Temperature-induced states of isolated F1-ATPase affect catalysis, enzyme conformation and high-affinity nucleotide binding sites

Isolated, nucleotide-depleted bovine-heart F1-ATPase exhibits a break in Arrhenius plot with a 2.7-fold increase in activation energy of ATP hydrolysis below 18-19 degrees C. Analysis of intrinsic tyrosine fluorescence and of the circular dichroism of F1-ATPase showed an abrupt and reversible conformational change occurring at the break temperature, characteristic of a structural tightening at low temperature. Analysis of catalytic nucleotide binding sites using fluorescent ADP analog, 3'-O-(1-naphthoyl)adenosine diphosphate did not show any significant change in affinity of nucleotide binding around the transition temperature but the bound fluorophore exerted a more restricted motion and slower rotation at temperature below the break, indicating a change in the mobility of groups in the close neighbourhood. It is concluded that, as a result of temperature, two kinetically distinct states of F1-ATPase are induced, due to a change in enzyme conformation, which influences directly the properties of catalytic nucleotide binding sites.     

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase.

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.     

Classification of nucleotide binding sites on mitochondrial F1-ATPase from yeast

Methods are described to classify nucleotide binding sites of the mitochondrial coupling factor F1 from yeast on the basis of their affinities and stability properties. High affinity sites or states for ATP and related adenine analogs and low affinity sites or states which bind a broad range of different nucleotide triphosphates are found. The results are discussed in terms of a two site, two cycle scheme, where binding of nucleotide at one site facilitates the release of nucleotide at a second site.     

1H-NMR studies on nucleotide binding to the catalytic sites of bovine mitochondrial F1-ATPase

The conformation of adenine nucleotides bound to bovine mitochondrial F1-ATPase was investigated using transfer nuclear Overhauser enhancement measurements. It is shown that all nucleotides investigated adopt a predominantly anti conformation when bound to the catalytic sites. Furthermore, the experiment suggests that 8-azido-ADP and 8-azido-ATP, which are predominantly in the syn conformation in solution, are in the anti conformation when bound to F1 catalytic sites.     

Evidence for a nucleotide binding site on the isolated beta subunit from Escherichia coli F1-ATPase. Interaction between nucleotide and aurovertin D binding sites

The nucleotide binding capacity and affinity of the isolated beta subunit from Escherichia coli F1-ATPase have been studied with radiolabeled ADP and ATP by an equilibrium dialysis technique. Each mole of beta subunit in the presence of EDTA bound 1 mol of ADP or ATP with Kd values of 25 microM and 50-100 microM, respectively. At a saturating concentration, aurovertin enhanced the affinity of ADP or ATP for the isolated beta subunit by 3-6-fold. The Kd values for the binding of ADP or ATP were also assessed through the enhancing effect of ADP on [14C]aurovertin binding (Issartel, J.-P., Klein, G., Satre, M., & Vignais, P.V. (1983) Biochemistry 22, 3485-3492); the Kd values determined by this approach were several times lower than in the absence of aurovertin, in agreement with results obtained by direct titration with radiolabeled ADP or ATP.     

When beef-heart mitochondrial F1-ATPase is inhibited by inhibitor protein a nucleotide is trapped in one of the catalytic sites.

Inactivation of the isolated ATPase portion of ATP synthase from beef-heart mitochondria (F1) by its natural inhibitor protein (IP) during steady-state ATP hydrolysis is accompanied by a trapping of 1 mol nucleotide/mol F1 in one of the catalytic sites. The trapped nucleotide is not released during incubation of IP-inhibited F1 in the presence of MgATP at pH 8.0 for at least 20 min, indicating a very low turnover rate of the IP.F1 complex. The ATP/ADP ratio of the trapped nucleotides is higher than that found for transitorily bound nucleotides under the same conditions but in the absence of IP. The IP impairs the acceleration of ATP hydrolysis and product release steps that results from the binding of ATP to an alternate catalytic site. It also inhibits ATP hydrolysis by a single catalytic site or shifts the equilibrium toward ATP formation from bound ADP and Pi. At high pH, an active acidic form of the free IP is transformed to the inactive basic one with a half-time of 3-4 s. This process seems to be prevented by IP binding to F1. The inactive basic form of IP does not compete with the active acidic IP for the binding to F1. The data do not favor the existence of a long-lived catalytically active IP.F1 intermediate during IP action on F1. The reactivation of IP-inhibited membrane-bound F1 by energization may be due to a conformational change in the IP.F1 complex allowing the transformation of IP into an inactive basic state that rapidly dissociates.     

