Feedback Regulation and Time Hierarchy of Oxidative Phosphorylation in Cardiac Mitochondria

To determine how oxidative ATP synthesis is regulated in the heart, the responses of cardiac mitochondria oxidizing pyruvate to alterations in [ATP], [ADP], and inorganic phosphate ([P_i]) were characterized over a range of steady-state levels of extramitochondrial [ATP], [ADP], and [P_i]. Evolution of the steady states of the measured variables with the flux of respiration shows that: (1) a higher phosphorylation potential is achieved by mitochondria at higher [P_i] for a given flux of respiration; (2) the time hierarchy of oxidative phosphorylation is given by phosphorylation subsystem, electron transport chain, and substrate dehydrogenation subsystems listed in increasing order of their response times; (3) the matrix ATP hydrolysis mass action ratio [ADP] × [P_i]/[ATP] provides feedback to the substrate dehydrogenation flux over the entire range of respiratory flux examined in this study; and finally, (4) contrary to previous models of regulation of oxidative phosphorylation, [P_i] does not modulate the activity of complex III.     

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.     

Simulation of state 4 --> state 3 transition in isolated mitochondria

The mathematical dynamic model of oxidative phosphorylation developed previously and in the accompanying paper was modified to involve isolated mitochondria conditions; it was also used to simulate state 4 --> state 3 transition in rat liver mitochondria incubated with succinate as respiratory substrate and glucose-hexokinase as an ADP-regenerating system. Changes in the respiration rate, protonmotive force and reduction level of ubiquinone and cytochrome c as well as the internal (i) and external (e) ATP/ADP ratio between state 4 and state 3 were calculated and compared with the experimental data. Flux control coefficients with respect to oxygen consumption flux for different reactions and processes of oxidative phosphorylation were simulated for different values of the respiration rate (state 4, state 3 and intermediate states). Flux control coefficients for the oxidation, phosphorylation and proton leak subsystems with respect to the oxidation, phosphorylation and proton leak fluxes for different values of the respiration rate were also calculated. These theoretical data were compared with the experimental results obtained in the frame of metabolic control analysis and the 'top-down' approach to this analysis. A good agreement was obtained. Simulated time courses of the respiration rate, the protonmotive force (Deltap) and other parameters after addition of a small amount of ADP to mitochondria in state 4 mimicked at least semiquantitatively the experimentally measured time courses of these parameters. It was concluded, therefore, that in the present stage, the model is able to reflect different properties of the oxidative phosphorylation system in a broad range of conditions fairly well, allows deeper insight into the mechanisms responsible for control and regulation of this process, and can be used for simulation of new experiments, thus inspiring experimental verification of the theoretical predictions.     

Theoretical studies on the regulation of oxidative phosphorylation in intact tissues

The theoretical studies on the regulation of oxidative phosphorylation that were performed with the aid of kinetic models of this process are overviewed. A definition of the regulation of the flux through a metabolic pathway is proposed and opposed to the control exerted by particular enzymes over this flux. Different kinetic models of oxidative phosphorylation proposed in the literature are presented, of which only the model proposed by myself and co-workers was extensively used in theoretical studies on the regulation and compensation in the oxidative phosphorylation system. These theoretical studies have led to the following conclusions: (1) in isolated mitochondria, an increase in the activity of an artificial ATP-using system stimulates mitochondria mainly via changes in [ADP], while changes in [ATP] and [P(i)] play only a minor role; (2) in non-excitable tissues (e.g. liver), hormones (acting via some cytosolic factor(s)) activate directly both ATP usage and at least some enzymes of the ATP-producing block; (3) in excitable tissues (e.g. skeletal muscle), neural signals stimulate (via some cytosolic factor(s)) in parallel all the steps of oxidative phosphorylation together with ATP usage and substrate dehydrogenation; (4) the decrease in the flux through cytochrome oxidase caused by a decrease in oxygen concentration is, at least partially, compensated by a decrease in Delta p and increase in the reduction level of cytochrome c. A theoretical prediction is formulated that there should exist and be observable a universal cytosolic factor/regulatory mechanism which directly activates (at least in excitable tissues) all complexes of oxidative phosphorylation during an increased energy demand.     

