THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 13, Issue of May 5, pp. 6165-6173.1967 Printed in U.S.A.

Upper and Lower Limits of the Charge Translocation Stoichiometry of Mitochondrial Electron Transport*

(Received for publication, August 14, 1986)

Andrew D. Beavis From the Department of Phurmacology, Medical College of Ohio, Toledo, Ohio 43699

The upper and lower limits of the mechanistic stoi- chiometry (n) of electric charge translocation coupled to mitochondrial electron transport have been deter- mined for the oxidation of succinate and B-hydroxy- butyrate using a recently described method (Beavis, A. D., and Lehninger, A. L. (1986) Eur. J. Biochem. 158, 307-314). This method requires no assumptions re- garding the magnitude of proton leakage or pump slip- page, but it takes advantage of the ability to predict the direction of change as the coupled fluxes are mod- ulated by specific means. In this study, the rates of K+ uptake ( JK) and O2 consumption ( Jo) were determined from simultaneous electrode measurements in the pres- ence of various concentrations of valinomycin or inhib- itors of electron flow. When valinomycin is varied, the rate of proton leakage or pump slippage should de- crease as Jo increases, with the result that the slope dJKjdJo will be greater than n. On the other hand, when an inhibitor of electron flow is varied, the rate of proton leakage or pump slippage should increase as Jo increases, with the result that the slope dJKldJ0

should be less than n. The data obtained using this approach indicate that n lies between 6.7 and 7.3 for succinate oxidation and between 10.2 and 11.7 for B- hydroxybutyrate (or NADH) oxidation. It is concluded that the mechanistic stoichiometry of charge separa- tion coupled to electron flow is 7 q+/O in the span from succinate to oxygen and 11 q+/O in the span from NADH to oxygen. These conclusions are fully consist- ent with the limits of the mechanistic ATP/O ratios previously determined for these spans (Beavis, A. D., and Lehninger, A. L. (1986) Eur. J. Biochem. 158, 315-322).

The stoichiometry of proton translocation coupled to elec- tron transport in mitochondria is the subject of much contro- versy (1-3). Early studies suggested that electron flow from NADH and succinate (or ubiquinone) to oxygen is accom- panied by the ejection of 6 H+/O and 4 H’/O, respectively (4, 5); however, it is now certain that these ratios are underesti- mates of the mechanistic stoichiometry. Current opinion is divided between stoichiometries of 8 H’/O (6-8), 10 H+/O (9, lo), 1 2 H+/O (11-17), and 13 H+/O (18) for NADH oxidation and 6 H+/O (3, 6-8) and 8 H+/O (11-21) for succinate oxi- dation. However, in a recent communication (22) in which a

* This work was supported by National Institutes of Health Grants GM05919 and HL36573 awarded by the National Institute of General Medical Sciences and National Heart, Lung, and Blood Institute, United States Public Health Service, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

new rationale (23) was used to determine the upper and lower limits of the mechanistic ATP/O ratios of oxidative phospho- rylation, it was suggested that none of these values may be correct and that the true values may be 11 H+/O for NADH and 7 H+/O for succinate oxidation. To test this hypothesis, this new approach has now been used to determine the upper and lower limits of the mechanistic K+/O stoichiometries.

Large variations in the observed H’/O ratio are not sur- prising when one considers the transient nature of net H’ ejection. Due to the small matrix volume of mitochondria, transmembrane gradients are generated very rapidly. In fact, net H’ ejection is difficult to detect unless a permeant cation is present to act as a “vectorial buffer” of A* (24, 25). Even under these conditions, net H’ ejection is limited to 20-30 nmol/mg, the limit of the pH buffering power of the matrix. During the rapid transitions generally employed in determi- nations of the H+/O ratio, a large pH gradient is generated. Protons rapidly reenter the matrix, either electroneutrally with permeant acids (26-28) or through cation/H+ antiport mechanisms (29-31), or electrophoretically through leak path- ways or indirectly by slippage of H’ pumps (32-35). The net result is that the observed H+/O ratio, as well as the rates of 0, consumption and H’ ejection, decline rapidly with time (19, 36, 37). This has led to the suggestions that the highest H+/O ratios reported are higher than the mechanistic stoi- chiometry due to an underestimation of the initial rate of oxygen consumption (38) and that the lowest H+/O values reported are lower than the mechanistic stoichiometry due to an underestimation of the H’ back leakage (21).

An alternative experimental approach is to measure the cation uptake which is coupled to H+ ejection rather than H+ ejection itself (13, 39-42). Under appropriate conditions, i.e. when endogenous Pi transport is blocked with N-ethylmaleim- ide, it has been shown that the H+/O ratio is equal to the K’/O ratio (13,43) or twice the Ca2+/0 ratio (44). This result is predicted by the very small quantity of net charge transport necessary to produce a large change in the membrane poten- tial (25). Measurement of K’ uptake has the advantage that it is possible to increase the extent of K+ uptake 10-fold or more by the inclusion of anions of permeant acids such as phosphate or acetate which will buffer the pH gradient. Be- cause the transition is greatly extended, the initial rates of oxygen consumption and K’ uptake can be more accurately determined. The disadvantage of measuring K+ uptake is that during these measurements mitochondria maintain a substan- tial membrane potential. Thus, electrophoretic H’ leakage cannot be assumed negligible at any point in the transition. For this reason, the method developed for the determination of the upper and lower limits of the mechanistic stoichiometry is particularly well suited to these measurements, since this procedure sidesteps the uncertainty of correcting for net H+ leakage (23).

6165

6166 q'/O Stoichiometry of Mitochondrial Electron Transport

EXPERIMENTAL PROCEDURES

Rat liver mitochondria were isolated in 0.25 M sucrose following the procedure of Schneider (45). The mitochondria were suspended in 0.25 M sucrose at a stock concentration of 100 mg of protein/ml. Protein was determined by the method of Murphy and Kies (46). Experiments in which oxygen uptake was measured using a Clark oxygen electrode (Yellow Springs Instrument Co.) were carried out in a closed thermostatted glass cell of 2.3-ml capacity into which the oxygen electrode (provided with a tightly stretched high sensitivity membrane), a K+ electrode (Beckman 39047), and an independent reference electrode (Microelectrodes Inc. MI402) were inserted. The reference electrode was filled with 3 M LiCl saturated with AgCI; LiCl was used in place of KC1 to avoid a drift in the electrode signal due to leakage of K+ from the reference cell into the reaction chamber. The outputs from the K+ and reference electrodes were fed into a Beckman Expandomatic SS2 pH meter connected to a Soltec dual- channel strip-chart recorder. In some experiments a fast-responding membraneless oxygen electrode, slightly modified from that described in Refs. 47 and 48, was used to determine the rate of oxygen con- sumption. In this case, the oxygen and pH electrodes were introduced into a water-jacketed Perspex chamber of 1.5-ml capacity, similar to that described in Ref. 49.

