The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

The bioenergetics of IF₁ transiently silenced cancer cells has been extensively investigated, but the role of IF₁ (the natural inhibitor protein of F₁F₀-ATPase) in cancer cell metabolism is still uncertain. To shed light on this issue, we established a method to prepare stably IF₁-silenced human osteosarcoma clones and explored the bioenergetics of IF₁ null cancer cells. We showed that IF₁-silenced cells proliferate normally, consume glucose, and release lactate as controls do, and contain a normal steady-state ATP level. However, IF₁-silenced cells displayed an enhanced steady-state mitochondrial membrane potential and consistently showed a reduced ADP-stimulated respiration rate. In the parental cells (i.e. control cells containing IF₁) the inhibitor protein was found to be associated with the dimeric form of the ATP synthase complex, therefore we propose that the interaction of IF₁ with the complex either directly, by increasing the catalytic activity of the enzyme, or indirectly, by improving the structure of mitochondrial cristae, can increase the oxidative phosphorylation rate in osteosarcoma cells grown under normoxic conditions.     

Modulation of nucleotide specificity of thermophilic F(o)F(1)-ATP Synthase by epsilon-subunit

The C-terminal two α-helices of the ε-subunit of thermophilic Bacillus F(o)F(1)-ATP synthase (TF(o)F(1)) adopt two conformations: an extended long arm ("up-state") and a retracted hairpin ("down-state"). As ATP becomes poor, ε changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TF(o)F(1) expressed in Escherichia coli, we compared TF(o)F(1) with up- and down-state ε in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TF(o)F(1) with the up-state ε was achieved by inclusion of hexokinase in the assay and TF(o)F(1) with the down-state ε was represented by εΔc-TF(o)F(1), in which ε lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TF(o)F(1) with the down-state ε synthesizes GTP at the same rate of ATP, whereas TF(o)F(1) with the up-state ε synthesizes GTP at a half-rate. Though rates are slow, TF(o)F(1) with the down-state ε even catalyzes UTP and CTP synthesis. Authentic TF(o)F(1) from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of εΔc-TF(o)F(1) suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ε-subunit modulates substrate specificity of TF(o)F(1).     

Temperature-sensitive reaction intermediate of F1-ATPase

F(1)-ATPase is a rotary molecular motor that makes 120 degrees stepping rotations, with each step being driven by a single-ATP hydrolysis. In this study, a new reaction intermediate of F(1)-ATPase was discovered at a temperature below 4 degrees C, which makes a pause at the same angle in its rotation as when ATP binds. The rate constant of the intermediate reaction was strongly dependent on temperature with a Q(10) factor of 19, implying that the intermediate reaction accompanies a large conformational change. Kinetic analyses showed that the intermediate state does not correspond to ATP binding or hydrolysis. The addition of ADP to the reaction mixture did not alter the angular position of the intermediate state, but specifically lengthened the time constant of this state. Conversely, the addition of inorganic phosphate caused a pause at an angle of +80 degrees from that of the intermediate state. These observations strongly suggest that the newly found reaction intermediate is an ADP-releasing step.     

Crystal Structures of Mutant Forms of the Yeast F1 ATPase Reveal Two Modes of Uncoupling

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F₁ ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and β_DP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.     

Mechanically driven proton conduction in single delta-free F0F1-ATPase

In order to observe mechanically driven proton flux in F(0)F(1)-ATPase coupled with artificial driven rotation on F(1) simultaneously, a double channel observation system was established. An artificial delta-free F(0)F(1)-ATPase was constructed with alpha(3), beta(3), epsilon, gamma, and c(n) subunits as rotator and a, b(2) as stator. The chromatophore was immobilized on the glass surface through biotin-streptavidin-biotin system, and the magnetic bead was attached to the beta subunit of delta-free F(0)F(1)-ATPase. The mechanically driven proton flux was indicated by the fluorescence intensity change of fluorescein reference standard (F1300) and recorded by a cooled digital CCD camera. The mechanochemical coupling stoichiometry between F(0) and F(1) is about 4.15 +/- 0.2H(+)/rev when the magnetic field rotated at 0.33 Hz (rps).     