Regulation of energy metabolism in liver

Energy metabolism in liver has to cope with the special tasks of this organ in intermediary metabolism. Main ATP-generating processes in the liver cell are the respiratory chain and glycolysis, whereas main ATP-consuming processes are gluconeogenesis, urea synthesis, protein synthesis, ATPases and mitochondrial proton leak. Mitochondrial respiratory chain in the intact liver cell is subject to control mainly by substrate (hydrogen donors, ADP, oxygen) transport and supply and proton leak/slip. Whereas hormonal control is mainly on substrate supply to mitochondria, proton leak/slip is supposed to play an important role in the modulation of the efficiency of oxidative phosphorylation.     

Regulation of oxidative phosphorylation in intact mammalian heart in vivo

A dynamic computer model of oxidative phosphorylation in intact heart was developed by modifying the model of oxidative phosphorylation in intact skeletal muscle published previously. Next, this model was used for theoretical studies on the regulation of oxidative phosphorylation in intact heart in vivo during transition between different work intensities. It is shown that neither a direct activation of ATP usage alone nor a direct activation of both ATP usage and substrate dehydrogenation, including the calcium-activated tricarboxylate acid cycle dehydrogenases, can account for the constancy of [ADP], [PCr], [P(i)] and [NADH] during a significant increase in oxygen consumption and ATP turnover encountered in intact heart in vivo. Only a direct activation of all oxidative phosphorylation complexes in parallel with a stimulation of ATP usage and substrate dehydrogenation enabled to reproduce the experimental data concerning the constancy of metabolite concentrations. The molecular background of the differences between heart and skeletal muscle in the kinetic behaviour of the oxidative phosphorylation system is also discussed.     

Quantitation and origin of the mitochondrial membrane potential in human cells lacking mitochondrial DNA.

Mammalian mitochondrial DNA (mtDNA) encodes 13 polypeptide components of oxidative phosphorylation complexes. Consequently, cells that lack mtDNA (termed rho degrees cells) cannot maintain a membrane potential by proton pumping. However, most mitochondrial proteins are encoded by nuclear DNA and are still imported into mitochondria in rho degrees cells by a mechanism that requires a membrane potential. This membrane potential is thought to arise from the electrogenic exchange of ATP4- for ADP3- by the adenine nucleotide carrier. An intramitochondrial ATPase, probably an incomplete FoF1-ATP synthase lacking the two subunits encoded by mtDNA, is also essential to ensure sufficient charge flux to maintain the potential. However, there are considerable uncertainties about the magnitude of this membrane potential, the nature of the intramitochondrial ATPase and the ATP flux required to maintain the potential. Here we have investigated these factors in intact and digitonin-permeabilized mammalian rho degrees cells. The adenine nucleotide carrier and ATP were essential, but not sufficient to generate a membrane potential in rho degrees cells and an incomplete FoF1-ATP synthase was also required. The maximum value of this potential was approximately 110 mV in permeabilized cells and approximately 67 mV in intact cells. The membrane potential was eliminated by inhibitors of the adenine nucleotide carrier and by azide, an inhibitor of the incomplete FoF1-ATP synthase, but not by oligomycin. This potential is sufficient to import nuclear-encoded proteins but approximately 65 mV lower than that in 143B cells containing fully functional mitochondria. Subfractionation of rho degrees mitochondria showed that the azide-sensitive ATPase activity was membrane associated. Further analysis by blue native polyacrylamide gel electrophoresis (BN/PAGE) followed by activity staining or immunoblotting, showed that this ATPase activity was an incomplete FoF1-ATPase loosely associated with the membrane. Maintenance of this membrane potential consumed about 13% of the ATP produced by glycolysis. This work has clarified the role of the adenine nucleotide carrier and an incomplete FoF1-ATP synthase in maintaining the mitochondrial membrane potential in rho degrees cells.     

