Mitochondrial F1-ATPase will bind and cleave ATP but only slowly release ADP after N,N'-dicyclohexylcarbodiimide or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatization

The ATPase from the inner mitochondrial membrane is known to be inhibited by modification of one of the three catalytic subunits with N,N'-dicyclohexylcarbodiimide (DCCD) or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. An experimental approach described in this paper shows that most of the residual ATPase activity observed after the usual DCCD or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole modification is due to the presence of unmodified enzyme, although the large fraction of modified enzyme retains a weak catalytic activity. This weak catalytic activity can be stimulated by methanol or dimethyl sulfoxide. When the modified enzymes are exposed to Mg2+ and [3H]ATP, about equal amounts of [3H]ATP and [3H]ADP appear at catalytic sites. The turnover rate for these enzymes is less than 1/1000 that of the native enzyme when it is calculated from the rate at which the enzyme becomes labeled at the catalytic sites with [3H]ATP and [3H]ADP during steady state hydrolysis. In addition, a higher ATP concentration is required for steady state turnover and, after ATP binding, the principal rate-limiting step is the capacity of the derivatized enzyme to undergo the binding changes necessary for the release of ADP and Pi. When the modified enzymes are not hydrolyzing ATP, they convert to form(s) that show a distinct lag in the replacement of bound nucleotides at catalytic sites. The replacement of bound nucleotides is still promoted by MgATP, even though the enzymes have been converted to sluggish forms. Contrary to a recent suggestion based on the study of the DCCD-modified enzyme (Soong, K.S., and Wang, J.H. (1984) Biochemistry 23, 136-141), our data provide evidence for the existence of catalytic cooperatively between at least two alternating sites in the modified enzyme and are consistent with continued sequential participation of all three sites.     

ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase

The enzyme F1-adenosine triphosphatase (ATPase) is a molecular motor that converts the chemical energy stored in the molecule adenosine triphosphate (ATP) into mechanical rotation of its gamma-subunit. During steady-state catalysis, the three catalytic sites of F1 operate in a cooperative fashion such that at every instant each site is in a different conformation corresponding to a different stage along the catalytic cycle. Notwithstanding a large amount of biochemical and, recently, structural data, we still lack an understanding of how ATP hydrolysis in F1 is coupled to mechanical motion and how the catalytic sites achieve cooperativity during rotatory catalysis. In this publication, we report combined quantum mechanical/molecular mechanical simulations of ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase. Our simulations reveal a dramatic change in the reaction energetics from strongly endothermic in betaTP to approximately equienergetic in betaDP. The simulations identify the responsible protein residues, the arginine finger alphaR373 being the most important one. Similar to our earlier study of betaTP, we find a multicenter proton relay mechanism to be the energetically most favorable hydrolysis pathway. The results elucidate how cooperativity between catalytic sites might be achieved by this remarkable molecular motor.     

The properties of hybrid F1-ATPase enzymes suggest that a cyclical catalytic mechanism involving three catalytic sites occurs

Maximal rates of ATP hydrolysis catalyzed by F1-ATPase enzymes are known to involve strong positive catalytic site cooperativity. There are three potential catalytic nucleotide-binding sites on F1. Two important and unanswered questions are (i) whether all three potential catalytic sites must interact cooperatively to yield maximal rates of ATP hydrolysis and (ii) whether a cyclical three-site mechanism operates as suggested by several authors. We have studied these two questions here by measuring the ATPase activities of hybrid enzymes containing normal beta-, gamma-, delta-, and epsilon-subunits together with different combinations of mutant and normal alpha-subunits. The mutant alpha-subunits were derived from uncA401, uncA447, and uncA453 mutant E. coli F1-ATPase, in which positive cooperativity between catalytic sites is strongly attenuated by defined mis-sense mutations. Our data show that three normal catalytic sites are required to interact in order to achieve maximal ATPase rates and suggest that a cyclical mechanism does operate. Hybrid enzyme containing one-third mutant alpha-subunit and two-thirds normal alpha-subunits had substantial but submaximal activity, showing that cooperativity between three sites in a noncyclical fashion, or between pairs of sites, can achieve effective catalysis.     