The solubility of oxygen corrected to 760 mm Hg was determined as previously described (22). The value obtained was 512 p~ 0 in the assay media, compared with 525 p M 0 in air-equilibrated water. The 100% signal from the oxygen electrode was determined by adding a volume of medium air-saturated at 25 "C to the open chamber (25 "C) and waiting until a stable signal was obtained. The zero signal was established by using dithionite or respiring mitochondria to consume all the dissolved oxygen. It was necessary to make frequent checks to ensure linearity of response of the Clark electrode, particularly when fitted with a stretched high-sensitivity membrane.

Unless otherwise indicated in the legends, the medium used for measurements with succinate as the substrate contained 120 mM LiC1, 10 mM succinate, 5 mM Pi, 3 mM MgC12, 0.5 mM EGTA,' 2 mM KCl, 4 p M rotenone, 1 pg of oligomycin per mg of protein, and 10 p M cytochrome c. The medium was equilibrated with air at 25 'C and adjusted to pH 7.1 with LiOH. When 8-hydroxybutyrate was used as the substrate the medium was essentially the same except for the omission of rotenone and succinate and the inclusion of the Li+ salts of DL-j3-hydroxybutyrate (10 mM) and malate (5 mM).

For each measurement, medium was added to the chamber followed by the inhibitor or uncoupler. Respiration was initiated by the addi- tion of 3-4 mg/ml rat liver mitochondria. When the mitochondria had consumed 30-40% of the oxygen in the system, valinomycin (0- 5 pl) was added to initiate K+ uptake. During state 4 respiration, the K+ electrode response was calibrated by adding 2 or 3 pulses of standard KC1 to a total of 1000 nmol of K+ and a 10-pl volume. The response of the electrode did not change during the course of an experiment, and so the average deflection obtained at a given K+ concentration was used to calculate the rate of K+ uptake at that concentration. When the extent of K+ uptake was calculated, in order to avoid any errors due to the nonlinear response of the electrode, the extents of the deflections from the K+ standard additions were used to determine the exponential equation which best fitted the K+ calibration curve. To determine the rate of 0 2 diffusion into the chamber, dithionite was added to medium in the sealed chamber to consume most of the dissolved oxygen. The time course of re-equili- bration was then followed. The first-order rate constant was deter- mined and used to calculate the rate of O2 back diffusion into the chamber. At 50-70% saturation, the range in which most measure- ments were made, rates of back diffusion were insignificant compared with the rates of 02 consumption.

To obtain upper and lower limits of the mechanistic stoichiometry, which are as close as possible to the mechanistic stoichiometry, conditions should be selected which yield maximal K+/O ratios. Ideally, K+ uptake should be measured under conditions where elec- trophoretic transmembrane movements of all other ions are mini- mized, e.g. influx of endogenous Ca2+ is eliminated by the addition of EGTA. Electrophoretic H+ leaks cannot be eliminated but can be minimized by selecting conditions to minimize the driving force. Thus, A* can be minimized by employing high concentrations of valinomycin and the highest concentration of K+ (2-3 mM) compat-

The abbreviations used are: EGTA, [ethylenebis(oxyethylene- nitri1o)ltetraacetic acid; HEPES, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid; RCR, respiratory control ratio.

ible with accurate electrode measurements of K+ uptake, and the pH gradient can be minimized by using high concentrations of a permeant acid which can be translocated into the matrix very rapidly.

Both acetic acid, which is readily permeant as such and requires no transport system, and phosphoric acid, which is translocated by a P;/H+ symporter (or Pi-/OH- antiporter) (50) were employed in this work. Addition of valinomycin to mitochondria respiring in media of high ionic strength containing 2-3 mM K+ plus 20 mM acetate results in the stimulation of respiration and the rapid accumulation of about 100 nmol of K+/mg of mitochondrial protein, followed by a sharp decrease in both K' uptake and respiration rates. Respiration cannot be stimulated again, even by uncouplers, unless the suspension is supplemented with cytochrome c, eg. addition of 10 p~ cytochrome c reactivates respiration allowing K+ accumulation to continue until a maximum of 200-300 nmol of K+/mg is accumulated. These obser- vations are interpreted in the following way. K+ acetate accumulation within the matrix leads to osmotic swelling and eventual rupture of the outer membrane. Cytochrome c, normally located between the two membranes, is then lost to the suspending medium, with the result that electron flow between the b-c, complex and cytochrome oxidase limits the overall rate of respiration.

When phosphate is used in place of acetate, K' uptake and osmotic swelling leads to uncoupling and K+ efflux prior to anaerobiosis. The tendency of phosphate to evoke K+ efflux can be lessened by increas- ing the concentration of succinate in the medium. In view of the fact that succinate can exchange for matrix phosphate on the dicarboxy- late carrier (50), this suggests that Pi exerts its effect in the matrix. This exchange also permits succinate to act as a vectorial buffer of ApH and consequently will also help maintain the concentration of succinate in the matrix as swelling proceeds. When j3-hydroxybutyr- ate was used as the substrate, 5 mM malate, which is oxidized very poorly by liver mitochondria, was included in the medium to prevent a large accumulation of Pi and help buffer matrix pH. This appeared to be effective, since the extent of K+ uptake was increased without significantly changing the stoichiometry. These observations suggest that a combination of matrix Pi and swelling may activate the K+/H+ antiporter (29-31). This could lead to an underestimate of the K+/O ratio; however, the following observations suggest that the K+/H+ antiport does not affect the initial K+/O ratio following the addition of valinomycin under the conditions used. 1) Mitochondria incubated with rotenone and malonate in place of succinate exhibit negligibly small rates of K+ efflux. 2) If K+ is omitted from the medium, the addition of valinomycin does not increase the steady state rate of respiration. Only a short burst of respiration is seen as a small amount of K' present in the mitochondrial suspending medium is taken up. 3) There appears to be a threshold level of matrix Pi necessary to activate the K+/H+ exchange mechanism. The extent of K+ (and presumably Pi) accumulation prior to anaerobiosis can be decreased by delaying the addition of valinomycin. Comparison of the subse- quent rates of K+ efflux under anaerobic conditions shows that if 50- 100 nmol of K+/mg have been taken up, the rate of K+ efflux is very low, whereas if 200-300 nmol of K+/mg have been taken up, the K+ efflux rate is much faster even at equal external K+ concentrations. It has been proposed by Garlid (29-31) that matrix M%+ acts as a brake on the K+/H+ antiporter and that phosphate and swelling activate the carrier by lowering the concentration of free M%+ within the matrix. To minimize Mg2+ loss from the mitochondria during stoichiometry measurements, 3 mM M%+ was included in the medium. Addition of M%+ also ensures inhibition of the intrinsic electropho- retic Li+ (and K+) transport which has been observed when chelators of Mg2+ such as EDTA and nucleoside triphosphates are added to mitochondria respiring in Mg+-free media (51).'

