Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Evidence for linkage of the energy-yielding and energy-consuming steps by freely diffusible intermediates and for an allosteric mechanism of respiratory control at coupling site 2

The three coupling segments of the respiratory chain of bovine heart mito-chondria were examined individually by steady-state kinetic methods to determine whether or not freely diffusible intermediates occur between the energy-yielding and energy-consuming steps involved in the oxidative phosphorylation of extramitochondrial ADP. The principal method employed was the dual inhibitor technique, for which an appropriate model is provided. The results indicate that in accordance with the chemiosmotic theory the intermediate reactants that link the energy-yielding rotenone-sensitive (Site 1), cytochromebc ₁ (Site 2), and cytochromeaa ₃ (Site 3) reactions of the respiratory chain to the energy-consuming ATP synthetase, AdN transport, and P_i transport reactions are freely diffusible (delocalized). Site 2 was found to differ from the others in regard to the mechanism by which the energy-linked respiratory chain reaction is controlled by the energy-consuming steps. Whereas the Site 1 and Site 3 respiratory chain reactions are controlled primarily by the thermodynamic mechanism of reaction reversal, the Site 2 respiratory reaction is controlled primarily by a kinetic mechanism in which an intermediate that links it to the energy-consuming steps inhibits it allosterically. From the effects of nigericin and valinomycin the allosteric intermediate appears to be the electrical component of the protonmotive force.     

Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria

The steady-state velocity dependence of the overall mitochondrial oxidative phosphorylation reaction on the concentrations of extramitochondrial ADP and P₁ and of several of the catalytic components was investigated, using the O₂ uptake step as the indicator reaction and conditions of saturation with O₂, malate, and pyruvate. The studies were carried out with tightly coupled bovine heart mitochondria incubated in the presence of hexokinase, glucose, and Mg²⁺. The data were corrected to conditions of hexokinase saturation with factors determined in hexokinase dependence studies. The concentrations of catalytic components were varied, in effect, by application of highly specific, tight-binding inactivators of the components. The principal objectives were (a) to distinguish individual reactions coupled by freely diffusible intermediate reactants, (b) to determine the relationships (coupling relationships) between these reactions in regard to how a change in the degrees to which one limits the rate of the overall reaction affects the degree to which the others limit the rate, and (c) to use the findings to determine how the individual reactions are coupled. The feasibility of achieving these objectives was suggested by the observations (a) that the initial steady-state velocity of the overall reaction varies in fairly close accord with a rectangular hyperbola (i.e., with Michaelis-Menten kinetics) whether it is a catalytic component or a substrate that is varied, (b) that apparent Michaelis constants of the substrates and catalytic components may be used as indicators of the coupling relationships between the individual reactions, and (c) that two types of coupling relationships exist between the individual reactions: sequential (characteristic of reactions linked in simple sequence) and nonsequential (mechanism uncertain), in which a change in the degree to which one individual reaction of a pair is rate limiting results in an inverse change and in no change, respectively, in the degree to which the other is rate limiting. Six individual reactions were distinguished: the energy-yielding rotenone-, antimycin-, and cyanide-sensitive steps of the respiratory chain and the energy-consuming P_i transport, phosphorylation, and AdN (adenine nucleotide) transport reactions. The results indicate (a) that the coupling relationship is sequential between the P_i transport and rotenone-sensitive reactions, the P_i transport and cyanide-sensitive reactions, the AdN transport and rotenone-sensitive reactions, the AdN transport and cyanide-sensitive reactions, and the AdN transport and phosphorylation reactions, and (b) that the coupling relationship is nonsequential between the AdN and P_i transport reactions, the P_i transport and phosphorylation reactions, the P_i transport and antimycin-sensitive reactions, and the AdN transport and antimycin-sensitive reactions. In the sequential group of individual reaction pairs, the individual reactions of all but the AdN transport-phosphorylation reaction pair appear to be linked in a partially nonsequential manner. It is proposed that the nonsequential and partially nonsequential coupling relationships come about as a result of one individual reaction of a pair removing freely diffusible intermediate reactants at two or more points which are situated symmetrically and unsymmetrically, respectively, about the other.     

Rate-controlling steps of oxidative phosphorylation in rat liver mitochondria. A synoptic approach of model and experiment

The contribution of different steps to the control of oxidative phosphorylation in isolated rat liver mitochondria was investigated by a combination of experiments and computer simulations. The parameters of the mathematical model of phosphorylating mitochondria were derived from experimental data. The model correctly describes the competition between ATP utilization inside and outside mitochondria for the ATP generated in mitochondria. On the basis of the good agreement between experiments and simulations, the contribution of different steps to the control of respiration was estimated by computing their control strengths, i.e., the influence of their activities on the rate of respiration. The rate-controlling influences vary depending on the load of oxidative phosphorylation. The predominant steps are: in the fully active state (State 3) — the hydrogen supply to the respiratory chain; in the resting state (State 4) — the proton leak of the mitochondrial inner membrane; in states of non-maximum ATP export — the adenine nucleotide translocator. Titrations of respiration with phenylsuccinate, antimycin, oligomycin and carboxyatractyloside completely support these conclusions.     