New probes of the F1-ATPase catalytic transition state reveal that two of the three catalytic sites can assume a transition state conformation simultaneously

MgADP in combination with fluoroscandium (ScFx) is shown to form a potently inhibitory, tightly bound, noncovalent complex at the catalytic sites of F(1)-ATPase. The F(1).MgADP.ScFx complex mimics a catalytic transition state. Notably, ScFx caused large enhancement of MgADP binding affinity at both catalytic sites 1 and 2, with little effect at site 3. These results indicate that sites 1 and 2 may form a transition state conformation. A new direct optical probe of F(1)-ATPase catalytic transition state conformation is also reported, namely, substantial enhancement of fluorescence emission of residue beta-Trp-148 observed upon binding of MgADP.ScFx or MgIDP. ScFx. Using this fluorescence signal, titrations were performed with MgIDP.ScFx which demonstrated that catalytic sites 1 and 2 can both form a transition state conformation but site 3 cannot. Supporting data were obtained using MgIDP-fluoroaluminate. Current models of the MgATP hydrolysis mechanism uniformly make the assumption that only one catalytic site hydrolyzes MgATP at any one time. The fluorometal analogues demonstrate that two sites have the capability to form the transition state simultaneously.     

Adenine nucleotide-binding sites on beef heart F1-ATPase. Conditions that affect occupancy of catalytic and noncatalytic sites

Beef heart mitochondrial F1 contains a total of six adenine nucleotide-binding sites including at least two different types of sites. Three "exchangeable" sites exchange rapidly during hydrolysis of MgATP, whereas three "nonexchangeable" sites do not (Cross, R. L. and Nalin, C. M. (1982) J. Biol. Chem. 257, 2874-2881). When F1 that has been stored as a suspension in (NH4)2SO4/ATP/EDTA/sucrose/Tris, pH 8.0, is pelleted, rinsed with (NH4)2SO4, dissolved, and desalted, it retains three bound adenine nucleotides. We find that two of these endogenous nucleotides are bound at nonexchangeable sites and one at an exchangeable site. The vacant nonexchangeable site is highly specific for adenine nucleotide and is rapidly filled by ADP upon addition of ADP or during ATP hydrolysis. ADP bound at this site can be removed by reprecipitating the enzyme with (NH4)2SO4. The single nucleotide retained by desalted F1 at an exchangeable site is displaced during hydrolysis of ATP, GTP, or ITP. The binding of PPi at two sites on the enzyme also promotes its dissociation. Neither procedure affects retention of nucleotide at the nonexchangeable sites. These observations, combined with the finding that PPi is much more easily removed from exchangeable sites than ADP, have led to the development of a procedure for preparing F1 with uniform and well-defined nucleotide site occupancy. This involves sequential exposure to MgATP, PPi, and high concentrations of Pi. Unbound ligand is removed between each step. The resulting enzyme, F1[3,0], has three occupied nonexchangeable sites and three vacant exchangeable sites. Evidence that nonexchangeable and exchangeable sites represent noncatalytic and catalytic sites, respectively, suggest that this form of the enzyme will prove useful in numerous applications, including transient kinetic measurements and affinity labeling of active site residues.     

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.     

Role of betaAsn-243 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F(1)-ATPase

In the catalytic mechanism of ATP synthase, phosphate (P(i)) binding and release steps are believed to be correlated to gamma-subunit rotation, and P(i) binding is proposed to be prerequisite for binding ADP in the face of high cellular [ATP]/[ADP] ratios. In x-ray structures, residue betaAsn-243 appears centrally located in the P(i)-binding subdomain of catalytic sites. Here we studied the role of betaAsn-243 in Escherichia coli ATP synthase by mutagenesis to Ala and Asp. Mutation betaN243A caused 30-fold impairment of F(1)-ATPase activity; 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibited this activity less potently than in wild type and P(i) protected from inhibition. ADP-fluoroaluminate was more inhibitory than in wild-type, but ADP-fluoroscandium was less inhibitory. betaN243D F(1)-ATPase activity was impaired by 1300-fold and was not inhibited by ADP-fluoroaluminate or ADP-fluoroscandium. 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole activated betaN243D F(1)-ATPase, and P(i) did not affect activation. We conclude that residue betaAsn-243 is not involved in P(i) binding directly but is necessary for correct organization of the transition state complex through extensive involvement in hydrogen bonding to neighboring residues. It is also probably involved in orientation of the "attacking water" and of an associated second water.     