RESULTS

Rationale-The theory for determining the upper and lower limits of the mechanistic q+/O stoichiometry has been pre- sented in detail in a previous communication (23) and, there- fore, will only be summarized here. This rationale exploits the ability to predict the direction of change in the rate of leakage or pump slippage as the fluxes of interest, K' uptake (&) and oxygen consumption (Jo), are modulated by specific means. For measurable net fluxes it is assumed that

A. D. Beavis, unpublished observations.

9’10 Stoichiometry of Mitochondrial Electron Transport

JK = d o - JL (1)

where n is the mechanistic stoichiometry and J k is the sum of the rates of H+ leakage and pump slippage, which for brevity shall hereafter simply be referred to as the rate of leakage or the leak. The slope of a plot of JK uersus JO is, therefore, given by

! 3 L n - - ark dJ0 dJ0

Thus, the slope differs from the mechanistic stoichiometry n by the quantity dJk/dJo , and, therefore, if one can find conditions under which dJk/dJo should be negative or posi- tive, one can establish upper or lower limits.

To determine the upper limit, J k must increase as JO decreases. This may be achieved by modulating the rates by varying the concentration of valinomycin. Under these con- ditions, the decrease in Jo results from a decrease in the rate of energy utilization and thus will be associated with an increase in A;, or its equivalent. Since A ~ H is the driving force for the leak, J k will increase and, therefore, dJk/dJo will be negative. To determine the lower limit, J k must de- crease as Jo decreases. This may be achieved by modulating the rates by systematically varying the kinetics of electron flow. As rates of electron flow decline, A i i ~ or its equivalent will decline (52), and, thus, J k will also decline, and, therefore, dJk/a’Jo will be positive.

Determination of the Upper Limit of the Mechanistic q+/O Ratio-Fig. 1 shows typical traces from an experiment in which succinate was used as the substrate and K+ uptake was initiated by the addition of various amounts of valinomycin at the points indicated. Although it is clear that rates of K’ and oxygen uptake each decline with time, the initial rates are essentially constant until approximately 500 nmol of K+ (100 nmol of K+/mg) have been accumulated. Measurements of state 4 rates (( J 0 ) J , taken prior to the addition of valino- mycin, revealed no significant increase in the intrinsic H+ leak during the course of the experiment.

Fig. 2A shows the relationship between the initial rates of K’ uptake and the rates of oxygen consumption from two separate experiments. In the first (data from Fig. l), phos- phate was used as the vectorial buffer of ApH and O2 was monitored with a fast-responding membraneless electrode (36). In the second, acetate was used as the permeant H+- carrying anion and O2 was monitored with a Clark electrode. The data from the first experiment fall on a straight line with

vo I t t t t f

FIG. 1. Modulation of rates of K+ uptake and electron flow by varying valinomycin concentration. Typical oxygen and K+ traces are shown from an experiment in which succinate was used as the substrate. Oxygen concentration was monitored with a mem- braneless oxygen electrode. The experiment was performed as de- scribed under “Experimental Procedures” with the following concen- trations of valinomycin (Val) added where indicated (a) 733, ( b ) 91.6, ( c ) 41.2, ( d ) 20.6, ( e ) 6.9 ng of valinomycin/mg of protein. Note that trace a was recorded at twice the chart speed indicated.

1600

A

m E

c

\ E

Y 000

. 1200 -

+

7

0 E - 400 !z

6

( J0lK ( n m o 1 O / m 1 n . m g l

6167

6 00

- m E

c “ 400

\ E

Y +

- 0 E 200 c v

2

0

B J

0 20 40 60

(J0IK (nmol O / m i n . m g ) FIG. 2. Determination of the upper limit of the mechanistic

K+/O ratio. The initial rate of K+ uptake, JK, following the addition of various concentrations of valinomycin is plotted versus the corre- sponding rate of oxygen consumption ( Jo)K. The upper limit is given by the slope of the graph. A, data obtained with succinate as the substrate with valinomycin varied from 0 to 1 pg/mg. 0, data taken from the experiment described in the legend to Fig. 1 in which the membraneless 0, electrode was used. The line fitted to this data is described by the equation JK = 7.29 (JO)K - 162. 0, data from a similar experiment in which phosphate was replaced by the lithium salts of HEPES (5 mM) and acetate (20 mM) and O2 was monitored with a Clark electrode. In this case the linear equation describing the relationship is JK = 7.76 (Jo)K - 217 (line not drawn). B, data obtained with 0-hydroxybutyrate as the substrate, using the Clark electrode with valinomycin varied from 0 to 60 ng/mg. The line fitted to the data is described by the equation J K = 11.7 (Jo)K - 80. Other details are described under “Experimental Procedures.”

a slope of 7.29, which, according to the rationale, is the upper limit of the mechanistic q+/O stoichiometry. In addition, the maximum K+/O flux ratio observed in this experiment is about 6.6 with a respiratory control ratio (Jo/( J&) of about 8.0. From this it follows that the K+/O ratio calculated using

6168 q’/O Stoichiometry of Mitochondrial Electron Transport

only the “extra oxygen” consumed ( Jo - ( J0)J is 7.5. I t should also be noted, however, that the stoichiometry remains greater than 6.0 until Jo drops below 126 nmol of O/min. mg. Stoichiometries greater than those found in this experiment were not observed even when K+ or valinomycin concenira- tions were raised to yield an RCR greater than 10. As shown, the data from the second experiment fit the same line quite well, although they are best described by a line with a slope of 7.76 (see legend). This was the highest slope observed in any experiment of this type, and slopes less than 7.0 were not observed.

