Regulation of ATP hydrolysis by the ε subunit, ζ subunit and Mg-ADP in the ATP synthase of Paracoccus denitrificans

F₁F_O-ATP synthase is a crucial metabolic enzyme that uses the proton motive force from respiration to regenerate ATP. For maximum thermodynamic efficiency ATP synthesis should be fully reversible, but the enzyme from Paracoccus denitrificans catalyzes ATP hydrolysis at far lower rates than it catalyzes ATP synthesis, an effect often attributed to its unique ζ subunit. Recently, we showed that deleting ζ increases hydrolysis only marginally, indicating that other common inhibitory mechanisms such as inhibition by the C-terminal domain of the ε subunit (ε-CTD) or Mg-ADP may be more important. Here, we created mutants lacking the ε-CTD, and double mutants lacking both the ε-CTD and ζ subunit. No substantial activation of ATP hydrolysis was observed in any of these strains. Instead, hydrolysis in even the double mutant strains could only be activated by oxyanions, the detergent lauryldimethylamine oxide, or a proton motive force, which are all considered to release Mg-ADP inhibition. Our results establish that P. denitrificans ATP synthase is regulated by a combination of the ε and ζ subunits and Mg-ADP inhibition.     

Effects of site-directed mutation on the function of the chloroplast ATP synthase ε subunit

The previous work in our lab showed that the spinach chloroplast ATP synthase ε mutant with 3 amino acid residues deleted from the N-terminus had much lower ability to inhibit ATP hydrolysis and block proton leakage in comparison to a mutant with 1 or 2 residues deleted from the N-terminus. The present study aimed at determining whether there is special importance in the structure and function of the N-terminal third residue of the chloroplast ε subunit. The leucine residue at the N-terminal third site (Leu3) of the spinach chloroplast ε subunit was replaced with Ile, Phe, Thr, Arg, Glu or Pro by site-directed mutagenesis, forming mutants εL3I, εL3F, εL3T, εL3R, εL3E and εL3P, respectively. These ε variants all showed lower abilities to inhibit ATP hydrolysis and to block proton leakage, as compared to the wild type ε subunit (εWT). The abilities of mutants εL3I and εL3F to restore the ATP synthesis activity of reconstituted membranes were higher than those of εWT, but the abilities of the other ε variants were lower than that of εWT. These results indicate that the hydrophobic and neutral characteristics of Leu3 of the chloroplast ε subunit are very important for its ability to inhibit ATP hydrolysis and block proton leakage, and for the ATP synthesis ability of ATP synthase.     

The ∈ subunit of the chloroplast ATP synthase of Pisum sativum

In contrast to the well-characterized spinach ( Spinacea oleracea) chloroplast ATP synthase (CF1–CFo), the properties of the chloroplast ATP synthase from pea (Pisum sativum ) have not been as intensively studied. Preliminary data suggested that the regulatory properties of the two enzymes differ. In the absence of activating treatments the ATPase activity of pea thylakoids in the dark was higher than that in spinach thylakoids. When assayed in the presence of sulfite, the MgATPase activity of pea thylakoids was inhibited to a maximum of 67% by tentoxin, indicating that the dark ATPase activity is in part catalyzed by CF1–CFo. The ATPase activity of purified pea CF1 was also higher than that of spinach CF1 in the absence of activating treatments. These differences could result from the different regulatory properties of the pea or subunit or both. The pea subunit was less effective in binding to or inhibiting the ATPase activity of pea o r spinach CF1 deficient in (CF1-). Spinach inhibited the ATPase activity of pea CF1- at lower concentrations than pea . The gene encoding the pea subunit was cloned and over-expressed. Recombinant pea did not restore low proton permeability to spinach thylakoid membranes reconstitituted with spinach CF1-, although pea was effective when tested with pea thylakoids reconstitituted with pea CF1-. These results confirm earlier suggestions that the C-terminal region of is important in -CF1 and -CFo interactions.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Conformational change of the α subunit of Escherichia coli F1 ATPase: ATP changes the trypsin sensitivity of the subunit

Conformational change in the α subunit of Escherichia coli proton-translocating ATPase was studied using trypsin. The subunit was cleaved with a small amount of trypsin (1 μg/mg subunit) to peptides of less than 8000 daltons. On the other hand, the subunit was cleaved to two main polypeptides (30,000 and 25,000 daltons) in the presence of sufficient ATP (1 mm-0.5 μm) to saturate the high-affinity site of the subunit. Analysis of digests of the subunit combined with fluorescent maleimide suggested that the subunit was digested in the middle of the polypeptide chain in the presence of the nucleotide. ADP and adenylyl imidodiphosphate had the same effect as ATP. These results suggest that the conformation of the subunit changed to form two trypsin-resistant domains upon binding of ATP to the high-affinity site.     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.     

Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae?

Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses of detergent mitochondrial extracts have provided evidence that the yeast ATP synthase could form dimers. Cross-linking experiments performed on a modified version of the i-subunit of this enzyme indicate the existence of such ATP synthase dimers in the yeast inner mitochondrial membrane. We also show that the first transmembrane segment of the eukaryotic b-subunit (bTM1), like the two supernumerary subunits e and g, is required for dimerization/oligomerization of ATP synthases. Unlike mitochondria of wild-type cells that display a well-developed cristae network, mitochondria of yeast cells devoid of subunits e, g, or bTM1 present morphological alterations with an abnormal proliferation of the inner mitochondrial membrane. From these observations, we postulate that an anomalous organization of the inner mitochondrial membrane occurs due to the absence of ATP synthase dimers/oligomers. We provide a model in which the mitochondrial ATP synthase is a key element in cristae morphogenesis.     

ATP synthase: what dictates the size of a ring?

Recent data suggest the source of F(0)F(1) ATP synthase determines a significant and surprising difference in the size of a putative rotating ring of integral membrane subunits of F(0); this can be correlated with biochemical data suggesting there is variation in the number of protons translocated per ATP synthesised.     

Multiple phosphorylation sites in the β subunit of thylakoid ATP synthase

Proteomic analyses of the β subunit of the plastid ATP synthase of barley (Hordeum vulgare L.) revealed that mature protein was not carboxy terminus processed and suggested the correction of the 274 codon (GAT to AAT) in the data bank that was confirmed by DNA sequencing. Six isoforms of the ATP synthase β subunit with pI ranging from 4.95 to 5.14 were resolved by two-dimensional electrophoresis (2-DE). Mass spectrometry analyses indicated that the six isoforms differ in their phosphorylation degree, which was confirmed by the disappearance of more acidic forms after incubation with the protein phosphatase calcineurin. Six Ser and/or Thr were detected as phosphorylated, among them the conserved Thr-179 that is also phosphorylated in the β subunit of human mitochondria. The results are discussed in relation with the proposed regulation of the ATP synthase by phosphorylation and 14-3-3 proteins.     

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

The interaction of the nucleotide-binding subunit B with subunit F is essential in coupling of ion pumping and ATP synthesis in A₁A_O ATP synthases. Here we provide structural and thermodynamic insights on the nucleotide binding to the surface of subunits B and F of Methanosarcina mazei Gö1 A₁A_O ATP synthase, which initiated migration to its final binding pocket via two transitional intermediates on the surface of subunit B. NMR- and fluorescence spectroscopy as well as ITC data combined with molecular dynamics simulations of the nucleotide bound subunit B and nucleotide bound B-F complex in explicit solvent, suggests that subunit F is critical for the migration to and eventual occupancy of the final binding site by the nucleotide of subunit B. Rotation of the C-terminus and conformational changes in subunit B are initiated upon binding with subunit F causing a perturbation that leads to the migration of ATP from the transition site 1 through an intermediate transition site 2 to the final binding site 3. This mechanism is elucidated on the basis of change in binding affinity for the nucleotide at the specific sites on subunit B upon complexation with subunit F. The change in enthalpy is further explained based on the fluctuating local environment around the binding sites.     

Covalent modification of the non-catalytic sites of the H+-ATPase from chloroplasts with 2-azido-[α-32P]ATP and its effect on ATP synthesis and ATP hydrolysis