A model of oxidative phosphorylation in mammalian skeletal muscle

A dynamic computer model of oxidative phosphorylation in oxidative mammalian skeletal muscle was developed. The previously published model of oxidative phosphorylation in isolated skeletal muscle mitochondria was extended by incorporation of the creatine kinase system (creatine kinase plus phosphocreatine/creatine pair), cytosolic proton production/consumption system (proton production/consumption by the creatine kinase-catalysed reaction, efflux/influx of protons), physiological size of the adenine nucleotide pool and some additional minor changes. Theoretical studies performed by means of the extended model demonstrated that the CK system, which allows for large changes in P(i) in relation to isolated mitochondria system, has no significant influence on the kinetic properties of oxidative phosphorylation, as inorganic phosphate only slightly modifies the relationship between the respiration rate and [ADP]. Computer simulations also suggested that the second-order dependence of oxidative phosphorylation on [ADP] proposed in the literature refers only to the ATP synthesis flux, but not to the oxygen consumption flux (the difference between these two fluxes being due to the proton leak). Next, time courses of changes in fluxes and metabolite concentrations during transition between different steady-states were simulated. The model suggests, in accordance with previous theoretical predictions, that activation of oxidative phosphorylation by an increase in [ADP] can (roughly) explain the behaviour of the system only at low work intensities, while at higher work intensities parallel activation of different steps of oxidative phosphorylation is involved.     

Intrinsic and extrinsic uncoupling of oxidative phosphorylation

This article reviews parameters of extrinsic uncoupling of oxidative phosphorylation (OxPhos) in mitochondria, based on induction of a proton leak across the inner membrane. The effects of classical uncouplers, fatty acids, uncoupling proteins (UCP1-UCP5) and thyroid hormones on the efficiency of OxPhos are described. Furthermore, the present knowledge on intrinsic uncoupling of cytochrome c oxidase (decrease of H(+)/e(-) stoichiometry=slip) is reviewed. Among the three proton pumps of the respiratory chain of mitochondria and bacteria, only cytochrome c oxidase is known to exhibit a slip of proton pumping. Intrinsic uncoupling was shown after chemical modification, by site-directed mutagenesis of the bacterial enzyme, at high membrane potential DeltaPsi, and in a tissue-specific manner to increase thermogenesis in heart and skeletal muscle by high ATP/ADP ratios, and in non-skeletal muscle tissues by palmitate. In addition, two mechanisms of respiratory control are described. The first occurs through the membrane potential DeltaPsi and maintains high DeltaPsi values (150-200 mV). The second occurs only in mitochondria, is suggested to keep DeltaPsi at low levels (100-150 mV) through the potential dependence of the ATP synthase and the allosteric ATP inhibition of cytochrome c oxidase at high ATP/ADP ratios, and is reversibly switched on by cAMP-dependent phosphorylation. Finally, the regulation of DeltaPsi and the production of reactive oxygen species (ROS) in mitochondria at high DeltaPsi values (150-200 mV) are discussed.     

Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production. respiration rate; parallel activation; oxidative phosphorylation; metabolic control analysis; flux control coefficient; muscle contraction     

Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria

Activation of oxidative phosphorylation by physiological levels of calcium in mitochondria from rat skeletal muscle was analysed using top-down elasticity and regulation analysis. Oxidative phosphorylation was conceptually divided into three subsystems (substrate oxidation, proton leak and phosphorylation) connected by the membrane potential or the protonmotive force. Calcium directly activated the phosphorylation subsystem and (with sub-saturating 2-oxoglutarate) the substrate oxidation subsystem but had no effect on the proton leak kinetics. The response of mitochondria respiring on 2-oxoglutarate at two physiological concentrations of free calcium was quantified using control and regulation analysis. The partial integrated response coefficients showed that direct stimulation of substrate oxidation contributed 86% of the effect of calcium on state 3 oxygen consumption, and direct activation of the phosphorylation reactions caused 37% of the increase in phosphorylation flux. Calcium directly activated phosphorylation more strongly than substrate oxidation (78% compared to 45%) to achieve homeostasis of mitochondrial membrane potential during large increases in flux.     