The role of the adenine nucleotide translocator in oxidative phosphorylation. A theoretical investigation on the basis of a comprehensive rate law of the translocator

A minimum model of adenine nucleotide exchange through the inner membrane of mitochondria is presented. The model is based on a sequential mechanism, which presumes ternary complexes formed by binding of metabolites from both sides of the membrane. The model explains the asymmetric kinetics of ADP-ATP exchange as a consequence of its electrogenic character. In energized mitochondria, a part of the membrane potential suppresses the binding of extramitochondrial ATP in competition with ADP. The remaining part of the potential difference inhibits the back exchange of internal ADP for external ATP. The assumption of particular energy-dependent conformational states of the translocator is not necessary. The model is not only compatible with the kinetic properties reported in the literature about the adenine nucleotide exchange, but it also correctly describes the response of mitochondrial respiration to the extramitochondrial ATP/ADP ratio under different conditions. The model computations reveal that the translocation step requires some loss of free energy as driving force. The size of the driving force depends on the flux rate as well as on the extra- and intramitochondrial ATP/ADP quotients. By both quotients the translocator controls the export of ATP formed by oxidative phosphorylation in mitochondria.     

Rapid isolation of muscle and heart mitochondria, the lability of oxidative phosphorylation and attempts to stabilize the process in vitro by taurine, carnitine and other compounds

We modified the isolation procedure of muscle and heart mitochondria. In human muscle, this resulted in a 3.4 fold higher yield of better coupled mitochondria in half the isolation time. In a preparation from rat muscle we studied factors that affected the stability of oxidative phosphorylation (oxphos) and found that it decreased by shaking the preparation on a Vortex machine, by exposure to light and by an increase in storage temperature. The decay was found to be different for each substrate tested. The oxidation of ascorbate was most stable and less sensitive to the treatments.When mitochondria were stored in the dark and the cold, the decrease in oxidative phosphorylation followed first order kinetics. In individual preparations of muscle and heart mitochondria, protection of oxidative phosphorylation was found by adding candidate stabilizers, such as desferrioxamine, lazaroids, taurine, carnitine, phosphocreatine, N-acetylcysteine, Trolox-C and ruthenium red, implying a role for reactive oxygen species and calcium-ions in the in vitro damage at low temperature to oxidative phosphorylation.In heart mitochondria oxphos with pyruvate and palmitoylcarnitine was most labile followed by glutamate, succinate and ascorbate.We studied the effect of taurine, hypotaurine, carnitine, and desferrioxamine on the decay of oxphos with these substrates. 1 mM taurine (n = 6) caused a significant protection of oxphos with pyruvate, glutamate and palmitoylcarnitine, but not with the other substrates. 5 mM L-carnitine (n = 6), 1 mM hypotaurine (n = 3) and 0.1 mM desferrioxamine (n = 3) did not protect oxphos with any of the substrates at a significant level.These experiments were undertaken in the hope that the in vitro stabilizers can be used in future treatment of patients with defects in oxidative phosphorylation. (Mol Cell Biochem 174: 61–66, 1997)     

Spatio-temporal regulation of glycolysis and oxidative phosphorylation in vivo in tumor and yeast cells.

Recent advances in the in vivo control and regulation of glycolysis and oxidative phosphorylation in yeast and tumor cells is revised. New insigths are presented from old and new experimental data interpreted in the light of powerful new technologies (e.g. NMR confocal microscopy) and quantitative techniques combined with mathematical modeling. Those new aspects are mainly concerned with the dynamical organization of glycolysis and oxidative phosphorylation which emerges from the multiple interactions between compartments and processes inside the cells. Those compartments may be of structural origin, e.g. plasma membrane defining the cell boundary, mitochondrial-cytoplasmic, or functional ones such as the alternative association-dissociation of enzymes to subcellular structures (e.g. mitochondria, cytoskeleton) with different kinetic properties in each state. A novel regulatory mechanism concerning polymerization-depolymerization of microtubular protein may add a new dimension to the in vivo physiological properties of cells. One main suggestion coming from the modulatory power of the polymeric status and concentration of cytoskeleton components is that it could function as an intracellular mechanism of synchronization between microscopic (local) to macroscopic (global) processes. How the cell "mixes" or switches on or off those regulatory steps or effectors under different physiological and environmental conditions and for different genetic backgrounds, is a main avenue of systematic research for the future.