A similar experiment was carried out with B-hydroxybutyr- ate as the substrate. The traces were qualitatively similar to those with succinate, except that net K’ efflux began after accumulation of approximately 130 nmol of K+/mg. Fig. 2B shows that the relationship between J K and Jo is linear. The slope is 11.7 and provides the upper limit of the mechanistic K+/O ratio for P-hydroxybutyrate. The maximum rate of respiration with P-hydroxybutyrate is lower than with succi- nate, and less valinomycin was necessary to induce the max- imum rate. In light of recent proposals that the mechanistic stoichiometry is 10 (9, lo), it is noteworthy that, at the highest rates of respiration in Fig. 2B, the observed flux ratio exceeded 10 (10.2) and that the flux ratio did not fall to 8.0, the lowest ratio currently favored (6-8), until respiration rates fell below 22 nmol of O/min. mg.

Determination of the Lower Limit of the Mechanistic q+/O Ratio-Fig. 3 shows some typical traces from an experiment in which succinate was used as the substrate and the rates of K’ uptake and electron flow were modulated by titrating with malonate, a competitive inhibitor of succinate oxidation. In this experiment the fast-responding membraneless oxygen electrode was used (36). Fig. 4A shows the initial rate of K+ uptake plotted versus the initial rate of oxygen consumption for the entire experiment. The relationship is linear, and the slope provides a lower limit of 6.53 for the mechanistic q+/O ratio for succinate oxidation. Similar results are obtained when antimycin is used to inhibit electron flow and the Clark oxygen electrode is employed to determine J o (see Figs. 4, A and B). As with succinate, the relationship between JK and Jo during P-hydroxybutyrate oxidation is found to be linear (Fig, 4B). The slope of the line provides a lower limit of 10.24 for the mechanistic q+/O stoichiometry for NADH oxidation.

An interesting feature of the data shown in Fig. 4 is that the curves intercept the axes close to the origin. This is

I p g m

FIG. 3. Modulation of rates of K* uptake and electron flow by varying malonate concentration. Typical oxygen and K’ traces are shown from an experiment in which succinate was the substrate, Oxygen concentration was monitored with the membrane- less electrode (36), and K+ uptake was initiated by the addition of 0.74 pg of valinomycin/mg of protein. The following concentrations of malonate were included in the media: (a) 0, (b) 0.i3, ( c ) 0.26, and ( d ) 0.66 mM. Other details are as described under “Experimental Procedures.”

1600

CI

?n E

1

\ E

Y 800

- 1200 .-

+

,“

0 E c

400

+i*

0 0

800

CI

?n E

c

\ E

Y 400

- 600

.-

+

- 0 E 1 - 200

l-Y

50 100 150 200

0 20 40 60 80

( & I K ( n m o l O / m i n . m g )

FIG. 4. Determination of the lower limit of the mechanistic K*/O ratio. The initial rate of K’ uptake, JK, following the addition of valinomycin is plotted versus the corresponding rate of oxygen consumption (J& measured in the presence of various concentra- tions of inhibitors of electron flow. The lower limit is given by the slope of the graph. A , data obtained using succinate as the substrate. 0, data from the experiment described in Fig. 3 in which the inhibitor was malonate. K+ uptake was initiated by the addition of 0.74 pg of valinomycin/mg of protein, and 0 2 concentration was monitored with the membraneless electrode. The line fitted to the data is described by the equation JK = 6.53 (Jo)K - 1.4. 0, data from an experiment in which the inhibitor was antimycin A (0-30 pmol/mg). K+ uptake was initiated by the addition of 0.13 pg of valinomycin/mg of protein, and 02 concentration was monitored with a Clark electrode. The line fitted to the data is described by the equation JK = 6.75 (J& - 71. B, data obtained with b-hydroxybutyrate as the substrate. Antimycin A (0-30 pmol/mg) was the inhibitor, and oxygen was monitored with the Clark electrode. K+ uptake was initiated by the addition of 50 ng of valinomycin/mg of protein. The line fitted to the data is described by the equation JK = 10.2 (J& - 17.6.

q+/O Stoichiometry of Mitochondrial Electron Transport 6169

probably related to the further observation that the depend- ence of the stoichiometry on the rate of respiration is very small when respiration is titrated with inhibitors of electron transport. For example, it was necessary to inhibit respiration by more than 80% before the K'/O flux ratio fell from 10.2 to 9.0 in the experiment shown in Fig. 4B. The constancy of stoichiometry observed when succinate oxidation is titrated with malonate argues against the criticism that the high values may result from incomplete inhibition of electron flow through site 1 from NADH. Direct evidence against this explanation was also obtained. When rotenone was deliber- ately omitted to maximize endogenous electron flow from NADH, no effect was seen on the K+/O flux ratio until the rate of electron flow from succinate was inhibited to a level comparable with the rate of endogenous electron flow from NADH. At this point, the stoichiometry was found to increase so that the slope of J K versus Jo decreased resulting in an underestimation of the lower limit (data not shown).

The intercept at J K = 0 also reveals something about the current-voltage characteristics of the net H' leakage in these experiments. This intercept equals the minimum rate of res- piration necessary to maintain the existing K+ gradient and must, therefore, be proportional to the rate of net H+ leakage. Since A ~ K is about 100 mV in .these experiments, the mem- brane potential required to hold K' at equilibrium must be at least 100 mV, about half the potential generated in state 4. In most experiments, the intercept at J K = 0 is much less than half the state 4 rate and often close to zero, indicating that the H' leak cannot be ohmic, Le. proportional to AbH+. It also follows that the rate of electroneutral K+/H+ exchange must be very low under these conditions, since this will contribute to the net H' leak in the presence of valinomycin.

From the findings that the stoichiometry is nearly inde- pendent of the rate of respiration and that the two oxygen electrodes give similar results, it seems unlikely that the initial rates of Jo and J K are significantly under- or overestimated. On the other hand, initial rates are not easily determined in the partially inhibited state, as both J K and Jo decrease continuously with time, and this invites two criticisms of the K'/O ratio as determined from initial rates. 1) It underesti- mates the true initial stoichiometry due to a decline in the K'/O ratio with time (36). 2) It overestimates the true initial stoichiometry due to an underestimation of the initial rate of oxygen consumption (3). To address these potential criti- cisms, the relationship between the extent of K' uptake and the extent of oxygen consumed was examined at specific time intervals following the addition of valinomycin. Fig. 5A con- tains an analysis of the four traces shown in Fig. 3. The closed circles represent data obtained from trace a of Fig. 3 (no malonate) and indicate that the stoichiometry remained un- changed at 6.17 for at least 6 s. The open circles represent data from trace b of Fig. 3 (0.13 mM malonate) and indicate that the stoichiometry remained unchanged at 6.45 for 14 s. Similar results were obtained with 0.26 and 0.66 mM malonate (open and closed squares). Thus, partial inhibition of electron flow extends the period over which the stoichiometry remains constant, permitting the conclusion that the observed high flux ratios (over 6 in each case) and the lower limit determined from the fluxes are not due to uncertainties in estimating rapidly changing initial rates.