ATP binding at noncatalytic sites of soluble chloroplast F1-ATPase is required for expression of the enzyme activity

The F1-ATPase from chloroplasts (CF1) lacks catalytic capacity for ATP hydrolysis if ATP is not bound at noncatalytic sites. CF1 heat activated in the presence of ADP, with less than one ADP and no ATP at non-catalytic sites, shows a pronounced lag in the onset of ATP hydrolysis after exposure to 5-20 microM ATP. The onset of activity correlates well with the binding of ATP at the last two of the three noncatalytic sites. The dependence of activity on the presence of ATP at non-catalytic sites is shown at relatively low or high free Mg2+ concentrations, with or without bicarbonate as an activating anion, and when the binding of ATP at noncatalytic sites is slowed 3-4-fold by sulfate. The latent CF1 activated by dithiothreitol also requires ATP at noncatalytic sites for ATPase activity. A similar requirement by other F1-ATPases and by ATP synthases seems plausible.     

Effect of the epsilon-subunit on nucleotide binding to Escherichia coli F1-ATPase catalytic sites.

The influence of the epsilon-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (beta-Trp-331). The interaction between epsilon and F1 was not affected by the mutation. Kd for binding of epsilon to betaY331W mutant F1 was approximately 1 nM, and epsilon inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the epsilon-depleted and epsilon-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. Kd1(MgATP) and Kd1(MgADP) were an order of magnitude higher in the absence of epsilon than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the epsilon-depleted and epsilon-replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of epsilon, Km equals Kd3. Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (Vmax) MgATP hydrolysis rates.     

Site-site interaction on mitochondrial F1-ATPase. Functional symmetry of the high-affinity nucleotide binding sites

Interactions between the high affinity binding sites on mitochondrial F1 were analysed by combined use of the nucleotide analogues 3'-O-(1-naphthoyl)-ADP (N-ADP) and 2'-3'-O-(2,4,6-trinitrophenyl)-ADP (TNP-ADP). The binding behaviour of F1 with respect to these ligands was studied by measuring the fluorescence of F1 and of TNP-ADP and the fluorescence anisotropy of N-ADP. A total of 3 high affinity binding sites can be occupied by TNP-ADP. By exchange experiments, it could be shown that binding of TNP-ADP to such a site considerably accelerates the dissociation of a ligand bound to a neighbouring site. These results support the notion that the functional behaviour of F1 is symmetric: during the catalytic cycle any individual site can successively assume different affinity states as has been predicted by hypotheses such as the binding change model.     

Adenine nucleotide binding sites on beef heart F1-ATPase. Asymmetry and subunit location

Previously we have shown that beef heart mitochondrial F1 contains a total of six adenine nucleotide binding sites. Three "catalytic" sites exchange bound ligand rapidly during hydrolysis of MgATP, whereas three "noncatalytic" sites do not. The noncatalytic sites behave asymmetrically in that a single site releases bound ligand upon precipitation of F1 with ammonium sulfate. In the present study, we find this same site to be the only noncatalytic site that undergoes rapid exchange of bound ligand when F1 is incubated in the presence of EDTA at pH 8.0. Following 1000 catalytic turnovers/F1, the site retains the unique capacity for EDTA-induced exchange, indicating that the asymmetric determinants are permanent and that the three noncatalytic sites on soluble F1 do not pass through equivalent states during catalysis. Measurements of the rate of ligand binding at the unique noncatalytic site show that uncomplexed nucleotide binds preferentially. At pH 7.5, in the presence of Mg2+, the rate constant for ADP binding is 9 X 10(3) M-1 s-1 and for dissociation is 4 X 10(-4) s-1 to give a Kd = 50 nM. The rate of dissociation is 10 times faster in the presence of EDTA or during MgATP hydrolysis, and it increases rapidly at pH below 7. EDTA-induced exchange is inhibited by Mg2+, Mn2+, Co2+, and Zn2+ but not by Ca2+ and is unaffected by dicyclohexylcarbodiimide modification. The unique noncatalytic site binds 2-azido-ADP. Photolysis results in the labeling of the beta subunit. Photolabeling of a single high-affinity catalytic site under conditions for uni-site catalysis also results in the labeling of beta, but a different pattern of labeled peptides is obtained in proteolytic digests. The results demonstrate the presence of two different nucleotide binding domains on the beta subunit of mitochondrial F1.     

