Studies on the mechanism of oxidative phosphorylation. Positive cooperativity in ATP synthesis

Kinetic and nucleotide binding studies have shown that submitochondrial particles from bovine heart possess three exchangeable binding sites for ADP or GDP. In order of decreasing affinity at neutral pH, these sites will be referred to as sites I, II, and III, and their respective dissociation constants as KI, KII, and KIII. In oxidative phosphorylation experiments in the presence of saturating amounts of inorganic phosphate, rapid ATP (or GTP) synthesis occurred only upon ADP (or GDP) binding to site III. The Eadie-Hofstee plots (v/[S] on the ordinate versus v on the abscissa) of the kinetics of ATP (or GTP) synthesis at variable ADP (or GDP) were, therefore, composed of an initial upward phase, indicating positive cooperativity with respect to substrate concentration, followed by a downward phase where rapid product formation took place. These data allowed calculation of KII from the upward phase and KIII (equivalent to apparent Km) from the downward phase. KI was estimated from Scatchard plots of binding data with radiolabeled ADP or GDP. Thus, together with our previous results, these findings have allowed characterization of the process of ATP or GTP synthesis by bovine-heart submitochondrial particles in terms of KI, KII, KIII, and kcat.     

Molecular mechanism of ATP synthesis in oxidative phosphorylation

The experiments described in this paper may perhaps point the way towards a reaction mechanism for oxidative phosphorylation. However, we are not yet in a position to write a detailed chemical equation, supported by experimental evidence, for the mechanism of ATP synthesis. Continued pursuit of some of the implications of these experiments will be very much dependent on information presently unavailable. For example, it would be of great value to have three-dimensional X-ray crystal structures for F0 as well as F1. It will also be important to know the pathway of proton translocation through the ATPase complex. We shall surely require entirely new experimental tools to probe many of these questions.     

On the mechanism of ATP synthesis in oxidative phosphorylation: A review

It is shown that the presently available evidence supports the existence of two entry points for water oxygen in the mitochondrial oxygen exchanges. This in turn provides support for the pseudorotation reaction mechanism of ATP synthesis which is the only mechanism of ATP synthesis proposed to date allowing for two entry points of water oxygen. It is also shown that the pseudorotation mechanism can resolve the apparent paradox of a P₁ ? H₂O exchange·separate from the reversal of phosphorylation yet dependent on the mechanism of P₁ activation. In addition an interpretation consistent with the experimental observations concerning As_i-induced stimulation of respiration and effects associated with oligomycin and aurovertin is shown to follow from the analysis of the oxygen exchanges. Implications of the pseudorotation mechanism for the mechanism of energy coupling in oxidative phosphorylation are discussed.     

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.     

The mode of inhibition of oxidative phosphorylation by efrapeptin (A23871). Evidence for an alternating site mechanism for ATP synthesis.

Results are presented that confirm and extend earlier findings that efrapeptin is a potent inhibitor of oxidative phosphorylation. Binding of efrapeptin is shown to be reversible, and a dissociation constant for the enzyme-inhibitor complex is estimated to be 10(-8) M under conditions for either ATP synthesis or hydrolysis. Fifty per cent inhibition of the ATP hydrolysis activity of submitochondrial particles is obtained at a ratio of 0.56 mol of inhibitor/mol of enzyme. Studies of efrapeptin binding under pseudo-first order conditions show that the onset of inhibition is first order with respect to efrapeptin. Combined with the inhibition titer, these results indicate that there is one inhibitor binding site per molecule of enzyme. Steady state velocity studies using a substrate regenerating system show that efrapeptin is competitive with both ADP and phosphate during ATP synthesis. However, during ATP hydrolysis, a distinctly different mode of inhibition is indicated with respect to ATP. Data are presented which suggest that ATP promotes the binding of efrapeptin to the enzyme. Indications that efrapeptin is a catalytic site inhibitor make these results difficult to reconcile with a simple mechanistic scheme involving a single independnet catalytic site for ATP synthesis and hydrolysis. Our results are discussed in terms of support for catalytic cooperativity between adjacent subunits as recently proposed by Kayalar et al. (Kayalar, C., Rosing, J., and Boyer, P. D. (1977) J. Biol. Chem. 252, 2486-2491).     

