Mechanism of ATP synthesis by mitochondrial ATP synthase from beef heart

Previous studies of the rate constants for the elementary steps of ATP hydrolysis by the soluble and membrane-bound forms of beef heart mitochondrial F₁ supported the proposal that ATP is formed in high-affinity catalytic sites of the enzyme with little or no change in free energy and that the major requirement for energy in oxidative phosphorylation is for the release of product ATP.The affinity of the membrane-bound enzyme for ATP during NADH oxidation was calculated from the ratio of the rate constants for the forward binding step (k ₊₁) and the reverse dissociation step (k _–1).k _–1 was accelerated several orders of magnitude by NADH oxidation. In the presence of NADH and ADP an additional enhancement ofk _–1 was observed. These energy-dependent dissociations of ATP were sensitive to the uncoupler FCCP.k ₊₁ was affected little by NADH oxidation. The dissociation constant (K _d ATP) increased many orders of magnitude during the transition from nonenergized to energized states.     

Coupling Proton Movement to ATP Synthesis in the Chloroplast ATP Synthase

The chloroplast F₀F₁-ATP synthase-ATPase is a tiny rotary motor responsible for coupling ATP synthesis and hydrolysis to the light-driven electrochemical proton gradient. Reversible oxidation/reduction of a dithiol, located within a special regulatory domain of the γ subunit of the chloroplast F₁ enzyme, switches the enzyme between an inactive and an active state. This regulatory mechanism is unique to the ATP synthases of higher plants and its physiological significance lies in preventing nonproductive depletion of essential ATP pools in the dark. The three-dimensional structure of the chloroplast F₁ gamma subunit has not yet been solved. To examine the mechanism of dithiol regulation, a model of the chloroplast gamma subunit was obtained through segmental homology modeling based on the known structures of the mitochondrial and bacterial γ subunits, together with de novo construction of the unknown regulatory domain. The model has provided considerable insight into how the dithiol might modulate catalytic function. This has, in turn, suggested a mechanism by which rotation of subunits in F₀, the transmembrane proton channel portion of the enzyme, can be coupled, via the ε subunit, to rotation of the γ subunit of F₁ to achieve the 120° (or 90°+30°) stepping action that is characteristic of F₁ γ subunit rotation.     

ADP and ATP binding to noncatalytic sites of thiol-modulated chloroplast ATP synthase

A modified ‘cold chase’ technique was used to study tight [¹⁴C]ADP and [¹⁴C]ATP binding to noncatalytic sites of chloroplast ATP synthase (CF₀F₁). The binding was very low in the dark and sharply increased with light intensity. Dissociation of labeled nucleotides incorporated into noncatalytic sites of CF₀F₁ or CF₁ reconstituted with EDTA-treated thylakoid membranes was also found to be light-dependent. Time dependence of nucleotide dissociation is described by the first order equation with a k _d of about 5 min⁻¹. The exposure of thylakoid membranes to 0.7–24.8 μM nucleotides leads to filling of up to two noncatalytic sites of CF₀F₁. The sites differ in their specificity: one preferentially binds ADP, whereas the other – ATP. A much higher ATP/ADP ratio of nucleotides bound at noncatalytic sites of isolated CF₁ dramatically decreases upon its reconstitution with EDTA-treated thylakoid membranes. It is suggested that the decrease is caused by conformational changes in one of the α subunits induced by its interaction with the δ subunit and/or subunit I–II when CF₁ becomes bound to a thylakoid membrane.     

Mussel and mammalian ATP synthase share the same bioenergetic cost of ATP

The molecular mechanism by which the membrane-embedded F_O sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to F_O c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H⁺ by coordinating the hydronium ion (H₃O⁺) in the ion binding site of F_O. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel F_O probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.     

