The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria

The F_oF₁ synthase produces ATP from ADP and inorganic phosphate. The γ subunit of F_oF₁ ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole F_oF₁ complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire F_oF₁ ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of F_oF₁ ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of F_oF₁ ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.     

Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V₁ moiety of Enterococcus hirae V-ATPase (EhV₁) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV₁ using single-molecule analysis employing a load-free probe. EhV₁ rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (V_max) of 107 revolutions/s and a Michaelis constant (K_m) of 154 μm at 26 °C. At all ATP concentrations tested, EhV₁ showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V₁-ATPase (TtV₁). At 10 μm ATP (⪡K_m), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10⁶ m⁻¹ s⁻¹. At 40 mm ATP (⪢K_m), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV₁. Our results identify the common properties of rotary catalysis of V₁-ATPases that are distinct from those of F₁-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.     

ATP binding and hydrolysis steps of the uni-site catalysis by the mitochondrial F1-ATPase are affected by inorganic phosphate

The effect of inorganic phosphate (P_i) on uni-site ATP binding and hydrolysis by the nucleotide-depleted F₁-ATPase from beef heart mitochondria (ndMF₁) has been investigated. It is shown for the first time that P_i decreases the apparent rate constant of uni-site ATP binding by ndMF₁ 3-fold with the K_d of 0.38 ± 0.14 mM. During uni-site ATP hydrolysis, P_i also shifts equilibrium between bound ATP and ADP + P_i in the direction of ATP synthesis with the K_d of 0.17 ± 0.03 mM. However, 10 mM P_i does not significantly affect ATP binding during multi-site catalysis.     

Modulation of nucleotide binding to the catalytic sites of thermophilic F1-ATPase by the ε subunit: Implication for the role of the ε subunit in ATP synthesis

Effect of ε subunit on the nucleotide binding to the catalytic sites of F₁-ATPase from the thermophilic Bacillus PS3 (TF₁) has been tested by using α₃β₃γ and α₃β₃γε complexes of TF₁ containing βTyr341 to Trp substitution. The nucleotide binding was assessed with fluorescence quenching of the introduced Trp. The presence of the ε subunit weakened ADP binding to each catalytic site, especially to the highest affinity site. This effect was also observed when GDP or IDP was used. The ratio of the affinity of the lowest to the highest nucleotide binding sites had changed two orders of magnitude by the ε subunit. The differences may relate to the energy required for the binding change in the ATP synthesis reaction and contribute to the efficient ATP synthesis.     

Met23Lys mutation in subunit gamma of FOF1-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force

H⁺-F_OF₁-ATP synthase couples proton flow through its membrane portion, F_O, to the synthesis of ATP in its headpiece, F₁. Upon reversal of the reaction the enzyme functions as a proton pumping ATPase. Even in the simplest bacterial enzyme the ATPase activity is regulated by several mechanisms, involving inhibition by MgADP, conformational transitions of the ε subunit, and activation by protonmotive force. Here we report that the Met23Lys mutation in the γ subunit of the Rhodobacter capsulatus ATP synthase significantly impaired the activation of ATP hydrolysis by protonmotive force. The impairment in the mutant was due to faster enzyme deactivation that was particularly evident at low ATP/ADP ratio. We suggest that the electrostatic interaction of the introduced γLys23 with the DELSEED region of subunit β stabilized the ADP-inhibited state of the enzyme by hindering the rotation of subunit γ rotation which is necessary for the activation.     

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.     

Nucleotide Binding States of Subunit A of the A-ATP Synthase and the Implication of P-Loop Switch in Evolution

The crystal structures of the nucleotide-empty (A_E), 5′-adenylyl-β,γ-imidodiphosphate (A_PNP)-bound, and ADP (A_DP)-bound forms of the catalytic A subunit of the energy producer A₁A_O ATP synthase from Pyrococcus horikoshii OT3 have been solved at 2.47 Å and 2.4 Å resolutions. The structures provide novel features of nucleotide binding and depict the residues involved in the catalysis of the A subunit. In the A_E form, the phosphate analog SO₄²⁻ binds, via a water molecule, to the phosphate binding loop (P-loop) residue Ser238, which is also involved in the phosphate binding of ADP and 5′-adenylyl-β,γ-imidodiphosphate. Together with amino acids Gly234 and Phe236, the serine residue stabilizes the arched P-loop conformation of subunit A, as shown by the 2.4-Å structure of the mutant protein S238A in which the P-loop flips into a relaxed state, comparable to the one in catalytic β subunits of F₁F_O ATP synthases. Superposition of the existing P-loop structures of ATPases emphasizes the unique P-loop in subunit A, which is also discussed in the light of an evolutionary P-loop switch in related A₁A_O ATP synthases, F₁F_O ATP synthases, and vacuolar ATPases and implicates diverse catalytic mechanisms inside these biological motors.     

Role of alphaPhe-291 residue in the phosphate-binding subdomain of catalytic sites of Escherichia coli ATP synthase

The role of αPhe-291 residue in phosphate binding by Escherichia coli F₁F₀-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F₁F₀ membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.     

Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na-ATPase

Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V₁; NtpA₃-B₃-D-G) and an integral membrane domain (V₀; NtpI-K₁₀) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V₁ portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 μM for ADP or 3.1 μM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA₃-B₃ heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA₃-B₃ forming V₁ (NtpA₃-B₃-D-G) complex independent of nucleotides. The V₁ formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V₁ complex was as high as that of native V₁-ATPase purified from the V₀V₁ complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V₁ complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V₁-ATPase complex.     

Fluorometric evidence for control of the activity of F1-adenosinetriphosphatase by ligand-induced conformation change

The effect of ATP on the fluorescence intensity of bovine heart F₁-adenosinetriphosphatase labeled at its essential Lys with 7-chloro-4-nitro-2,1,3-benzoxadiazole (N-NBD-F₁) has been examined in solutions containing different concentrations of ADP. The fluorescence of N-NBD-F₁ is unaffected by ATP in the absence of ADP. But when increasing amounts of ATP are added to a solution of N-NBD-F₁ containing 0.37 or 1.0 mM ADP, the fluorescence of N-NBD-F₁ first decreases and then increases continually as the concentration of ATP is further raised. Parallel measurements of the suppression of the fluorescence of N-NBD-F₁ and the inhibition of the ATPase activity of the unlabeled enzyme by ADP in the presence of ATP show a quantitative correlation between the changes in fluorescence and in ATPase activity. The data are consistent with the model for F₁-ATPase with one principal catalytic subunit for ATP hydrolysis and synthesis, and two auxiliary subunits which control the conformation and hence the catalytic activity of through interaction between all the subunits.     

A Biological Molecular Motor, Proton-Translocating ATP Synthase: Multidisciplinary Approach for a Unique Membrane Enzyme

Proton-translocating ATP synthase (F_oF₁) synthesizes ATP from ADP and phosphate, coupled with an electrochemical proton gradient across the biological membrane. It has been established that the rotation of a subunit assembly is an essential feature of the enzyme mechanism and that F_oF₁ can be regarded as a molecular motor. Thus, experimentally, in the reverse direction (ATP hydrolysis), the chemical reaction drives the rotation of a c _10-14 subunit assembly followed by proton translocation. We discuss our very recent results regarding subunit rotation in Escherichia coli F_oF₁ with a combined biophysical and mutational approach.     

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.     

Relationships of inosine triphosphate and bicarbonate effects on F1 ATPase to the binding change mechanism

Two interesting previously reported properties of mitochondrial F₁ ATPase have been confirmed and have been examined by¹⁸O exchange measurements to assess if they are consistent with sequential participation of catalytic sites during ATP hydrolysis. These are the ability of HCO ₃ ^– to increase reaction rate with apparent loss of cooperative interaction between subunits and the ability of ITP to accelerate the hydrolysis of a low concentration of ATP. The effect of HCO ₃ ^– was tested at concentrations of ATP lower than previous measurements. The activation disappeared when ATP was reduced to 0.1 µM. The HCO ₃ ^– activation at higher ATP concentrations did not change the extent of reversal of the cleavage of tightly bound ATP at the catalytic site, as measured by the average number of water oxygens incorporated with each P_i formed when 5 or 10 µM ATP is hydrolyzed. The data are consistent with sequential site participation with HCO ₃ ^– acceleration of ADP departure after a binding change that stops¹⁸O exchange and loosens ADP binding.When ITP concentration was lowered during net ITP hydrolysis by F₁ ATPase an increase in water oxygen incorporation into P_i formed is observed, as noted previously for ATP hydrolysis. The acceleration of the cleavage of a constant low concentration of [-¹⁸O]ATP by concomitant hydrolysis of increasing concentrations of ITP was accompanied by a decrease in water oxygen incorporation with each P_i formed from the ATP. These results add to evidence for the binding change mechanism for F₁ ATPase with sequential participation of catalytic sites.     

Mechanical Modulation of ATP-binding Affinity of V1-ATPase

V₁-ATPase is a rotary motor protein that rotates the central shaft in a counterclockwise direction hydrolyzing ATP. Although the ATP-binding process is suggested to be the most critical reaction step for torque generation in F₁-ATPase (the closest relative of V₁-ATPase evolutionarily), the role of ATP binding for V₁-ATPase in torque generation has remained unclear. In the present study, we performed single-molecule manipulation experiments on V₁-ATPase from Thermus thermophilus to investigate how the ATP-binding process is modulated upon rotation of the rotary shaft. When V₁-ATPase showed an ATP-waiting pause, it was stalled at a target angle and then released. Based on the response of the V₁-ATPase released, the ATP-binding probability was determined at individual stall angles. It was observed that the rate constant of ATP binding (k_on) was exponentially accelerated with forward rotation, whereas the rate constant of ATP release (k_off) was exponentially reduced. The angle dependence of the k_off of V₁-ATPase was significantly smaller than that of F₁-ATPase, suggesting that the ATP-binding process is not the major torque-generating step in V₁-ATPase. When V₁-ATPase was stalled at the mean binding angle to restrict rotary Brownian motion, k_on was evidently slower than that determined from free rotation, showing the reaction rate enhancement by conformational fluctuation. It was also suggested that shaft of V₁-ATPase should be rotated at least 277° in a clockwise direction for efficient release of ATP under ATP-synthesis conditions.     