Adenine nucleotide binding sites on beef heart F1-ATPase. Specificity of cooperative interactions between catalytic sites

Cooperative interactions between nucleotide binding sites on beef heart mitochondrial F1-ATPase have been studied by measuring substrate-promoted release of 5'adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) from a single high affinity site. The site is initially loaded by incubating F1 with an equimolar amount of the nonhydrolyzable ATP analog. When unbound [3H]AMP-PNP is removed and the complex diluted to a concentration below the Kd, release of ligand shows an apparent absolute requirement for medium ADP. Release is biphasic with the extent of release during the initial rapid phase dependent on the concentration of medium ADP. Although phosphate alone has no effect, it enhances the rapid phase of ADP-promoted release over 2-fold with a half-maximal effect at 60 micrometers P1. The binding of efrapeptin (A23871) to the F1.AMP-PNP complex completely prevents ADP-promoted dissociation. Although AMP-PNP release also occurs in the presence of medium ATP, the F1.AMP-PNP complex does not dissociate if an ATP-regenerating system of sufficient capacity to prevent accumulation of medium ADP is added. Consistent with an inability of nucleoside triphosphate to promote release is the failure of medium, nonradioactive AMP-PNP to affect retention of the 3H-labeled ligand. The stability of F1.AMP-PNP complex in the absence of medium nucleotide and the highly specific ability of ADP plus P1 to promote rapid release of the ATP analog are interpreted as support for an ATP synthesis mechanism that requires substrate binding at one catalytic site for product release from an adjacent interacting site.     

The process in which nucleotide is buried into the active site of heavy meromyosin

The process in which nucleotide is buried into the active site of heavy meromyosin was studied with stopped-flow apparatus by monitoring the time-course of the large fluorescence increase of 1,N6-ethenoadenosine triphosphate (epsilon-ATP) when it binds from acrylamide-containing solutions. We have recently reported that free epsilon-ATP fluorescence is effectively quenched by acrylamide while bound epsilon-ATP is resistant to quenching by acrylamide. In the present study it was found that in the first step the phosphate moiety binds at a high rate, while the adenine moiety is still on the rim of the active site; the adenine moiety is then pulled into a crevice, and finally epsilon-ATP hydrolysis occurs.     

Interaction between aurovertin and adenine nucleotide binding sites on mitochondrial F1-ATPase and the isolated beta subunit

The effect of aurovertin on the binding parameters of ADP and ATP to native F1 from beef heart mitochondria in the presence of EDTA has been explored. Three exchangeable sites per F1 were titrated by ADP and ATP in the absence or presence of aurovertin. Curvilinear Scatchard plots for the binding of both ADP and ATP were obtained in the absence of aurovertin, indicating one high affinity site (Kd for ADP = 0.6-0.8 microM; Kd for ATP = 0.3-0.5 microM) and two lower affinity sites (Kd for ADP = 8-10 microM; Kd for ATP = 7-10 microM). With a saturating concentration of aurovertin capable of filling the three beta subunits of F1, the curvilinearity of the Scatchard plots was decreased for ATP binding and abolished for ADP binding, indicating homogeneity of ADP binding sites in the F1-aurovertin complex (Kd for ADP = 2 microM). When only the high affinity aurovertin site was occupied, maximal enhancement of the fluorescence of the F1-aurovertin complex was attained with 1 mol of ADP bound per mol of F1 and maximal quenching for 1 mol of ATP bound per mol of F1. When the F1-aurovertin complex was incubated with [3H]ADP followed by [14C]ATP, full fluorescence quenching was attained when ATP had displaced the previously bound ADP. In the case of the isolated beta subunit, both ADP and ATP enhanced the fluorescence of the beta subunit-aurovertin complex. The Kd values for ADP and ATP in the presence of EDTA were 0.6 mM and 3.7 mM, respectively; MgCl2 decreased the Kd values to 0.1 mM for both ADP and ATP. It is postulated that native F1 possesses three equivalent interacting nucleotide binding sites and exists in two conformations which are in equilibrium and recognize either ATP (T conformation) or ADP (D conformation). The negative interactions between the nucleotide binding sites of F1 are strongest in the D conformation. Upon addition of aurovertin, the site-site cooperativity between the beta subunits of F1 is decreased or even abolished.     

Tightly bound adenosine diphosphate, which inhibits the activity of mitochondrial F1-ATPase, is located at the catalytic site of the enzyme

The binding of one ADP molecule at the catalytic site of the nucleotide depleted F1-ATPase results in a decrease in the initial rate of ATP hydrolysis. The addition of an equimolar amount of ATP to the nucleotide depleted F1-ATPase leads to the same effect, but, in this case, inhibition is time dependent. The half-time of this process is about 30 s, and the inhibition is correlated with Pi dissociation from the F1-ATPase catalytic site (uni-site catalysis). The F1-ATPase-ADP complex formed under uni-site catalysis conditions can be reactivated in two ways: (i) slow ATP-dependent ADP release from the catalytic site (tau 1/2 20 s) or (ii) binding of Pi in addition to MgADP and the formation of the triple F1-ATPase-MgADP-Pi complex. GTP and GDP are also capable of binding to the catalytic site, however, without changes in the kinetic properties of the F1-ATPase. It is proposed that ATP-dependent dissociation of the F1-ATPase-GDP complex occurs more rapidly, than that of the F1-ATPase-ADP complex.     

Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase

4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with [alpha-32P]azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi [Issartel et al. (1987) J. Biol. Chem. 262, 13538-13544], the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of [32P]Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of [alpha-32P]azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aurovertin fluorescence changes of the mitochondrial F1-ATPase during multi- and uni-site ATP hydrolysis

The aurovertin-F1 complex was used to monitor fluorescence changes of the mitochondrial adenosine triphosphatase during multi- and uni-site ATP hydrolysis. It is known that the fluorescence intensity of the complex is partially quenched by addition of ATP or Mg2+ and enhanced by ADP (Chang, T., and Penefsky, H. S. (1973) J. Biol. Chem. 248, 2746-2754). In the present study low concentrations of ATP (0.03 mM) induced a marked fluorescence quenching which was followed by a fast fluorescence recovery. This recovery could be prevented by EDTA or an ATP regenerating system. The rate of ATP hydrolysis by the aurovertin-F1 complex and the reversal of the ATP-induced fluorescence quenching were determined in these various conditions. ITP hydrolysis also resulted in fluorescence quenching that was followed by a recovery of fluorescence intensity. Under conditions for single site catalysis, fluorescence quenching was observed upon the addition of ATP. This strongly indicates that fluorescence changes in the aurovertin-F1 complex are due to the binding and hydrolysis of ATP at a catalytic site. Therefore the resulting ADP molecule bound at this catalytic site possibly induces the fluorescence recovery observed.     

Total number and differentiation of nucleotide binding sites on mitochondrial F1-ATPase. An approach by photolabeling and equilibrium binding studies

In this study 3'-O-[3-(4-azido-2-nitrophenyl)propionyl]-ADP was used as a photoaffinity analog for nucleotide binding sites on nucleotide-depleted F1-ATPase. Catalytic and binding properties of the labeled enzyme were investigated. The analog behaves as a competitive inhibitor in the dark (Ki = 50 microM). Photoirradiation of F1 in the presence of the analog leads to inactivation depending linearly on the incorporation of label. Complete inactivation is achieved at a stoichiometry of 3 mol/mol F1. The label is distributed between alpha and beta subunits in a ratio of 30%:70%. Although three sites were blocked covalently by photolabeling, three reversible sites of much higher affinity than the labeled sites were preserved. Mild alkaline treatment of photoinactivated enzyme leads to almost complete reactivation which is due to hydrolysis of the 3'-ester bond and release of the ADP moiety from the covalently bound analog. The conclusions drawn are as follows. The total number of sites which can be simultaneously occupied by nucleotides on F1 is six. Adopting the finding [Grubmeyer, C. & Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718-3727] that the high-affinity sites are the catalytic ones which can be covalently labeled by 3'-O-[5-azidonaphthoyl(1)]-ADP [Lübben, M., Lücken, U., Weber, J. & Sch?fer, G. (1984) Eur. J. Biochem. 143, 483-490], it appears likely that azidonitrophenylpropionyl-ADP is a specific photolabel for the lower-affinity sites on nucleotide-depleted F1. This means that both types of sites can be differentiated by specific photoaffinity analogs. The labeled low-affinity sites interact with the catalytic sites, abolishing enzyme turnover, when steadily occupied by ADP kept in place by the covalently linking residue, which by itself has no inhibitory effect on the enzyme.     

Rotary catalysis of the stator ring of F1-ATPase

F₁-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α₃β₃ ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a “dictator” completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F₁-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Structure of bovine mitochondrial F(1)-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis

The crystal structure of a novel aluminium fluoride inhibited form of bovine mitochondrial F(1)-ATPase has been determined at 2 A resolution. In contrast to all previously determined structures of the bovine enzyme, all three catalytic sites are occupied by nucleotide. The subunit that did not bind nucleotide in previous structures binds ADP and sulfate (mimicking phosphate), and adopts a "half-closed" conformation. This structure probably represents the posthydrolysis, pre-product release step on the catalytic pathway. A catalytic scheme for hydrolysis (and synthesis) at physiological rates and a mechanism for the ATP-driven rotation of the gamma subunit are proposed based on the crystal structures of the bovine enzyme.