STIMULATION OF BRAIN ACETYL-CoA HYDROLASE BY ATP AND ATP ANALOGUES

The effects of ATP and ATP analogues on the brain acetyl-CoA hydrolase (EC 3.1.2.1) were studied. The enzyme was stimulated reversibly by ATP-Mg²⁺ the presence of Mg²⁺ being absolutely required for the stimulation. The stimulatory effect of ATP was highly specific since adenine nucleotides other than ATP had no stimulatory effects and nucleoside triphosphates other than ATP stimulated the enzyme much less than ATP in the following order: ATP > ITP CTP, UTP GTP. A phosphate modified analogue of ATP, AMP-PNP had a similar stimulatory effect to that of ATP. Other ATP analogues such as AMP-PCP and AMPCPP showed less stimulatory effect than ATP. The order of the stimulatory effects of these ATP analogues was: ATP > AMP-PNP > AMP-PCP > AMPCPP. The concentrations needed for half-maximal stimulation of ATP, AMP-PNP and AMP-PCP were approx 0.11 mm , 0.22 mm, and 0.22 mm , respectively. Double reciprocal plots demonstrated that ATP as well as AMP-PNP produced a significant decrease in the apparent K_m, value for acetyl-CoA and an increase in V_max indicating that these nucleotides increased the affinity for acetyl-CoA through binding at a site other than the catalytic site. The data described above suggest that the rate of hydrolysis of acetyl-CoA may be regulated by the concentration of ATP in the micro-environment of the enzyme.     

Actin polymerization and ATP hydrolysis

This paper surveys several aspects of the consequences of ATP hydrolysis associated with actin polymerization, and their physiological implications. ATP hydrolysis occurs on F-actin in two subsequent reactions, cleavage of ATP followed by the slower release of P_i. The latter reaction is linked to a conformation change of the actin subunit that causes a destabilization of the actin-actin interactions in the filament, i.e., a structural change of the filament. The nature of the nucleotide bound to terminal subunits therefore affects the dynamics of actin filaments. It is shown that this regulation is different at the two ends, terminal F-ADP-P_i subunits being present at steady state at the barbed end, while F-ADP-subunits are present at the pointed end. While cleavage of ATP on F-actin is irreversible, P_i release is reversible, which allows the regulation of filament dynamics by cellular P_i. The nature of the divalent metal ion — Ca²⁺ or Mg²⁺ — tightly bound to actin, in direct interaction with ATP, also affects the conformation of actin and the rate of ATP hydrolysis, therefore regulating actin dynamics. Finally, the rate of nucleotide exchange on G-actin is relatively slow, which allows the critical concentration to increase with the number of filaments in ATP, a property largely used by the cell via the action of severing proteins.     

Neuronal cell surface ATP synthase mediates synthesis of extracellular ATP and regulation of intracellular pH

The ATP synthase is known to play important roles in ATP generation and proton translocation within mitochondria. Here, we now provide evidence showing the presence of functional ecto‐ATP synthase on the neuronal surface. Immunoblotting revealed that the α, β subunits of ATP synthase F₁ portion are present in isolated fractions of plasma membrane and biotin‐labelled surface protein from primary cultured neurons; the surface distribution of α, β subunits was also confirmed by immunofluorescence staining. Moreover, α and β subunits were also found in fractions of plasma membrane and lipid rafts isolated from rat brain, and flow cytometry analysis showed α subunits on the surface of acutely isolated brain cells. Activity assays showed that the extracellular ATP generation of cultured neurons could be compromised by α, β subunit antibodies and ATP synthase inhibitors. pH_i (intracellular pH) analysis demonstrated that at low extracellular pH, α or β subunit antibodies decreased pH_i of primary cultured neurons. Therefore, ATP synthase on the surface of neurons may be involved in the machineries of extracellular ATP generation and pH_i homoeostasis.     

