Thermodynamics of Oxidative Phosphorylation in Bovine Heart Submitochondrial Particles*

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (AG,) of the adenine nucleotide system. AG, is the Gibbs free energy of ATP synthesis and is defined by the relationship where AG’, is the standard free energy of ATP hydrolysis. Studies of the effects of AG, on NADH respiration and the reduction of NAD+ by succinate show that increasing values of AG, cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between AG, values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measure-ments indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a AG,, equal to 10.7 to 11.4 kcal/mol. The reversible coupling between electron transfer, ATP synthesis, and the electrochemical proton gradient

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (AG,) of the adenine nucleotide system. AG, is the Gibbs free energy of ATP synthesis and is defined by the relationship AG, = -AG10 + RTln([ATPI/[ADPIIPil) where AG', is the standard free energy of ATP hydrolysis. Studies of the effects of AG, on NADH respiration and the reduction of NAD+ by succinate show that increasing values of AG, cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between AG, values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a AG,, equal to 10.7 to 11.4 kcal/mol. The reversible coupling between electron transfer, ATP synthesis, and the electrochemical proton gradient has been extensively analyzed in intact mitochondria (l-9). Under some experimental conditions, oxidative phosphorylation is close enough to thermodynamic equilibrium that the rates of electron flow and ATP synthesis are controlled by the overall Gibbs free energy of the phosphorylation system rather than by the concentration of a single substrate such as ADP (6,(8)(9)(10)(11)(12). The Gibbs free energy of ATP hydrolysis, against which phosphorylation of ADP occurs, is -15 to -16 kcal/mol in the extramitochondrial medium (6,8,(12)(13)(14). ATP synthesis, however, actually occurs on the inner side of the mitochondrial * This work was supported by Research Grant HL14483 from the National Institutes oi-Health-awarded to P. C. H. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. membrane. The transport of ATP, ADP, and Pi in and out of the matrix space may cause the high AG, of the external medium (15-17) since AG, values in the range of 11 to 12 kcal/mol have been observed for the intramitochondrial space (6,8,14).
In this paper we report studies of the effect of the thermodynamic phosphorylation potential, or Gibbs free energy of ATP synthesis, on the rates of forward and reverse electron transfer in phosphorylating submitochondrial particles. These provide a simplified experimental system compared to intact mitochondria because the membrane orientation is inverted, placing the ATPase in direct contact with the external medium.
Analytical Procedures -Respiration rates were measured with a Clark oxveen electrode in a closed chamber.

Control of Forward Electron
Transfer by AG, -The influence of AG, on respiration was studied by recording the respiratory rates of submitochondrial particles before and after the addition of mixtures of ATP and ADP producing different AG, values in the medium (Fig. 1). In order to observe the maximum effect on respiration, poly(L-lysine) was included in the medium to inhibit the oxidation of exogenous cytochrome c (23). However, the inhibition of oxygen uptake by poly(L-lysine) was less than 10% with the ETP, used in these experiments, suggesting that this submitochondrial particle preparation consists of nearly homogeneous inverted membranes (23). The altered respiratory rates remained constant for at least 1 min following the adenine ' The abbreviations used are: ETP, , electron transport particles prepared from heavy layer bovine heart mitochondria; Hepes, 4-(2hydroxyethylj-1-piperazineethanesulfonic acid.

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Phosphorylation Thermodynamics in Submitochondrial Particles nucleotide addition. Analysis of the respiratory rates as a function of AG,, showed that similar rates were obtained by either varying the ATP/ADP ratio at constant Pi concentration, or by varying the Pi concentration at a constant ATP/ ADP ratio (Fig. 2). However, at P, concentrations greater than 10 mM additional stimulation of respiration occurred due to a salt effect (24). The AG,, values were calculated from the added ATP, ADP, and Pi concentrations and the appropriate value of AG',, which was -7.3 ? 0.1 kcalimol for the conditions of these experiments (25). Low AG,, values caused a stimulation while high AG,, values produced an inhibition relative to the respiratory rate observed before the addition of adenine nucleotides. The rate of respiration changed approximately 3-fold between AG,, values of 7.6 and 13.0 kcabmol. Addition of an uncoupler stimulated respiration &fold relative to the rate observed in the absence of added adenine nucleotides. According to the observed relationship, the AG,, which caused no change in the rate of respiration, the "null point," corresponded to 10.6 kcal/mol (Fig. 2). For five independent experiments with different ETP, preparations, the null point AC,, occurred at 10.7 * 0.3 kcabmol.
Control of Reverse Electron Transfer by AG, -The energylinked reduction of NAD+ by succinate was examined to determine the effect of AG,, on reverse electron transfer. The rate of NAD+ reduction was recorded following the addition of various ATP plus ADP mixtures. An approximately linear dependence of the rate of this reaction on AG, was observed between values of 8.5 and 13.0 kcal/mol (Fig. 3). In contrast to respiration, reverse electron transfer proceeded faster at high AG,, values and slower at low AG,, values. The rate of NAD+ reduction increased 3-fold between AG,, values of 8.5 and 13.0 kcal/mol. For comparison, the rate of NAD+ reduction driven by respiration (phenazine methosulfate plus ascorbate) was determined in the presence of an optimum level of the oxidation-reduction mediator phenazine methosulfate. This respiration-driven rate was equivalent to that produced by a AG,, of approximately 10.3 kcal/mol based on the relationship shown in Fig. 3 NADH respiration (Fig. 4). Vigorous aeration was necessary in order to prevent anaerobiosis during the experiment.
For calculation of the AG,, values, the change in Pi concentration was determined from the changes in ADP and ATP levels, ignoring a small formation of AMP by adenylate kinase. Starting with only ATP added to the medium, the AG,, declined rapidly from an initial value of 12.6 kcal/mol to a constant value of 11.2 kcabmol. Starting with only ADP present, the AG,, rose from approximately 9.0 kcabmol to a 600. . Respiration was measured at 36" as described for Fig. 1  The reactions were quenched at the indicated times and the actual AG, was determined from enzymatic ATP and ADP analyses as described under "Experimental Procedures." value of 11.6 kcal/mol. In both cases 2 to 3 min were required in order to establish a constant AG,. These steady state values of AG,, achieved from the initial presence of either ATP or ADP agree well and indicate that the AG,, produced by NADH respiration corresponded to about 11.4 kcal/mol for this ETP, preparation.