Theoretical studies on control of oxidative phosphorylation in muscle mitochondria at different energy demands and oxygen concentrations

The mathematical dynamic model of oxidative phosphorylation in muscle mitochondria developed previously was used to calculate the flux control coefficients of particular steps of this process in isolated mitochondria at different amounts of hexokinase and oxygen concentrations. The pattern of control was completely different under different conditions. For normoxic concentration, the main controlling steps in state 4, state 3.5 and state 3 were proton leak, ATP usage (hexokinase) and complex III, respectively. The pattern of control in state 4 was not changed at hypoxic oxygen concentration, while in state 3.5 and state 3 much of the control was shifted from other steps to cytochrome oxidase. The implications of the theoretical results obtained for the regulation of oxidative phosphorylation in intact muscle are discussed.     

Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion

Proton leak exerts stronger control over ATP/ADP in mitochondria from clonal pancreatic beta-cells (INS-1E) than in those from rat skeletal muscle, due to the higher proton conductance of INS-1E mitochondria [Affourtit and Brand (2006) Biochem. J. 393, 151-159]. In the present study, we demonstrate that high proton leak manifests itself at the cellular level too: the leak rate (measured as myxothiazol-sensitive, oligomycin-resistant respiration) was nearly four times higher in INS-1E cells than in myoblasts. This relatively high leak activity was decreased more than 30% upon knock-down of UCP2 (uncoupling protein-2) by RNAi (RNA interference). The high contribution of UCP2 to leak suggests that proton conductance through UCP2 accounts for approx. 20% of INS-1E respiration. UCP2 knock-down enhanced GSIS (glucose-stimulated insulin secretion), consistent with a role for UCP2 in beta-cell physiology. We propose that the high mitochondrial proton leak in beta-cells is a mechanism which amplifies the effect of physiological UCP2 regulators on cytoplasmic ATP/ADP and hence on insulin secretion.     

An extended dynamic model of oxidative phosphorylation.

The presented model based on an earlier one (Korzeniewski, B. and Froncisz, W. (1989) Studia Biophys. 132, 173-187) simulates concentration changes in time of chemical compounds and thermodynamic forces during respiration of cell suspension in a closed chamber. A set of differential equations solved numerically describes the utilization of oxygen up to anaerobiosis and the behaviour of the system after a sudden pulse of oxygen. Flux control coefficients for most important reactions (enzymes) of oxidative phosphorylation were calculated. A good qualitative and (when a direct comparison is possible) quantitative agreement with experimental results can be observed. The following conclusions can be drawn from the simulation: (1) Wilson's steady state model is not in contradiction with sharing of the control over the respiration between some steps and displacement of the ATP/ADP carrier from equilibrium. (2) The overshoot characteristics of the delta microH+ time-course after reoxygenation can be explained without using the lag-phase kinetics of ATP-synthetase. (3) A 'hot region' (sharp changes of many parameters) can be distinguished when the oxygen concentration approaches zero; only cytochrome oxidase is clearly sensitive on oxygen concentration in all its range. (4) Control over oxidative phosphorylation is shared mainly between inputs of the system (ATP utilization and substrate dehydrogenation) and the proton leak.     

Oxidative phosphorylation by in situ synaptosomal mitochondria from whole brain of young and old rats