Mitochondria as ATP consumers in cellular pathology

ATP provided by oxidative phosphorylation supports highly complex and energetically expensive cellular processes. Yet, in several pathological settings, mitochondria could revert to ATP consumption, aggravating an existing cellular pathology. Here we review (i) the pathological conditions leading to ATP hydrolysis by the reverse operation of the mitochondrial F_oF₁-ATPase, (ii) molecular and thermodynamic factors influencing the directionality of the F_oF₁-ATPase, (iii) the role of the adenine nucleotide translocase as the intermediary adenine nucleotide flux pathway between the cytosol and the mitochondrial matrix when mitochondria become ATP consumers, (iv) the role of the permeability transition pore in bypassing the ANT, thereby allowing the flux of ATP directly to the hydrolyzing F_oF₁-ATPase, (v) the impact of the permeability transition pore on glycolytic ATP production, and (vi) endogenous and exogenous interventions for limiting ATP hydrolysis by the mitochondrial F_oF₁-ATPase.     

Structural Changes during ATP Hydrolysis Activity of the ATP Synthase from Escherichia coli as Revealed by Fluorescent Probes

F₁F₀-ATPase complexes undergo several changes in their tertiary and quaternary structureduring their functioning. As a possible way to detect some of these different conformationsduring their activity, an environment-sensitive fluorescence probe was bound to cysteineresidues, introduced by site-directed mutagenesis, in the subunit of the Escherichia colienzyme. Fluorescence changes and ATP hydrolysis rates were compared under variousconditions in F₁ and in reconstituted F₁F₀. The results are discussed in terms of possible modes ofoperation of the ATP synthases.     

Effect of arylazido aminopropionyl ATP (ANAPP3), a putative ATP antagonist,on ATP responses of isolated guinea pig smooth muscle

Arylazido aminopropionyl ATP (ANAPP₃), a photoaffinity analogue of adenosine 5′-triphosphate, photoactivated with visible light (+hv), specifically and irreversibly antagonized ATP contractions of the guinea pig vas deferens. ANAPP₃ (30 μM) antagonized responses to exogenously added ATP in untreated, and in tissues pretreated with indomethacin (2.9 μM) and 6-(2-hydroxy-5-nitrobenzyl)-thio guanosine (10 μM). It was of interest to see if this pharmacological antagonist of ATP could be used to assess the validity of the purinergic nerve hypothesis by allowing a differentiation between, or proof of the identity of, responses to ATP and the non-adrenergic inhibitory transmitter in guinea pig stomach fundus. After photoactivation (+hv) in the organ bath and subsequent washout, ANAPP₃ (30 and 100 μM) failed to antagonize relaxant responses to ATP (1.0 – 1000 μM) in fundic strips. In addition ANAPP₃ failed to antagonize ATP-induced inhibition of the twitch response in electrically stimulated guinea pig ileum longitudinal muscle strips. We conclude that ANAPP₃ does not antagonize all actions of ATP, which may limit its usefulness in assessing the above hypothesis. Results with this compound suggest that ATP excitatory receptors may differ from those mediating relaxation and other ATP actions.     

Enhanced-acceptor fluorescence-based single cell ATP biosensor monitors ATP in heterogeneous cancer populations in real time

Current methods to monitor cellular ATP do not provide spatial or temporal localization of ATP in single cells in real time or they display imperfect specificity to ATP. Here, we have developed a single cell, Enhanced Acceptor Fluorescence (EAF)-based ATP biosensor to visualize ATP in real time. This biosensor utilizes a modified mimic of the ε-subunits of the Bacillus subtilis F₀F₁ synthase and is coupled to the EAF fluorophores pairs, GFP and YFP. The sensor was then used to monitor ATP in a heterogeneous glioblastoma multiform cancer cell population. We anticipate that this innovative technology and our better understanding of the ATP machinery will have substantial influence on future translational studies.     