Fig. 5B contains the results of a similar experiment in which P-hydroxybutyrate was used as the substrate. In this experiment the incubation medium contained 10 mM malo- nate in place of malate to avoid endogenous succinate oxida- tion. Malonate was found to inhibit /3-hydroxybutyrate oxi- dation significantly, resulting in low rates of respiration. 'l'ns

180

0)

f 150

Q h

120

90

60

30

0

I

0 5 I0 15 20 25 30

(n rno l 0 consurned)/rng

I20

100

80

60

40

20

0 0 2 4 6 8 10 12

(nrnol 0 consurned) / rng

FIG. 5. Comparison of the extent of K+ uptake and oxygen consumed in individual traces. The amount of K+ taken up is plotted versus the amount of oxygen consumed at specific time intervals following the addition of valinomycin. A, data from the traces in Fig. 3 with succinate as the substrate. 0, measurements from trace a (no malonate) made at 1.5-s intervals; slope = 6.17, using first 4 points. 0, measurements from trace b (0.13 mM malonate) made at 1.5-s intervals; slope = 6.45, using first 9 points. ., measure- ments from trace c (0.26 mM malonate) made at 1.5-s intervals; slope = 6.35, using first 9 points. 0, measurements from trace d (0.66 mM malonate) made at 2-s intervals; slope = 6.21, using first 12 points. For clarity of presentation, the intercepts on the abscissa have been assigned arbitrary values. B, data using j3-hydroxybutyrate as sub- strate. 10 mM malonate replaced malate in the standard medium. Data from three traces (0, 0, and A) are presented with the points

the data is 10.25. on each trace taken at 2-s intervals. The slope of the line fitted to

extents of O2 and K' uptake were determined at 2-s intervals on each trace. Despite a large decrease in the rate with time, the K'/O ratio remained constant at 10.25 for at least 30 s. These analyses show that the lower limits determined from

6170

0 +\ Y

q’/O Stoichiometry of Mitochondrial Electron Transport

B

A 7 -

6 -

5 -

4 -

3 -

2 -

1 -

0 ‘ 0.0 .5

1 /RCR

1

0 +\ Y

0 ‘ 0.0 .5 1.0

1 /RCR

12

10

8 8

0 +\ 6 Y

0 +\ 6 Y

4 4

2 2

C C

the rate measurements cannot be explained by difficulties in determining rates and that the observed K+/O ratios exceed 6.0 for succinate and exceed 10.0 for P-hydroxybutyrate. Fur- thermore, the observation that the stoichiometry does not decline until considerable quantities of K’ have been taken up indicates that the rate of leakage does not increase signif- icantly and thus cannot lead to underestimation of initial value of JK.

Attempt to Extrapolate the K+/O Ratio to “Zero Leak”- Uncoupling agents permit the rate of H+ leakage to be mod- ulated directly and thus permit examination of the effect of varying “state 4” respiration on the K+/O flux ratio. The potential use of this procedure to extrapolate the observed stoichiometry to “zero leak” (or infinite RCR) has been dis- cussed in detail in a previous communication (23). In brief, it was concluded that the difference between the current-voltage characteristics of the endogenous leak and that catalyzed by carbonyl cyanide p-trifluoromethoxyphenylhydrazone would

1.0 lead to an extrapolated stoichiometry equal to or greater than the mechanistic stoichiometry. Since this procedure has proved useful in determination of the mechanistic ATP/O ratio (22), it,has now been applied to K’/O measurements,

Fig. 6A shows the results of an experiment in which oxygen uptake was monitored with the fast-responding electrode and K+ uptake was initiated by the addition of 0.74 pg of valino- mycin/mg. With this dose of valinomycin, the relationship between K’/O and l/RCR is complex, with a steep segment at high RCRs which may reflect the nonohmic nature of intrinsic H+ leakage (32-35, 53-56). Although these data are not suitable for extrapolating the K+/O ratio to 1/RCR = 0, they are consistent with the upper and lower limits obtained. The maximum K’/O flux ratio observed is 6.7 at a respiratory control ratio of 7.2. The K’/O ratio calculated from the “extra-oxygen’’ consumed is 7.8, which is lower than 8.0, whereas the maximum observed ratio is greater than 6.0. When a lower concentration of valinomycin is used (0.13 pg/ mg), a somewhat different result is obtained (Fig. 6B) . As expected, using a suboptimal concentration of valinomycin, the maximum observed RCR (about 4.4) and K+/O ratio (about 5.8) are lower; however the relationship between the K+/O ratio and 1/RCR is essentially linear as was the rela- tionship between the ATP/O ratio and 1/RCR observed pre- viously (22). With these data, extrapolation to 1/RCR = 0 is feasible, yielding a K+/O ratio of 7.5. Fig. 6C contains the results of a similar experiment in which 8-hydroxybutyrate was used as the substrate. A linear relationship is observed between the K+/O ratio and 1/RCR, which extrapolates to a K+/O ratio of 11.1 at 1/RCR = 0. Thus, these data support the conclusion that the upper limit is less than 12.0 q+/O.

1 /RCR

FIG. 6. Use of carbonyl cyanide p-trifluoromethoxyphen- ylhydrazone in extrapolation of the K+/O ratio to infinite RCR. The K+/O ratio (JK/J0) is plotted uersus the 1/RCR ((J&/ Jo). K+ uptake was initiated by the addition of a fixed dose of valinomycin after 35-40% of the 0, had been consumed. Various doses of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (0- 0.14 nmol/mg) were added to the medium prior to the addition of the mitochondria, and the rate of respiration prior to the addition of valinomycin was determined, (J&. A, succinate was the substrate, 0.74 pg of valinomycin/mg of protein was used, and 0 2 was monitored with the membraneless electrode. B, succinate was the substrate, 0.13 pg of valinomycin/mg of protein was used, and 0 2 was monitored with the Clark electrode. The equation of the line fitted to the data is K+/O = 7.49 - 7.55/RCR. C, p-hydroxybutyrate was the substrate, 50 ng of valinomycin/mg of protein was used, and 02 was monitored with the Clark electrode. The equation of the line fitted to the data is K+/O = 11.1 - 11.05/RCR.

q+/O Stoichiometry of Mitochondrial Electron Transport 6171

DISCUSSION

The upper limits of the mechanistic q+/O stoichiometry for succinate oxidation and NADH oxidation deduced from this study are 7.3 and 11.7, respectively. These values are incon- sistent with the stoichiometries of 8 and 12 or 13 proposed by others (11-21). On the other hand, the lower limits obtained, 6.7 for succinate and 10.2 for NADH, are higher than the stoichiometries proposed by Wikstrom and others (3, 6-10, 44). The only integral q+/O ratios consistent with these limits are 11.0 for NADH oxidation and 7.0 for succinate oxidation. These stoichiometries are completely consistent with the up- per and lower limits of the mechanistic ATP/O ratios for these spans, as previously determined (22). Thus, with an H+/ ATP ratio of 4 (16-19) the mechanistic ATP/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.