Incubation of the isolated H⁺-ATPase from chloroplasts, CF₀F₁, with 2-azido-[α-³²P]ATP leads to the binding of this nucleotide to different sites. These sites were identified after removal of free nucleotides, UV-irradiation and trypsin treatment by separation of the tryptic peptides by ion exchange chromatography. The nitreno-AMP, nitreno-ADP and nitreno-ATP peptides were further separated on a reversed phase column, the main fractions were subjected to amino acid sequence analysis and the derivatized tyrosines were used to distinguish between catalytic (β-Tyr362) and non-catalytic (β-Tyr385) sites. Several incubation procedures were developed which allow a selective occupation of each of the three non-catalytic sites. The non-catalytic site with the highest dissociation constant (site 6) becomes half maximally filled at 50 μM 2-azido-[α-³²P]ATP, that with the intermediate dissociation constant (site 5) at 2 μM. The ATP at the site with the lowest dissociation constant had to be hydrolyzed first to ADP before a replacement by 2-azido-[α-³²P]ATP was possible. CF₀F₁ with non-covalently bound 2-azido-[α-³²P]ATP and after covalent derivatization was reconstituted into liposomes and the rates of ATP synthesis as well as ATP hydrolysis were measured after energization of the proteoliposomes by ΔpH/Δϕ. Non-covalent binding of 2-azido-ATP to any of the three non-catalytic sites does not influence ATP synthesis and ATP hydrolysis, whereas covalent derivatization of any of the three sites inhibits both, the degree being proportional to the degree of derivatization. Extrapolation to complete inhibition indicates that derivatization of one site (either 4 or 5 or 6) is sufficient to block completely multi-site catalysis. The rates of ATP synthesis and ATP hydrolysis were measured as a function of the ADP and ATP concentration from uni-site to multi-site conditions with covalently derivatized and non-derivatized CF₀F₁. Uni-site ATP synthesis and ATP hydrolysis were not inhibited by covalent derivatization of any of the non-catalytic sites, whereas multi-site catalysis is inhibited. These results indicate that multi-site catalysis requires some flexibility between β- and α-subunits which is abolished by covalent derivatization of β-Tyr385 with a 2-nitreno-adenine nucleotide. Conformational changes connected with energy transduction between the F₀-part and the F₁-part are either not required for uni-site ATP synthesis or they are not impaired by the derivatization of any of the three β-Tyr385.     

Do modulators of the mitochondrial K(ATP) channel change the function of mitochondria in situ?

Pharmacological opening of mitochondrial cardiac ATP-sensitive potassium (K(ATP)) channels has the chance to be a promising but still controversial cardioprotective mechanism. Physiological roles of mitochondrial K(ATP) channels in the myocardium remain unclear. We studied the effects of diazoxide, a specific opener of these channels, on the function of rat mitochondria in situ in saponin-permeabilized fibers using an ionic medium that mimics the cytosol. In the presence of NADH-producing substrates (malate + glutamate), neither 100 microm diazoxide nor 100 microm glibenclamide (a K(ATP) channel blocker) changed the mitochondrial respiration in the absence or presence of ADP. Because the K(ATP) channel function could be modified by changes in adenine nucleotide concentrations near the mitochondria, we studied the effects of diazoxide and glibenclamide on the functional activity of mitochondrial kinases. Both diazoxide and glibenclamide did not change the in situ ADP sensitivity in the presence or absence of creatine (apparent K(m) values for ADP were, respectively, 59 +/- 9 and 379 +/- 45 microm). Similarly, stimulation of the mitochondrial respiration with AMP in the presence of ATP due to adenylate kinase activity was not affected by the modulators of K(ATP) channels. However, when succinate was used as substrate, diazoxide significantly inhibited basal respiration by 22% and maximal respiration by 24%. Thus, at a cardioprotective dose, the main functional effect of diazoxide depends on respiratory substrates and seems not to be related to K(ATP) channel activity.     

Is the ATP — dependent protection of lysosomes against osmotic lysis a function of the lysosomal proton pump

Summary Rat liver lysosomes have been used to characterize further the effects of ATP on lysosomal stability during incubation at 37°C at hypo-osmolarity. As previously reported, when the osmotically-supporting solute is the salt of a strong base (K⁺), ATP protects against lysis during incubation. However, if the osmotically-supporting solute is the salt of a weak base, e.g. Tris HCl or NH₄Cl, ATP actually promotes lysis during incubation. Thus, ATP can exert destabilizing as well as protective effects on lysosomes. The destabilizing effect is eliminated by protonophores. The protective effect in the presence of potassium salts is not eliminated by protonophores. Moreover, when incubation is in the presence of a salt of a weak base, protonophores actually cause an ATP-dependent protective effect to be established. The destabilizing effect occurs at 37°C, but not at 0°C. The Mg^–+-dependence of the destabilizing effect was found to be similar to that found earlier for the ATP-dependent protective effect, insofar as only 1 mM MgCl₂ in the presence of 1 mM EDTA is sufficient for nearly maximal stimulation of both effects. The destabilizing effect may result from a H^– ion gradient across the lysosomal membrane which is maintained by the lysosomal ATP-dependent proton pump. The protective effect, on the other hand, does not depend on such a gradient being maintained; on the contrary, protonophores appear to act as enablers of the protective effect. The question that remains to be answered is: does the protective effect derive in some way from the same ATP-driven mechanism which constitutes the proton pump? Some possible answers to this question are considered.Abbreviations Mops 2-(N-morpholine)-propanesulfonic acid - CCCP Carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-Dinitrophenol - EDTA Ethylenediaminetetracetic acid     