Synaptosomes, isolated from the whole brain of young (3 months) and old (24 months) rats were used to study the major bioenergetic systems of neuronal mitochondria in situ, within the synaptosome. Approximately 85% of the resting oxygen consumption of synaptosomes from both young and old rats was a result of proton leak (and possibly other ion cycling) across the mitochondrial inner membrane. There were no significant differences between synaptosomes from the young and old rats in the kinetic responses of the substrate oxidation system, the mitochondrial proton leak and the phosphorylation system to changes in the proton electrochemical gradient. Flux control coefficients of 0.71, 0.27 and 0.02 were calculated for substrate oxidation system, phosphorylation system and the proton leak, respectively, at maximal ATP producing capacity in synaptosomes from young animals. The corresponding values calculated for synaptosomes from old animals were 0.53, 0.43 and 0.05. Thus substrate oxidation had greatest control over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain, with proton leak, having little control under maximal ATP producing capacity. The uncoupled rate of oxygen consumption, in the presence of the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), was significantly lower (p = 0.0124) in synaptosomes from old rats (6.08 +/- 0.42, n = 11) when compared with those from the young rats (7.87 +/- 0.48, n = 8). Thus, there is an impaired flux through the substrate oxidation system is synaptosomes from old rats, as compared to synaptosomes from the young animals. These in situ results may have important implications for the interpretation of theories that age-dependent impairment of mitochondrial energy production may result in increased susceptibility to neurodegeneration.     

Uncoupling protein-1 (UCP1) contributes to the basal proton conductance of brown adipose tissue mitochondria

Proton leak pathways uncouple substrate oxidation from ATP synthesis in mitochondria. These pathways are classified as basal (not regulated) or inducible (activated and inhibited). Previously it was found that over half of the basal proton conductance of muscle mitochondria was catalyzed by the adenine nucleotide translocase (ANT), an abundant mitochondrial anion carrier protein. To determine whether ANT is the unique protein catalyst, or one of many proteins that catalyze basal proton conductance, we measured proton leak kinetics in mitochondria isolated from brown adipose tissue (BAT). BAT can express another mitochondrial anion carrier, UCP1, at concentrations similar to ANT. Basal proton conductance was measured under conditions where UCP1 and ANT were catalytically inactive and was found to be lower in mitochondria from UCP1 knockout mice compared to wild-type. Ablation of another abundant inner membrane protein, nicotinamide nucleotide transhydrogenase, had no effect on proton leak kinetics in mitochondria from liver, kidney or muscle, showing that basal proton conductance is not catalyzed by all membrane proteins. We identify UCP1 as a second protein propagating basal proton leak, lending support to the hypothesis that basal leak pathways are perpetrated by members of the mitochondrial anion carrier family but not by other mitochondrial inner membrane proteins.     

Brown adipose tissue mitochondria: recoupling caused by substrate level phosphorylation and extramitochondrial adenosine phosphates.

1. Uncoupled oxidative phosphorylation in isolated guinea pig brown-adipose-tissue mitochondria is reflected by a low phosphorylation state of adenosine phosphates in the mitochondrial matrix and in the extramitochondrial space during oxidation of succinate or glycerol 1-phosphate in the presence of serum albumin and 100 muM ADP. Recoupling of respiration and phosphorylation in the mitochondria is indicatdd by a dramatic increase in the phosphorylation state of adenine nucleotides in both compartments, when substrates inducing substrate level phosphorylation are respired. In this case ATP/ADP ratios in the extramitochondrial compartment are 10-15 times higher than in the mitochondrial matrix. 2. Recoupling mediated by substrate level phosphorylation depends on the presence of extramitochondrial adenosine phosphate and on intact adenine nucleotide translocation. In the presence of substrate level phosphorylation the amount of extramitochondrial ADP required to restore energy coupling can be extremely low (20 muM ADP or 10 nmol ADP/mg mitochondrial protein respectively). If substrate level phosphorylation is prevented by rotenone or in the presence of atractyloside, 20-50 times higher amounts of extramitochondrial adenine nucleotides are necessary to cause coupled oxidative phosphorylation. The recoupling effect of ATP is significantly stronger than that of ADP. 3. GDP (100 muM) causes a rapid increase of the ATP/ADP ratio in both compartments which is independent of substrate level phosphorylation as well as of the extramitochondrial adenosine phosphate concentration and the adenine nucleotide carrier. 4. The amount of extramitochondrial adenosine phosphate in guinea pig brown-adipose-tissue (18 nmol/mg mitochondrial protein or 2.5 mM respectively) would suffice for recoupling of oxidative phosphorylation mediated by substrate level phosphorylation under conditions in vitro; this suggests that substrate level phosphorylation is of essential importance in brown fat in vivo with respect to energy conditions in the tissue during different states of thermogenesis.     