Anomalous oxygen-18 exchange during ATP synthesis in oxidative phosphorylation

The synthesis of ATP from highly enriched [18O]Pi by submitochondrial particles driven by succinate oxidation produces distributions of 18O-labeled ATP species that deviate from the distributions predicted by a simple model for the exchange. Control experiments indicate no change in isotopic distribution when [18O]ATP is synthesized from [18O]ADP by adenylate kinase, which is bound to the submitochondrial particles. The observed deviations are in the opposite direction from that produced by heterogeneity due to multiple pathways for ATP synthesis. Two types of complex models can account for the observed deviations. One model has nonequivalence of the Pi oxygens during the exchange reaction, due to incomplete randomization of the Pi oxygens during the reversible cycles of hydrolysis and synthesis of bound ATP. The other model assumes that, during each turnover, a slow transition must occur between a high-exchange and a low-exchange pathway.     

Studies on the mechanism of oxidative phosphorylation. Flow-force relationships in mitochondrial energy-linked reactions

The relationship between the steady-state level of membrane potential (delta psi) and the rates of energy production and consumption has been studied in mitochondria and submitochondrial particles. The energy-linked reactions investigated were oxidative phosphorylation (with NADH, succinate, and beta-hydroxybutyrate as respiratory substrates) and nucleoside triphosphate-driven transhydrogenation from NADH to NADP and uphill electron transfer from succinate to NAD. Results have shown the following. 1) Attenuation of the rates of the energy-producing reactions results in a parallel change in the rates of the energy-consuming reactions with little or no change in the magnitude of steady-state delta psi. 2) At low rates of energy production and consumption, steady-state delta psi decreases. However, this is due largely to the energy leak of the system which lowers static-head delta psi when the rate of energy production is slow. 3) When the rate of energy production and static-head delta psi are held constant, and the rate of energy consumption is diminished by partial inhibition or the use of suboptimal conditions (e.g. subsaturating substrate concentrations), then even a small decrease in the rate of energy consumption results in an upward adjustment of the level of steady-state delta psi. The lower the rate of energy input, the greater the upward adjustment of steady-state delta psi upon suppression of the rate of energy consumption. 4) The above results have been discussed with regard to the role of bulk-phase delta mu H+ or delta psi in the mitochondrial energy transfer reactions.     

Thermodynamic limits to the ATP/site stoichiometries of oxidative phosphorylation by rat liver mitochondria

From measurements of reactants, products, and the oxidation-reduction state of cytochrome c + c1 during 3-hydroxybutyrate-supported oxidative phosphorylation by rat liver mitochondria at static head (state 4), we determined the free energy change of ATP formation from ADP and Pi (phosphorylation potential or delta GP) and the oxidation-reduction free energy changes (redox potentials or delta GR values) across Sites 1 + 2 (delta GR1 + 2), across Site 3 (delta GR3), and across Sites 1 + 2 + 3 (delta GR). At pH 7.4, -delta GR1 + 2/delta GP, -delta GR3/delta GP, and -delta GR/delta GP were maximally 1.80, 1.56, and 3.37. These can be taken as thermodynamic upper limits to the ATP/Sites 1 + 2, ATP/Site 3, and ATP/O stoichiometry of 3-hydroxybutyrate-supported oxidative phosphorylation. The theory of linear nonequilibrium thermodynamics were employed to estimate lower limits to the ATP/site stoichiometries. The lower limit is given by the expression, q2(-delta GRsite/delta GP). The degree of coupling, q, was 0.977 as determined from the dependence of respiratory rate on delta GP. Determined in this way, lower limits of the ATP/Sites 1 + 2, ATP/Site 3, and ATP/O stoichiometries were 1.67, 1.44, and 3.11, respectively. ADP addition to mitochondria incubated at static head lowered delta GP by 1.1 kcal/mol and stimulated respiration by a factor of about 2.5 but caused negligible changes in delta GR1 + 2 and delta GR3. This observation demonstrates that the respiratory reactions from substrate to cytochrome c and from cytochrome c to oxygen both move away from thermodynamic equilibrium with delta GP during the transition from resting to active oxidative phosphorylation. The findings are discussed in terms of current schemes of chemiosmotic coupling.     