The q+/O stoichiometries of sites 1 and 3 can also be deduced from these values, especially since different labora- tories generally agree that the 4+/2e- ratio is 2 in the span from succinate (or ubiquinone) to cytochrome c (site 2) (3). It follows that the q+/O ratio must be 5 from cytochrome c to oxygen (site 3). If 0 2 is reduced on the matrix side of the membrane, so that the vectorial translocation of 2e- from cytochrome c to O2 is equivalent to the ejection of 2q+, it also follows that the H+/O ratio is 3 for site 3. This is one less than the value of 4 favored by some (16, 18, 36, 37) and one more than the value of 2 favored by others (3, 6, 43, 44, 57, 58). Results consistent with this conclusion are reported in detail in the following paper (59). From the present data, it also follows that the q+/2e- (or H+/2e-) ratio in the span from NADH to ubiquinone (site 1) must be 11 - 7 = 4. These data, therefore, support the stoichiometry of 4H’/2e- previously reported from the laboratories of Lehninger (11-15), Azzone (16, 17), Hinkle (9), and Wikstrom (10) but do not support values of 2 (6-8), 3 (60), and 5 (20) which are still favored by others.

Low H+/O values reported in the literature have been criticized for underestimating the mechanistic stoichiometry due to the electroneutral or electrophoretic back leakage of protons into the mitochondria (21). Proton leakage is gener- ally dealt with in one of three ways. 1) The leak is assumed to be zero and the observed stoichiometry taken as an indi- cation of the mechanistic stoichiometry. 2) The leak is as- sumed to be equal to the “leak” observed in state 4 and, therefore, the “extra-oxygen” consumed during H’ ejection or cation accumulation is used to estimate the mechanistic stoi- chiometry (39, 42). 3) The extent of H’ leakage is estimated in classical “OZ pulse” experiments by extrapolating back the H+ influx which occurs after the oxygen has been consumed (4, 25, 44). Since the mechanistic stoichiometry is probably underestimated by the first method and overestimated by the second, these procedures can be used to provide upper and lower limits. The third procedure attempts to correct for leakage; however, this procedure has been criticized because it does not correct for electrophoretic leakage which occurs during the pulse (61). The method used in the present work avoids the uncertainty of correcting data for leakage by refin- ing the procedures for determining upper and lower limits of the mechanistic stoichiometry. No assumptions regarding the size of the H’ leak or its mechanism are required. A similar approach has been applied to refined oxygen pulse experi- ments by Costa et al. (19) to determine upper and lower limits of the H+/O ratio; however, these authors extrapolated rapidly decaying rates to zero time. Technical aspects of this proce- dure have been questioned (62) and defended (21); however, the assumption that the decay in JH and Jo may be described by a single exponential function from zero time remains

questionable (63), especially in light of recent reports which show that Jo declines in distinct phases following an oxygen pulse (21, 64). Apart from these considerations, it is interest- ing to note that when Costa et al. (19) extrapolate the fluxes to “level flow,” where H+ leakage should be minimal, the flux ratio is typically 7.25. If the mechanistic stoichiometry were 8.0, the value favored by these workers, it can be calculated that the rate of H’ leakage at zero time would have to account for the consumption of 22 nmol of O/min.mg. Since this is close to the rate of respiration in state 4, one would have to conclude that there is no difference in the rate of leakage between static head (state 4) and level flow. This conclusion is contrary to most experimental observations (32-35,53-56).

The most frequent and serious technical criticism leveled against the highest H+/O stoichiometries has been that the rates of oxygen consumption have been underestimated due to the slow response of the Clark-type electrode (3, 38, 58, 65). The present protocols minimize this problem. The pH gradient is buffered by the addition of permeant acids, the extent and duration of cation accumulation are maximized, and initial rates decline much less quickly than during meas- urements of H+ ejection. Three observations suggest that respiration rates are not underestimated in this work. 1) Inhibiting respiration has a negligible effect on the K+/O stoichiometry. 2) The K+/O stoichiometry remains constant for an extended period. 3) Measurements made with the fast- responding oxygen electrode lead to the same conclusions as those made with a Clark electrode.

Quantitation of oxygen consumption obviously requires accurate knowledge of solubility of oxygen in the assay me- dium. It is, therefore, surprising that, apart from Hinkle’s suggestion that the major systematic error in ATP/O meas- urements may be calibration of the oxygen electrode (9), the value assumed for oxygen solubility has never been considered as a potential source of error in stoichiometric studies. The oxygen concentration determined in these studies, 512 p~ 0, is consistent with the value of 498 p~ (based on a value of 516 pM 0 assumed for pure water) for 0.15 M KC1 in Ref. 25, but is significantly higher than the value of 474 p~ 0 (66) which was previously assumed (67, 68). This difference ac- counts for the difference between the conclusions drawn here and those drawn in preliminary reports of the upper and lower limit determinations of both the ATP/O ratios (67) and K+/O ratios (68).

Reynafarje et al. (69) have devised a new procedure to determine 0 2 solubility and have obtained values about 6% lower than those used here. The reasons for this discrepancy offered by Reynafarje et al. (69) do not, however, seem ade- quate. They propose that previous measurements have over- estimated the 0, content of media by overestimating the extent of NADH oxidation which can be incomplete due to the presence of slowly oxidized a-NADH. This problem was avoided in the present work by determining the concentration of oxidizable NADH enzymatically. The second criticism was that the rate of NADH oxidation slows substantially as NAD+ accumulates in the medium. This effect was not observed in the present work, possibly due to the fact that NADH was oxidized by NADH-cytochrome c reductase evidenced by the requirement for cytochrome c to observe rapid oxidation of NADH. The most probable sources of error in the measure- ments of Reynafarje et al. (69) lie in their calibration proce- dures. The H+ trace, which is used to determine the rate of NADH oxidation, is calibrated by the addition of only 1 pI of standard acid, and it is difficult to be certain of the accuracy of this addition. In contrast the 0, trace was calibrated by the addition of 50-p1 pulses of oxygenated medium. Since this

6172 q+/O Stoichiometry of Mitochondrial Electron Transport

represents 3% of the volume of the reaction chamber, signif- icant loss of oxygenated medium will occur when the second pulse (or even first pulse if mixing is fast) is added to the chamber. This could lead to a 3% underestimate of the oxygen content of the medium. Use of the lower values obtained by Reynafarje et al. (69) would raise the limits deduced in this and the following communication (59), but not sufficiently to alter the conclusions.