Using F0F1-ATPase motors as micro-mixers accelerates thrombolysis

We have developed a novel micro-mixer using a biological molecular ATP motor. The micro-mixer was constructed from arrays of chromatophore-embedded δ-free F₀F₁-ATPases, where the δ-free F₁ part acted as a rotator to mix solutions, and the F₀ part was driven by light. Confocal microscope studies indicated that the micro-mixer did not touch directly on the fibrin labeled with FITC. The nanomechanical force generated by the motor induced drug movement in the solution and accelerated the fibrinolysis process. All results strongly suggest that the micro-mixers generated a nanomechanical force which accelerated the fibrinolysis process in the presence of lower concentrations of lumbrokinase.     

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F₁F₀-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F₁F₀-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

The structure of bovine F1-ATPase inhibited by ADP and beryllium fluoride

The structure of bovine F1-ATPase inhibited with ADP and beryllium fluoride at 2.0 angstroms resolution contains two ADP.BeF3- complexes mimicking ATP, bound in the catalytic sites of the beta(TP) and beta(DP) subunits. Except for a 1 angstrom shift in the guanidinium of alphaArg373, the conformations of catalytic side chains are very similar in both sites. However, the ordered water molecule that carries out nucleophilic attack on the gamma-phosphate of ATP during hydrolysis is 2.6 angstroms from the beryllium in the beta(DP) subunit and 3.8 angstroms away in the beta(TP) subunit, strongly indicating that the beta(DP) subunit is the catalytically active conformation. In the structure of F1-ATPase with five bound ADP molecules (three in alpha-subunits, one each in the beta(TP) and beta(DP) subunits), which has also been determined, the conformation of alphaArg373 suggests that it senses the presence (or absence) of the gamma-phosphate of ATP. Two catalytic schemes are discussed concerning the various structures of bovine F1-ATPase.     

F0F1-ATPase as biosensor to detect single virus

F(0)F(1)-ATPase within chromatophore was constructed as a biosensor (immuno-rotary biosensor) for the purpose of capturing single virus. Capture of virus was based on antibody-antigen reaction. The detection of virus based on proton flux change driven by ATP-synthesis of F(0)F(1)-ATPase, which was indicated by F1300, was directly observed by a fluorescence microscope. The results demonstrate that the biosensor loading of virus particles has remarkable signal-to-noise ratio (3.8:1) compared to its control at single molecular level, and will be convenient, quick, and even super-sensitive for detecting virus particles.     

Interaction of beef-heart mitochondrial F1-ATPase with immobilized ATP in the presence of dimethylsulfoxide

Dimethylsulfoxide [Me₂SO, 30% (v/v)] promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F₁-ATPase. The effects of this solvent on the interaction of beef-heart mitochondrial F₁ with the immobilized ATP of Agarose-hexane-ATP were studied. In the presence of Me₂SO, F₁ bound less readily to the immobilized ATP, but once bound was more difficult to elute with exogenous ATP. This suggests that not only was the binding affinity for adenine nucleotide at the first binding site affected but that adenine nucleotide binding affinity at the second and/or third sites, which interact cooperatively with the first site to release bound nucleotide, was also affected. A reduction in the binding of [³H]ADP to these sites was shown. A change in the conformation of F₁ in 30% (v/v) Me₂SO was demonstrated by crosslinking and by the increased resistance of the enzyme to cold denaturation.     

