The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation

Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.     

Studies on the mechanism of oxidative phosphorylation. ADP promotion of GDP phosphorylation

The process of ATP or GTP synthesis by bovine heart submitochondrial particles involves the binding of ADP or GDP to 3 exchangeable sites I, II, and III, and only upon substrate occupation of site III does rapid ATP or GTP synthesis take place. The dissociation constants determined for ADP were KADPI less than or equal to 10(-8) M, KADPII approximately 10(-7) M, and KADPIII (equivalent to apparent KADPm), approximately 3 x 10(-6) M in the low Km mode and KADPIII approximately 150 x 10(-6) M in the high Km mode. For GDP, these constants were KGDPI approximately 10(-6)-10(-5) M, KGDPII approximately 10(-4) M, and KGDPIII approximately 10(-3) M when NADH was the respiratory substrate (Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Biol. Chem. 265, 82-88). Because of its low affinity for the above binding sites, GDP at micromolar concentrations does not lead to GTP synthesis. However, as shown in this paper, micromolar [GDP] undergoes phosphorylation in the presence of micromolar concentrations of ADP. Under these conditions, both ATP and GTP are synthesized. GDP inhibits ATP synthesis with KGDPi congruent to 7 microM, while ADP promotes GTP synthesis in a reaction that requires inorganic phosphate (apparent KPim = 2-3 mM) and is inhibited by uncouplers and inhibitors of the ATP synthase complex. The ADP-promoted GTP synthesis exhibited an "apparent" KGDPm = 4 microM and an "apparent" Vmax = 11 nmol of GTP (min.mg of protein)-1. These results were interpreted to mean that (a) micromolar [ADP] occupies sites I and II, allowing site III to bind and phosphorylate GDP, and (b) the KGDPm and Vmax calculated under these conditions represent values for the low Km-low Vmax mode of GTP synthesis, which in the absence of ADP is not detectable because of the positive cooperativity phase of GTP synthesis with the high KGDPII approximately 10(-4) M.     

Non-equilibrium thermodynamic sensitivity of oxidative phosphorylation.

A new method was developed to analyse the dynamic properties of oxidative phosphorylation, in particular the sensitivity of the phosphate potential with respect to fluctuating cellular ATP utilization. This treatment is based on the eigenvalue sensitivity analysis of an experimentally supported non-equilibrium thermodynamic model of oxidative phosphorylation. Such an analysis allows direct access to the kinetic information, while circumventing the awkward conventional numerical integration of a set of nonlinear differential equations. This procedure revealed, for the parameters characteristic for liver of starved rats in vivo, that the sensitivity of oxidative phosphorylation to a fluctuating ATP utilization is minimal at a degree of coupling q = 0.95. This means that the phosphate potential is highly buffered with respect to fluctuating energy demands at the degree of coupling. This value of q agrees well with the degree of coupling qeef, at which net ATP production of oxidative phosphorylation--at optimal efficiency--occurs in the most economic way. This simultaneous maximization of kinetic stability and economic thermodynamic efficiency at the same degree of coupling appears to be a coincidence.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

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.     

Theoretical studies on the control of the oxidative phosphorylation system.

The dynamic model developed in our previous publications [1,2] was used to calculate the flux control coefficients of oxidation, phosphorylation and proton leak fluxes for isolated mitochondria and for three modes of work of intact cells (hepatocytes). The results obtained were compared with experimental data, especially those measured in the frame of the 'top-down approach' of the metabolic control theory. A good agreement for mitochondria and for intact cells was found. The control of the oxygen consumption flux is shared between the ATP utilization (main controlling factor), substrate dehydrogenation, proton leak and, in some conditions, the ATP/ADP carrier. The phosphorylation subsystem seemed to be controlled mainly by itself, while the proton leak was influenced by all three subsystems. It was also shown that the large relative change in the enzyme activity during inhibitor titration of mitochondria or cells could lead to the overestimation of some flux control coefficient values in experimental measurements. An influence of some hormones (glucagon, vasopressin, adrenaline and others) on the mitochondrial respiration was also simulated. Our results suggest that these hormones stimulate the substrate dehydrogenation as well as the phosphorylation system (ATP usage and, possibly, the ATP/ADP carrier).     

Several nongenotoxic carcinogens uncouple mitochondrial oxidative phosphorylation.

A number of plasticizers and lipid-lowering drugs induce peroxisomes and cause hepatocellular carcinoma in rodents by mechanisms which remain unknown. In this study, seven structurally dissimilar peroxisome proliferating agents were shown to uncouple oxidative phosphorylation in isolated rat liver mitochondria. For example, perfluorooctanoate (0.5 mM) increased succinate-induced (state 4) mitochondrial respiration by over 50% while stimulation of state 3 respiration with ADP was minimal (i.e., uncoupling occurred). Interestingly, compounds which are potent carcinogens in vivo (e.g., Wy-14,643 and perfluorooctanoate) were more powerful uncouplers of oxidative phosphorylation in vitro than weak tumor-causing agents (e.g., valproate). Uncoupling also occurred in vivo. Basal rates of oxygen uptake in perfused livers from chronically treated rats were increased from 137 +/- 7 mumol g-1/h in pair-fed controls to 153 +/- 5 mumol g-1/h after 2.5 months of feeding Wy-14,643 (0.1% w/v in diet). Concomitantly, rates of urea synthesis from ammonia, a process highly dependent on ATP supply, were reduced almost completely from 104 +/- 10 mumol g-1/h to 13 +/- 6 mumol g-1/h. Bile flow, another energy-dependent process, was also reduced significantly by treatment with Wy-14,643 in vivo for 24 h. Taken together, these data indicate that energy supply for cellular processes such as urea synthesis and bile flow was disrupted in vivo due to uncoupling of oxidative phosphorylation by Wy-14,643. It is proposed that peroxisomal proliferators accumulate in the liver where they uncouple mitochondrial oxidative phosphorylation and interfere with cellular energetics.