen, varies with pH, remaining always lower than 1, and is unaffected by N-ethylmaleimide. 2-Heptyl-4-hydroxyquinoline N-oxide and 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole, at concentrations which inhibit in the presence of antimycin the oxygen-induced reduction of b cytochromes, cause also a marked depression of the H+ release associated with aerobic oxidation of terminal respiratory carriers. Aerobic oxidation of the cytochrome system in mitochondria and of isolated b-c1 complex and cytochrome c oxidase results in scalar proton release from ionizable groups (redox Bohr effects). In mitochondria and submitochondrial particles, about 70% of the oxidoreductions of the components of the cytochrome system are linked to scalar proton transfer by ionizable groups. In isolated b-c1 complex scalar proton transfer, resulting from redox Bohr effect, amounts to 0.9H+ per Fe-S protein (190 muT). In isolated cytochrome c oxidase, Bohr protons amount to 0.8 per haem a + a3. The results presented indicate that the H+ release from mitochondria during oxidation of terminal respiratory carriers derives from residual antimycin-insensitive electron flow in the quinone-cytochrome c span and from redox Bohr effects in the b-c1 complex and cytochrome c oxidase. There is no sign of proton pumping by cytochrome oxidase during its transition from the reduced to the active 'pulsed' state and the first one or two turnovers.