Involvement of phosphate-modified ATP analogs in the reactions of oxidative phosphorylation.

Various analogs of adenosine 5'-triphosphate with a modified terminal phosphate group have been tested in energy-requiring reactions with intact mitochondria and submitochondrial particles. It is shown that the fluorophosphate analog ATP(gamma F) is a strong inhibitor of mitochondrial respiration and of energy requiring reactions which involve the participation of high energy intermediates, generated aerobically by the respiratory chain. On the other hand, ATP(gamma F) does not affect the ATPase activity of intact or disrupted mitochondria and is less effective in inhibiting ATP-driven reactions. The imidophosphate analog AMP-P(NH)P also inhibits the partial reactions of oxidative phosphorylation, but does not affect ATP synthesis from ADP and Pi. In contrast to ATP(gamma F), it is strong inhibitor of both soluble and membrane-bound mitochondrial ATPases. The biological implication of the complementary effects of ATP(gamma F) and AMP-P(NH)P on mitochondria-catalysed reactions is discussed while suggesting the use of such nucleotide analogs as specific tools for the study of ATP-forming and ATP-utilizing reactions in mitochondria.     

Kinetics of oxidative phosphorylation in Paracoccus denitrificans. 1. Mechanism of ATP synthesis at the active site(s) of F0F1-ATPase

(1) The rate of ATP synthesis during NADH-driven aerobic respiration has been measured in plasma membrane vesicles from Paracoccus denitrificans as a function of the concentration of the substrates, ADP and inorganic phosphate (Pi). In both cases, the response of the reaction to changes in the degree of saturation of the F0F1-ATPase generated a perfect Micaelian dependence which allowed the determination of the corresponding Michaelis constants, KmADP and KmPi. (2) These kinetic parameters possess a real mechanistic significance, as concluded from the partial reduction of the rate of phosphorylation by the energy-transfer inhibitor venturicidin and the consequent analysis of the results within the framework of the theory of metabolic control. (3) The same membrane vesicles, which catalyze very high rates of ATP synthesis, have been shown to support much lower rates of the exchange ATP in equilibrium Pi and negligible rates of ATP hydrolysis. Under similar conditions, the preparations are also capable of generating phosphorylation potentials, delta Gp, of 60-61 kJ.mol-1. (4) These properties have allowed analysis of the synthetic reaction in the presence of significant concentrations of the product, ATP, using integrated forms of the Michaelis-Menten rate equations. (5) It has been shown that ATP produces pure competitive product inhibition of the forward reaction with a value of KiATP = 16 +/- 1 microM, thus indicating that the affinity of the nucleotide for the active site(s) of the F0F1-ATPase, during net ATP synthesis, is significantly higher than previously thought. (6) The order of binding of the substrates, ADP and Pi, to the active site(s) has been determined as random. (7) At very low concentrations of ADP, a second and much smaller Michaelis constant for this substrate has been identified, with an estimated value of KmADP approximately equal to 50 nM, associated with a maximal rate of only 2% of that measured at a higher range of concentrations. (8) The results obtained are discussed in relation to the presence of two or three equivalent catalytic sites operating in the cooperative manner explicitly described by the binding change mechanism.     

氧化磷酸化作用中ATP的产生数目释疑

就高中新课标教材《生物1:分子与细胞》教学中普遍质疑的"对于细胞呼吸的能量转移过程,应如何计算ATP的产量?"做一简要概述。这一问题的研究在生物化学史上是个重要内容。随着研究的深入,可以获得的解释将越来越合理。