The stoichiometry of 8 H+/O for succinate is also subject to criticism on thermodynamic grounds. There does not ap- pear to be enough energy available from electron flow during state 4 respiration to drive the ejection of 8 H+/O (see Refs. 70 and 71). Most workers now accept that well coupled mitochondria oxidizing succinate can maintain a A;LH of at least 200 mV in state 4 (58, 70, 72). To accommodate a Ab, of this magnitude, the affinity of electron flow (Ao = 2AE) between succinate and oxygen must be at least 1200 mV if n = 6 H+/O, 1400 mV if n = 7 H+/O, and 1600 mV if n = 8 H+/O. Since the total affinity is about 1520 mV (58, 70), an H+/O ratio of 8 appears impossible. On the other hand, an H+/O ratio of 7 is thermodynamically compatible with values of A;, up to about 220 mV, which is close to the upper limit of reported values (see Ref. 72 for review).

Thermodynamic criticisms have been challenged by Costa et al. (19) on the grounds that the role of the bulk phase A,& in oxidative phosphorylation is uncertain, leaving open the possibility that A;, might not be a quantitative measure of the intermediate high-energy state. These arguments are in- appropriate, because the thermodynamic constraint in both H+/O and K+/O measurements applies to the net reaction in which electron flow is coupled to K+/H+ exchange. The K+ for H+ exchange, in turn, is measured in the bulk phase, and the thermodynamic restriction is that the energy available from the flow of 2e- cannot be less than ~ ( A / . L H + ApK) where A / . L ~ and ApH refer to the phases of measurement. Thus, as always, the thermodynamic constraint on the stoichiometry is independent of both the transport mechanism and the nature of the "high-energy intermediate."

In conclusion, the maximum observed K+/O ratios in this work are not very much higher than the H+/O stoichiometries of 6 and 10 proposed by some (9, lo), and the maximum calculated K+/O ratios, using extra oxygen, are not very much lower than the values of 8 and 12 favored by others (11-21). On the other hand, the upper and lower limits obtained exclude both of these sets of values and point to the inter- mediate H+/O ratios of 7 and 11 for succinate and NADH oxidation, respectively. Taken at face value the limits ob- tained in this work would remove from contention the stoi- chiometries of 6 and 8 for succinate oxidation; however, the closeness of the limits to these extreme values together with a certain degree of experimental uncertainty makes it difficult to rule out the extreme values completely. To help draw firm conclusions, a better understanding of the characteristics of the H+ leak or pump slippage would be useful. In addition, determination of the limits of the q+/O ratio of cytochrome c oxidase would also help since it should be experimentally easier to distinguish values of 4, 5, and 6 than it is to distinguish values of 6, 7, and 8. The results of such a study are presented in the following paper.

Acknowledgment-The early stages of this work were carried out in the laboratory of the late Albert L. Lehninger in the Department of Biological Chemistry at The Johns Hopkins University School of Medicine. For the last 10 years of his life, Dr. Lehninger was a major force in the quest to determine the true proton-motive stoichiometries of mitochondrial respiration, and his generous support and encour- agement during the course of this work were greatly appreciated.

REFERENCES 1. Mitchell, P. (1979) Science 2 0 6 , 1148-1158 2. Mitchell, P. (1979) Eur. J. Biochem. 95, 1-20 3. Wikstrom, M., and Krab, K. (1980) Curr. Top. Bioenerg. 10 , 51-

4. Mitchell, P., and Moyle, J. (1967) Biochem. J. 105 , 1147-1162 5. Mitchell, P., and Moyle, J. (1979) Biochem. SOC. Trans. 7 , 887-

6. Brand, M. D., Harper, W. G., Nicholls, D. G., and Ingledew, W.

7. Brand, M. D. (1979) Biochem. SOC. Trans. 7,874-880 8. Al-Shawi, M. K., and Brand, M. D. (1981) Biochem. J. 200,539-

546 9. Hinkle, P. C. (1981) in Chemiosmotic Proton Circuits in Biological

Membranes (Skulachev, V. P., and Hinkle, P. C., eds) pp. 49- 58, Addison-Wesley Publishing Co., Reading, MA

101

894

J. (1978) FEBS Lett. 9 5 , 125-129

10. Wikstrom, M. (1984) FEBS Lett. 169,300-304 11. Lehninger, A. L., Reynafarje, B., and Alexandre, A. (1977) in

Structure and Function of Energy Transducing Membranes (van Dam, K., and van Gelder, B. F., eds) pp. 95-106, Elsevier/ North-Holland Biomedical Press, Amsterdam

12. Vercesi, A., Reynafarje, B., and Lehninger, A. L. (1978) J . Biol. Chem. 253,6379-6385

13. Reynafarje, B., and Lehninger, A. L. (1978) J. Biol. Chem. 253 ,

14. Villalobo, A., and Lehninger, A. L. (1979) J. Bwl. Chem. 254 ,

15. Lehninger, A. L., Reynafarje, B., Alexandre, A., and Villalobo, A.

16. Pozzan, T., Miconi, V., Di Virgilio, F., and Azzone, G. F. (1979)

17. Azzone, G. F., Pozzan, T., and Di Virgilio, F. (1979) J. Biol. Chem.

18. Alexandre, A., Reynafarje, B., and Lehninger, A. L. (1978) Proc.

19. Costa, L. E., Reynafarje, B., and Lehninger, A. L. (1984) J. Biol.

20. Lemasters, J. J. (1984) J. Biol. Chem. 259, 13123-13130 21. Lehninger, A. L., Reynafarje, B., Hendler, R. W., and Shrager,

22. Beavis, A. D., and Lehninger, A. L. (1986) Eur. J. Biochem. 158 ,

23. Beavis, A. D., and Lehninger, A. L. (1986) Eur. J. Biochem. 158,

24. Chappell, J. B., and Haarhoff, K. N. (1967) in Biochemistry of Mitochondria (Slater, E. C., Kaniuga, Z., and Wojtczak, L., eds) pp. 75-91, Academic Press, Orlando, FL

25. Mitchell, P., Moyle, J., and Mitchell, R. (1979) Methods Enzymol.

26. Brand, M. D., Reynafarje, B., and Lehninger, A. L. (1976) J. Biol.

27. Brand, M. D., Reynafarje, B., and Lehninger, A. L. (1976) Proc.

28. Reynafarje, B., Brand, M. D., and Lehninger, A. L. (1976) J. Biol.

29. Garlid, K. D. (1980) J. Biol. Chem. 2 5 5 , 11273-11279 30. Dordick, R. S., Brierley, G. P., and Garlid, K. D. (1980) J. Biol.