Properties of noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1

Nucleotide binding properties of two vacant noncatalytic sites of thioredoxin-activated chloroplast coupling factor 1 (CF₁) were studied. Kinetics of nucleotide binding to noncatalytic sites is described by the first-order equation that allows for two nucleotide binding sites that differ in kinetic features. Dependence of the nucleotide binding rate on nucleotide concentration suggests that tight nucleotide binding is preceded by rapid reversible binding of nucleotides. ADP binding is cooperative. The preincubation of CF₁ with Mg²⁺ produces only slight effect on the rate of ADP binding and decreases the ATP binding rate. The ATP and ADP dissociation from noncatalytic sites is described by the first-order equation for similar sites with dissociation rate constants k₋₂(ADP)=1.5×10⁻¹ min⁻¹ and k₋₂(ATP)≅10⁻³ min⁻¹, respectively. As follows from the study, the noncatalytic sites of CF₁ are not homogeneous. One of them retains the major part of endogenous ADP after CF₁ precipitation with ammonium sulfate. Its other two sites can bind both ADP and ATP but have different kinetic parameters and different affinity for nucleotides.     

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.     

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.     

Introduction: A Close Look at the Vacuolar ATPase

The vacuolar ATPases (V-type ATPases) are a family of ATP-dependent ion pumps and found in two principal locations, in endomembranes and in plasma membranes. This family of ATPases is responsible for acidification of intracellulare compartments and, in certain cases, ion transport across the plasma membrane of eucaryotic cells. V-ATPases are composed of two distinct domains: a catalytic V₁ sector, in which ATP hydrolysis takes place, and the membrane-embedded sector, V₀, which functions in ion conduction. In the past decade impressive progress has been made in elucidating the properties structure, function and moleculare biology. These knowledge sheds light also on the evolution of V-ATPases and their related families of A-(A₁A₀-ATPase) and F-type (F₁F₀-ATPases)ATPases.     

Characterization of the relationship between ADP- and epsilon-induced inhibition in cyanobacterial F1-ATPase

The ATPase activity of chloroplast and bacterial F(1)-ATPase is strongly inhibited by both the endogenous inhibitor ε and tightly bound ADP. Although the physiological significance of these inhibitory mechanisms is not very well known for the membrane-bound F(0)F(1), these are very likely to be important in avoiding the futile ATP hydrolysis reaction and ensuring efficient ATP synthesis in vivo. In a previous study using the α(3)β(3)γ complex of F(1) obtained from the thermophilic cyanobacteria, Thermosynechococcus elongatus BP-1, we succeeded in determining the discrete stop position, ～80° forward from the pause position for ATP binding, caused by ε-induced inhibition (ε-inhibition) during γ rotation (Konno, H., Murakami-Fuse, T., Fujii, F., Koyama, F., Ueoka-Nakanishi, H., Pack, C. G., Kinjo, M., and Hisabori, T. (2006) EMBO J. 25, 4596-4604). Because γ in ADP-inhibited F(1) also pauses at the same position, ADP-induced inhibition (ADP-inhibition) was assumed to be linked to ε-inhibition. However, ADP-inhibition and ε-inhibition should be independent phenomena from each other because the ATPase core complex, α(3)β(3)γ, also lapses into the ADP-inhibition state. By way of thorough biophysical and biochemical analyses, we determined that the ε subunit inhibition mechanism does not directly correlate with ADP-inhibition. We suggest here that the cyanobacterial ATP synthase ε subunit carries out an important regulatory role in acting as an independent "braking system" for the physiologically unfavorable ATP hydrolysis reaction.     

无机磷影响叠氮钠对ATP合成酶合成活性的抑制

利用ADP和放射性磷直接合成ATP的方法,研究了无机磷(P_i)和叠氮钠对猪心线粒体ATP合成酶（F₁F_O-ATPase）ATP合成活性的影响.结果发现无机磷除作为合成ATP的底物参与F₁F_O-ATPase的合成反应外,还对F₁F_O-ATPase的合成活性呈现抑制作用,在1 mmol/L ADP存在时,随着P_i浓度由0.01～10 mmol/L增加,抑制合成作用越来越强.与叠氮钠在低浓度时(小于1 mmol/L)只抑制ATP水解,不影响ATP合成的观点不同.实验结果显示0.1 mmol/L叠氮钠表观激活F₁F_O-ATPase的ATP合成活性,且激活程度与反应体系中所加P_i的浓度呈负相关.当固定P_i浓度（0.1 mmol/L）后,随着叠氮钠浓度的增加表观激活程度也在变化,叠氮钠与磷浓度相等时表观激活程度最大,直至叠氮钠浓度接近0.5 mmol/L时,开始呈现表观抑制现象,叠氮钠浓度高于1 mmol/L之后,就出现解偶联现象.