The nonlinear chemo-mechanic coupled dynamics of the F 1 -ATPase molecular motor

The ATP synthase consists of two opposing rotary motors, F₀ and F₁, coupled to each other. When the F₁ motor is not coupled to the F₀ motor, it can work in the direction hydrolyzing ATP, as a nanomotor called F₁-ATPase. It has been reported that the stiffness of the protein varies nonlinearly with increasing load. The nonlinearity has an important effect on the rotating rate of the F₁-ATPase. Here, considering the nonlinearity of the γ shaft stiffness for the F₁-ATPase, a nonlinear chemo-mechanical coupled dynamic model of F₁ motor is proposed. Nonlinear vibration frequencies of the γ shaft and their changes along with the system parameters are investigated. The nonlinear stochastic response of the elastic γ shaft to thermal excitation is analyzed. The results show that the stiffness nonlinearity of the γ shaft causes an increase of the vibration frequency for the F₁ motor, which increases the motor’s rotation rate. When the concentration of ATP is relatively high and the load torque is small, the effects of the stiffness nonlinearity on the rotating rates of the F₁ motor are obvious and should be considered. These results are useful for improving calculation of the rotating rate for the F₁ motor and provide insight about the stochastic wave mechanics of F₁-ATPase.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.     

The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF1

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α_DP- and β_DP-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β_E-subunits. The nucleotide binding site in β_E-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.     

Torque generation and utilization in motor enzyme F0F1-ATP synthase: half-torque F1 with short-sized pushrod helix and reduced ATP Synthesis by half-torque F0F1

ATP synthase (F(0)F(1)) is made of two motors, a proton-driven motor (F(0)) and an ATP-driven motor (F(1)), connected by a common rotary shaft, and catalyzes proton flow-driven ATP synthesis and ATP-driven proton pumping. In F(1), the central γ subunit rotates inside the α(3)β(3) ring. Here we report structural features of F(1) responsible for torque generation and the catalytic ability of the low-torque F(0)F(1). (i) Deletion of one or two turns in the α-helix in the C-terminal domain of catalytic β subunit at the rotor/stator contact region generates mutant F(1)s, termed F(1)(1/2)s, that rotate with about half of the normal torque. This helix would support the helix-loop-helix structure acting as a solid "pushrod" to push the rotor γ subunit, but the short helix in F(1)(1/2)s would fail to accomplish this task. (ii) Three different half-torque F(0)F(1)(1/2)s were purified and reconstituted into proteoliposomes. They carry out ATP-driven proton pumping and build up the same small transmembrane ΔpH, indicating that the final ΔpH is directly related to the amount of torque. (iii) The half-torque F(0)F(1)(1/2)s can catalyze ATP synthesis, although slowly. The rate of synthesis varies widely among the three F(0)F(1)(1/2)s, which suggests that the rate reflects subtle conformational variations of individual mutants.     

The coupling of the relative movement of thea andc subunits of the F0 to the conformational changes in the F1-ATPase

F₀F₁-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F₀F₁-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed.     

Cooperativity Between the Enzymatic Sites of F1-ATPase Revisited by the Use of HPLC Methods

The fundamental question of the cooperativity between the enzymatic sites of F₁-ATPase is examined in the light of new measurements of the enzymatic rate of ATP hydrolysis by CF₁, the enzyme isolated from spinach chloroplasts. The experimental data, obtained with a chromatographic method, fit a model that involves two kinds of independent enzymatic sites working with metal-free ATP, with no need of cooperativity between the sites. Binding measurements between ADP or ATP and CF₁ by the chromatographic method of Hummel and Dreyer (1962) also support this conclusion. The present data and interpretation are in agreement with those reported recently (Reynafarje and Pedersen, 1996) which show that the first order rate constant of ATP hydrolysis by MF₁, the analogous enzyme from mitochondria, is virtually constant under experimental conditions involving either unisite or multisite hydrolysis of ATP. The present data and interpretation are discussed together with those reported previously, in particular with regard to the methods that were used to support the commonly accepted opposite viewpoint.     

Mechanism of F1-ATPase studied by the genetic approach

E. coli F₁-ATPase has been studied mainly by the genetic approach. Mutations in either the or subunit modified the kinetics of multisite and uni-site hydrolysis of ATP. The mechanism of F₁-ATPase and the essential amino acid residues of subunits are discussed.Abbreviations used: P_i, inorganic phosphate; DCCD, dicyclohexylcarbodiimide.     

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.