Regulation of mitochondrial pyruvate carboxylation in isolated hepatocytes by acute insulin treatment.

The effect of acute insulin treatment of hepatocytes on pyruvate carboxylation in both isolated mitochondria and cells rendered permeable by filipin was examined. Challenging the cells with insulin alone had no effect on either the basal rate of pyruvate carboxylation or gluconeogenesis, although it did suppress the responses to both glucagon and catecholamines. Insulin treatment was unable to antagonize the enhanced rate of pyruvate carboxylation caused by stimulation of the cells with either angiotensin or vasopressin. Neither insulin nor the gluconeogenic hormones altered the total extractable pyruvate carboxylase activity in the isolated mitochondria, suggesting that the effect of hormones at the level of the isolated intact organelle was mediated via alterations in the intramitochondrial concentrations of effector molecules, notably ATP and the [ATP]/[ADP] ratio and substrate availability. The alterations in pyruvate carboxylation correlate well with glucose synthesis in terms of sensitivity to effector molecules, putative second messengers and time of onset of the response, indicating that alterations in the flux through this enzyme are compatible with it being an important site in the control of gluconeogenesis from C3 precursors.     

Control of energy transformation of mitochondria. Analysis by a quantitative model

A mathematical model of control of energy transformation in mitochondria is presented. The considered processes are: the proton translocation by the respiratory chain, the production of ATP by ATPase, the translocation of adenine nucleotides and of phosphate by their translocators, and a passive backflow of protons through the mitochondrial membrane. The mathematical equations expressing the steady-state kinetics of these processes and the relations between them were derived on the basis of current experimental data. The model predicts fairly well the values of the proton electrochemical gradient, of the ATP/ADP ratios within and outside mitochondria and of the distribution of phosphate between both compartments in different metabolic states of mitochondria. From the general agreement of model computations with experimental data, it is suggested that the electron flux through the respiratory chain is immediately controlled by the energy back-pressure of the proton electrochemical gradient, that the ATPase reaction is near equilibrium in phosphorylating mitochondria but that the adenine nucleotide exchange across the mitochondrial membrane requires some loss of energy. The latter is caused by an inhibition of the translocator by ATP from the outer side or by ADP from the inner side depending on the actual ATP/ADP in both compartments. It explains that no fixed relation exists between the rate of respiration and the phosphorylation state of extramitochondrial adenine nucleotides. The relation is modified by the concentration of phosphate and by intramitochondrial energy utilization.     

Interrelationships between hydrogen-supplying reactions, respiration rate and extramitochondrial adenine nucleotide pattern

1. The influence of a diminished hydrogen supply on the regulation of oxidative phosphorylation of isolated rat liver mitochondria in dependence on the extramitochondrial (ATP)/(ADP) ratio was investigated. 2. The hydrogen supply was diminished by using various (beta-hydroxybutyrate)/(acetoacetate) ratios as a redox buffer and the results were compared with those of experiments using perifusion of immobilized mitochondria with non-saturating substrate concentrations. 3. In both experimental approaches the influence of a diminished hydrogen pressure on the maximum (ATP)/(ADP) ratio at minimum flux was low. An extreme decrease in the (beta-hydroxybutyrate)/(acetoacetate) ratio by more than two orders of magnetitude causes the (APT)/(ADP) ratio to decrease by about 50%. 4. The load capacity of oxidative phosphorylation (maximum flux) is considerably decreased by diminished hydrogen pressure. 5. The borderline cases of purely kinetic and thermodynamic limitations of hydrogen supply were calculated by computer simulation with respect to the regulating behaviour of oxidative phosphorylation and changes in the control strength of adenine nucleotide translocator and hydrogen supply in the overall reaction. 6. A prevalent thermodynamic influence of hydrogen supply on oxidative energy transformation in the cell is discussed in the light of experimental data.