DISCUSSION
The direct chemical determination of the AG,, normally produced by NADH respiration (11.4 kcalimol, Fig. 4) agrees reasonably well with the indirect estimates from null point experiments (average 10.7 kcal/mol). The null point experimental design gives a value of the AG,, that is a characteristic of the mechanism of oxidative phosphorylation in these inverted vesicles and is not simply a result of membrane damage or "loose coupling." The high degree of stimulation of respiration by uncouplers as well as the stimulation by ADP much in excess of that previously observed for submitochondrial particles (26) indicates that ETP,, are in fact "tightly coupled." The finding that similar values of the characteristic AG,, are determined from studies of either respiration or reverse electron transfer indicates that the characteristic AG,, is approached by electron transfer in either direction. A similar characteristic value, 10.2 kcal/mol, has been reported for the AGp formed during succinate oxidation by submitochondrial vesicles prepared by sonication of rat liver mitochondria (27). The latter vesicles, however, do not exhibit significant respiratory control (27).
The value of AG,, characteristic of oxidative phosphorylation in inverted submitochondrial particles, approximately 11 kcal/ mol, is similar to values measured for the internal compartment of intact mitochondria.
Values of 11.2 to 11.8 kcal/mol (61, 11.8' kcal/mol (14), and 11.0" kcal/mol (8) have been reported for the intramitochondrial AG,, of rat liver. As shown in the following paper, the electrochemical proton gradient of submitochondrial particles also responds to changes in AG,. Accordingly, a AG,, of 11 kcal/mol would correspond to an p Recalculated from the actual reported value of 12.9 kcabmol (14) using a more appropriate AG', of -7.7 kcahmol for the intramitochondrial space (6,25). 3 Calculated assuming a AG',, of -7.7 kcal/mol (6,25) and the reported difference between internal and external concentration ratios of 3.3 kcal/mol (8).
electrochemical proton gradient equivalent to approximately 240 mV, based on a stoichiometry of 2H+/ATP for the reversible ATPase. Such a stoichiometry has been measured during the hydrolysis of ATP by both submitochondrial particles (20) and mitochondria (28,29). A value of 240 mV is also similar to values of the electrochemical proton gradient measured in intact mitochondria by different techniques (2, 7). The higher external AG,, values, 15 to 16 kcal/mol, observed in suspensions of intact mitochondria (6,8,(12)(13)(14) are apparently a consequence of the molecular mechanisms of the adenine nucleotide and phosphate transport systems (15)(16)(17). Electrogenic transport of ATP4-outwards in exchange for ADI'-driven by the membrane potential (30) and electroneutral phosphoric acid transport caused by the pH gradient (31,32) can account for the observed values of the extramitochondrial AG,, (17). The measured difference between the AG,, values of the internal and external compartments (AAG,,), approximately 4 kcalimol (6, 8, 141, would be accommodated by an electrochemical proton gradient (Ak,+) of about 170 mV since such a mechanism involves the electrogenic transport of one proton and AAG,, = nFAh+.
Alternatively, a Ah+ of 240 mV could support a AAG,, corresponding to about 5.5 kcal/mol.
In regard to the control of electron transfer, the finding that the AG,, can modulate the rate of respiration in submitochondrial particles is consistent with observations that the external AG,, can regulate the rate of respiration of intact mitochondria (6,8,9,11). Because both the internal and external AG,, are interrelated through the electrochemical proton gradient as a consequence of the mechanisms of adenine nucleotide and phosphate transport, either or both will change in response to metabolic perturbations.