31. Shi, G.-Y., Jung, D. W., Garlid, K. D., and Brierley, G. P. (1980)

32. Pietrobon, D., Azzone, G. F., and Walz, D. (1981) Eur. J. Biochem.

33. Pietrobon, D., Zoratti, M., and Azzone, G. F. (1983) Biochim.

34. Zoratti, M., Favaron, M., Pietrobon, D., and Azzone, G. F. (1986)

35. Pietrobon, D., Zoratti, M., Azzone, G. F., and Caplan, R. S. (1986)

36. Reynafarje, B., Alexandre, A., Davies, P., and Lehninger, A. L.

37. Reynafarje, B., Costa, L. E., and Lehninger, A. L. (1986) J. Biol.

38. Wikstrom, M., and Krab, K. (1979) Biochim. Biophys. Acta 5 4 9 ,

39. Rossi, C. S., and Lehninger, A. L. (1964) J. Biol. Chem. 239 ,

40. Rossi, E., and Azzone, G. F. (1969) Eur. J. Biochem. 7, 418-426

6331-6334

4352-4358

(1980) Ann. N. Y. Acad. Sci. 341,585-592

J. Biol. Chem. 254,10200-10205

254,10206-10212

Natl. Acad. Sci. U. S. A. 75,5296-5300

Chem. 259,4802-4811

R. I. (1985) FEBS Lett. 192,173-178

315-322

307-314

55,627-640

Chem. 251,5670-5679

Natl. Acad. Sci. U. S. A. 73 , 437-441

Chem. 251,7442-7451

Chem. 255,10299-10305

J. Biol. Chem. 255,10306-10311

117,389-394

Biophys. Acta 723,317-321

Biochemistry 25,760-767

Biochemistry 25 , 767-775

(1982) Proc. Natl. Acad. Sci. U. S. A. 7 9 , 7218-7222

Chem. 261,8254-8262

177-222

3971-3980

q+/O Stoichiometry of Mitochondrial Electron Transport 6173

41. Azzone, G. F., and Massari, S. (1971) Eur. J. Biochem. 19, 97- 56. Brown, G. C., and Brand, M. D. (1986) Biochem. J. 234, 75-81 107 57. Azzone, G. F., Pozzan, T., Massari, S., and Bragadin, M. (1978)

42. Brand, M. D., Chen, C-H., and Lehninger, A. L. (1976) J. Biol. Biochim. Biophys. Acta 501,296-306 Chem. 251,968-974 58. Wikstrom, M., Krab, K., and Saraste, M. (1981) Annu. Reu.

43. West, I. C., Mitchell, R., Moody, A. J., and Mitchell, P. (1986) Biochem. 50,623-655 Biochem. J. 236, 15-21 59. Beavis, A. D. (1987) J. Biol. Chem. 262,6174-6181

44. Mitchell, R., West, I. C., Moody, A. J., and Mitchell, P. (1986) 60. De Jonge, P. C., and Westerhoff, H. V. (1982) Biochem. J. 204, Biochim. Biophys. Acta 849,229-235 515-523

45. Schneider, W. C. (1957) in Manometric Techniques (Umbreit, W. 61. Heinz, E., Westerhoff, H. V., and van Dam, K. (1981) Eur. J. W., Burris, R., and Stanfer, J. F., eds) p. 188, Burgess Publish- Biochem. 115, 107-113 ing Co., Minneapolis, MN 62. Krab, K., Soos, J., and Wikstrom, M. (1984) FEBS Lett. 178,

46. Murphy, J. B., and Kies, M. W. (1960) Biochim. Biophys. Acta 187-192

47. Davies, P. W., and Grenell, R. G. (1962) J. Neurophysiol. (Be- 64. Lehninger, A. L., Reynafaje, B., and Costa, L. (1985) J. Znorg.

48. Davies, P. W. (1962) in Physical Techniques in Biological Research 65. Papa, S., Capuano, F., Markert, M., and Altamura, N. (1980)

45,382-384 63. Krab, K., and Wikstrom, M. (1986) FEBS Lett. 201, 198-204

thesda) 25,651-683 Bwchem. 23,335-340

FL (Nastuk, W. L., ed) Vol. 2, Chapter 3, Academic Press, Orlando, FEBS Lett. 1 1 1,243-248

66. Chappell, J. B. (1964) Biochem. J. 90, 225-237 49. Affolter, H., and Sigel, E. (1979) Anal. Biochem. 97, 315-319 67. Beavis, A. D. (1980) Fed. Proc. 39, 2056 50. Chappell, J. B. (1968) Br. Med. Bull. 24, 150-157 68. Beavis, A. D. (1981) Fed. Proc. 40, 1563 51. Wehrle, J. P., Jurkowitz, M., Scott, K. M., and Brierley, G . P. 69. Reynafaje, B., Costa, L. E., and Lehninger, A. L. (1985) Anal.

(1976) Arch. Biochem. Biophys. 174, 312-323 Biochem. 145,406-418 52. Zoratti, M., Pietrobon, D., and Azzone, G. F. (1982) Eur. J. 70. Wikstrom, M., and Saraste, M. (1984) in Bioenergetics (Ernster,

Biochem. 126,443-451 L., ed) pp. 49-94, Elsevier Scientific Publishing Co., Amster- 53. Nicholls, D. G. (1974) Eur. J. Biochem. 50, 305-315 dam 54. O’Shea, P. S., Petrone, G., Casey, R. P., and Azzi, A. (1984) 71. Murphy, M. P., Brown, G. C., and Brand, M. D. (1985) FEBS

55. Krishnamoorthy, G., and Hinkle, P. C. (1984) Biochemistry 23, 72. Ferguson, S. J., and Sorgato, M. C. (1982) Annu. Reu. Biochem. Bwchem. J. 219, 719-726 Lett. 187, 16-20

1640-1645 51,185-217