Modeling the ATP Production in Mitochondria

We revisit here the mathematical model for ATP production in mitochondria introduced recently by Bertram, Pedersen, Luciani, and Sherman (BPLS) as a simplification of the more complete but intricate Magnus and Keizer’s model. We identify some inaccuracies in the BPLS original approximations for two flux rates, namely the adenine nucleotide translocator rate J _ANT and the calcium uniporter rate J _uni. We introduce new approximations for such flux rates and then analyze some of the dynamical properties of the model. We infer, from exhaustive numerical explorations, that the enhanced BPLS equations have a unique attractor fixed point for physiologically acceptable ranges of mitochondrial variables and respiration inputs, as one would indeed expect from homeostasis. We determine, in the stationary regime, the dependence of the mitochondrial variables on the respiration inputs, namely the cytosolic concentration of calcium Ca_c and the substrate fructose 1,6-bisphosphate FBP. The same dynamical effects of calcium and FBP saturations reported for the original BPLS model are observed here. We find out, however, a novel nonstationary effect, which could be, in principle, physiologically interesting: some response times of the model tend to increase considerably for high concentrations of calcium and/or FBP. In particular, the larger the concentrations of Ca_c and/or FBP, the larger the necessary time to attain homeostasis.     

Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase

Living organisms rely on the F_oF₁ ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the F_o motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F₁ ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single F_oF₁ molecules embedded in lipid bilayer nanodiscs, we now report the observation of F_o-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with F_o stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation.     

ATP Photosynthetic vesicles for light-driven bioprocesses

We prepared ATP photosynthetic vesicles from inside-out membranes of Escherichia coli cells that express delta-rhodopsin (a novel light-driven H⁺ transporter) and TF₀F₁-ATP synthase (a thermo-stable ATP synthase). These vesicles showed light-dependent ATP synthesis. Furthermore, coupling the ATP photosynthetic vesicles with an ATP-hydrolyzing hexokinase enabled light-dependent glucose consumption. The ATP photosynthetic vesicles indicate their potential to applied to light-driven ATP-regenerating bioprocess for various ATP-hydrolyzing bioproductions.     

Influence of the redox state and the activation of the chloroplast ATP synthase on proton-transport-coupled ATP synthesis/hydrolysis

The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, E^ox_i. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, E^red_a. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme E^red_i is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, v^ox_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, v^red_max = 400 ATP per CF₀F₁ per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis v^red_max = −90 ATP per CF₀F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase.     

Endotoxemia impairs heart mitochondrial function by decreasing electron transfer, ATP synthesis and ATP content without affecting membrane potential

Acute endotoxemia (LPS, 10 mg/kg ip, Sprague Dawley rats, 45 days old, 180 g) decreased the O₂ consumption of rat heart (1 mm³ tissue cubes) by 33% (from 4.69 to 3.11 μmol O₂/min. g tissue). Mitochondrial O₂ consumption and complex I activity were also decreased by 27% and 29%, respectively. Impaired respiration was associated to decreased ATP synthesis (from 417 to 168 nmol/min. mg protein) and ATP content (from 5.40 to 4.18 nmol ATP/mg protein), without affecting mitochondrial membrane potential. This scenario is accompanied by an increased production of O₂^●− and H₂O₂ due to complex I inhibition. The increased NO production, as shown by 38% increased mtNOS biochemical activity and 31% increased mtNOS functional activity, is expected to fuel an increased ONOO⁻ generation that is considered relevant in terms of the biochemical mechanism. Heart mitochondrial bioenergetic dysfunction with decreased O₂ uptake, ATP production and contents may indicate that preservation of mitochondrial function will prevent heart failure in endotoxemia.     

Cell-free expression and assembly of ATP synthase

Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F₁F_o-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry α₃β₃γδ?ab₂c₁₃. Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems.     

Voltage- and [ATP]-dependent Gating of the P2X2 ATP Receptor Channel

P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X₂ channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this “activation” phase quantitatively. We characterized the conductance–voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel''s transition between a closed and an open state, we showed that the gating of P2X₂ is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X₂ even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate “gating.” We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X₂-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].