The rate-limiting step of sulfiredoxin is associated with the transfer of the γ-phosphate of ATP to the sulfinic acid of overoxidized typical 2-Cys peroxiredoxins

The eukaryotic sulfiredoxin (Srx) catalyzes the reduction of overoxidized typical 2-Cys peroxiredoxins PrxSO₂ via ATP/Mg²⁺-dependent phosphorylation of the sulfinic acid group, followed by formation of a PrxSO-SSrx thiolsulfinate intermediate. Using real-time kinetics of wild-type and C84A Srxs, and pH-rate profiles with ATP/Mg²⁺ analogues, we show that the rate-limiting step of the reaction is associated with the chemical process of transfer of the γ-phosphate of ATP to the sulfinic acid, in contrast to that described by Jönsson et al. [7]. Two pK_apps of 6.2 and 7.5 were extracted from the bell-shaped pH-rate profile, corresponding to the γ-phosphate of ATP, and to an acid–base catalyst, respectively.     

ATP binding to the ϵ subunit of thermophilic ATP synthase is crucial for efficient coupling of ATPase and H+ pump activities

ATP binding to the ? subunit of F1-ATPase, a soluble subcomplex of TFoF1 (FoF1-ATPase synthase from the thermophilic Bacillus strain PS3), affects the regulation of F1-ATPase activity by stabilizing the compact, ATPase-active, form of the ? subunit [Kato, S., Yoshida, M. and Kato-Yamada, Y. (2007) J. Biol. Chem. 282, 37618-37623]. In the present study, we report how ATP binding to the ? subunit affects ATPase and H+ pumping activities in the holoenzyme TFoF1. Wild-type TFoF1 showed significant H+ pumping activity when ATP was used as the substrate. However, GTP, which bound poorly to the ? subunit, did not support efficient H+ pumping. Addition of small amounts of ATP to the GTP substrate restored coupling between GTPase and H+ pumping activities. Similar uncoupling was observed when TFoF1 contained an ATP-binding-deficient ? subunit, even with ATP as a substrate. Further analysis suggested that the compact conformation of the ? subunit induced by ATP binding was required to couple ATPase and H+ pumping activities in TFoF1 unless the ? subunit was in its extended-state conformation. The present study reveals a novel role of the ? subunit as an ATP-sensitive regulator of the coupling of ATPase and H+ pumping activities of TFoF1.     

Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ε Subunit

The conserved residue Gly47 of the chloroplast ATP synthase ε subunit was substituted with Leu, Arg, Ala and Glu by site-directed mutagenesis. This process generated the mutants εG47L, εG47R, εG47A and εG47E, respectively. All the ε variants showed lower inhibitory effects on the soluble CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In reduced conditions, εG47E and εG47R had a lower inhibitory effect on the oxidized CF1(-ε) Ca^2 -ATPase compared with wild-type ε. In contrast, εG47L and εG47Aincreased the Ca^2 -ATPase activity of soluble oxidized CF1(-ε). The replacement of Gly47 significantly impaired the interaction between the subunit ε and γ in an in vitro binding assay. Further study showed that all ε variants were more effective in blocking proton leakage from the thylakoid membranes. This enhanced ATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to a level that was more efficient than that achieved by wild-type ε. These results indicate that the conserved Gly47 residue of the ε subunit is very important for maintaining the structure and function of the ε subunitand may affect the interaction between the ε subunit, β subunit of CF1 and subunit Ⅲ of CF0, therebyregulating the ATP hydrolysis and synthesis, as well as the proton translocation role of the subunit Ⅲ of CF0.