Purine but not pyrimidine nucleotides support rotation of F(1)-ATPase

The binding change model for the F(1)-ATPase predicts that its rotation is intimately correlated with the changes in the affinities of the three catalytic sites for nucleotides. If so, subtle differences in the nucleotide structure may have pronounced effects on rotation. Here we show by single-molecule imaging that purine nucleotides ATP, GTP, and ITP support rotation but pyrimidine nucleotides UTP and CTP do not, suggesting that the extra ring in purine is indispensable for proper operation of this molecular motor. Although the three purine nucleotides were bound to the enzyme at different rates, all showed similar rotational characteristics: counterclockwise rotation, 120 degrees steps each driven by hydrolysis of one nucleotide molecule, occasional back steps, rotary torque of approximately 40 piconewtons (pN).nm, and mechanical work done in a step of approximately 80 pN.nm. These latter characteristics are likely to be determined by the rotational mechanism built in the protein structure, which purine nucleotides can energize. With ATP and GTP, rotation was observed even when the free energy of hydrolysis was -80 pN.nm/molecule, indicating approximately 100% efficiency. Reconstituted F(o)F(1)-ATPase actively translocated protons by hydrolyzing ATP, GTP, and ITP, but CTP and UTP were not even hydrolyzed. Isolated F(1) very slowly hydrolyzed UTP (but not CTP), suggesting possible uncoupling from rotation.     

F1-ATPase, the C-terminal end of subunit gamma is not required for ATP hydrolysis-driven rotation

ATP hydrolysis by the isolated F(1)-ATPase drives the rotation of the central shaft, subunit gamma, which is located within a hexagon formed by subunits (alphabeta)(3). The C-terminal end of gamma forms an alpha-helix which properly fits into the "hydrophobic bearing" provided by loops of subunits alpha and beta. This "bearing" is expected to be essential for the rotary function. We checked the importance of this contact region by successive C-terminal deletions of 3, 6, 9, 12, 15, and 18 amino acid residues (Escherichia coli F(1)-ATPase). The ATP hydrolysis activity of a load-free ensemble of F(1) with 12 residues deleted decreased to 24% of the control. EF(1) with deletions of 15 or 18 residues was inactive, probably because it failed to assemble. The average torque generated by a single molecule of EF(1) when loaded by a fluorescent actin filament was, however, unaffected by deletions of up to 12 residues, as was their rotational behavior (all samples rotated during 60 +/- 19% of the observation time). Activation energy analysis with the ensemble revealed a moderate decrease from 54 kJ/mol for EF(1) (full-length gamma) to 34 kJ/mol for EF(1)(gamma-12). These observations imply that the intactness of the C terminus of subunit gamma provides structural stability and/or routing during assembly of the enzyme, but that it is not required for the rotary action under load, proper.     

Catalytic properties of chloroplast F1-ATPase modified at catalytic or noncatalytic sites by 2-azido adenine nucleotides

When heat-activated F1-ATPase from chloroplasts was repeatedly exposed to Mg2+ and 2-azido-ATP, followed by separation from medium nucleotides and photolysis, a total of two sites per enzyme, both catalytic and noncatalytic, were labeled. In a coupled assay with pyruvate kinase about half the activity was lost when one site per enzyme was modified. However, increased modification resulted in no further loss of activity. In contrast, methanol-sulfite activation of the enzyme showed a loss of most of the catalytic capacity when one site per enzyme was modified. Predominant labeling of either one catalytic or one noncatalytic site caused a loss of most of the activity in either assay. An indication that the enzyme modified at one site retained some catalytic activity was verified by measurement of the [18O]Pi species formed when [gamma-18O]ATP was hydrolyzed by partially derivatized enzyme. With either catalytic or noncatalytic site modification, the distributions of [18O]Pi species formed showed that the modified enzyme had different catalytic characteristics. An interpretation is that with modification by azido nucleotides at either catalytic or noncatalytic sites, capacity for rapid catalysis is largely lost but the remaining sites retain weak modified catalytic properties.     

Epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, also inhibits F(0)F(1)-ATPase

Since the report by Sternweis and Smith (Sternweis, P. C., and Smith, J. B. (1980) Biochemistry 19, 526-531), the epsilon subunit, an endogenous inhibitor of bacterial F(1)-ATPase, has long been thought not to inhibit activity of the holo-enzyme, F(0)F(1)-ATPase. However, we report here that the epsilon subunit is exerting inhibition in F(0)F(1)-ATPase. We prepared a C-terminal half-truncated epsilon subunit (epsilon(DeltaC)) of the thermophilic Bacillus PS3 F(0)F(1)-ATPase and reconstituted F(1)- and F(0)F(1)-ATPase containing epsilon(DeltaC). Compared with F(1)- and F(0)F(1)-ATPase containing intact epsilon, those containing epsilon(DeltaC) showed uninhibited activity; severalfold higher rate of ATP hydrolysis at low ATP concentration and the start of ATP hydrolysis without an initial lag at high ATP concentration. The F(0)F(1)-ATPase containing epsilon(DeltaC) was capable of ATP-driven H(+) pumping. The time-course of pumping at low ATP concentration was faster than that by the F(0)F(1)-ATPase containing intact epsilon. Thus, the comparison with noninhibitory epsilon(DeltaC) mutant shed light on the inhibitory role of the intact epsilon subunit in F(0)F(1)-ATPase.     

F1-ATPase rotates by an asymmetric, sequential mechanism using all three catalytic subunits

F1-ATPase, the catalytic part of FoF1-ATP synthase, rotates the central gamma subunit within the alpha3beta3 cylinder in 120 degrees steps, each step consuming a single ATP molecule. However, how the catalytic activity of each beta subunit is coordinated with the other two beta subunits to drive rotation remains unknown. Here we show that hybrid F1 containing one or two mutant beta subunits with altered catalytic kinetics rotates in an asymmetric stepwise fashion. Analysis of the rotations reveals that for any given beta subunit, the subunit binds ATP at 0 degrees, cleaves ATP at approximately 200 degrees and carries out a third catalytic event at approximately 320 degrees. This demonstrates the concerted nature of the F1 complex activity, where all three beta subunits participate to drive each 120 degrees rotation of the gamma subunit with a 120 degrees phase difference, a process we describe as a 'sequential three-site mechanism'.     

The metal-independent type IIs restriction enzyme BfiI is a dimer that binds two DNA sites but has only one catalytic centre

BfiI is a novel type IIs restriction endonuclease that, unlike all other restriction enzymes characterised to date, cleaves DNA in the absence of Mg(2+). The amino acid sequence of the N-terminal part of BfiI has some similarities to Nuc of Salmonella typhimurium, an EDTA-resistant nuclease akin to phospholipase D. The dimeric form of Nuc contains a single active site composed of residues from both subunits. To examine the roles of the amino acid residues of BfiI that align with the catalytic residues in Nuc, a set of alanine replacement mutants was generated by site-directed mutagenesis. The mutationally altered forms of BfiI were all catalytically inactive but were still able to bind DNA specifically. The active site of BfiI is thus likely to be similar to that of Nuc. BfiI was also found by gel-filtration to be a dimer in solution. Both gel-shift and pull-down assays indicated that the dimeric form of BfiI binds two copies of its recognition sequence. In reactions on plasmids with either one or two copies of its recognition sequence, BfiI cleaved the DNA with two sites more rapidly than that with one site. Yet, when bound to two copies of its recognition sequence, the BfiI dimer cleaved only one phosphodiester bond at a time. The dimer thus seems to contain two DNA-binding domains but only one active site.     

Structural insight into the cooperativity between catalytic and noncatalytic sites of F1-ATPase

F1-ATPase, the catalytic sector of Fo-F1 ATPases-ATPsynthases, displays an apparent negative cooperativity for ATP hydrolysis at high ATP concentrations which involves noncatalytic and catalytic nucleotide binding sites. The molecular mechanism of such cooperativity is currently unknown. To get further insights, we have investigated the structural consequences of the single mutation of two residues: Q173L in the alpha-subunit and Q170Y in the beta-subunit of the F1-ATPase of the yeast Schizosaccharomyces pombe. These residues are localized in or near the Walker-A motifs of each subunit and their mutation produces an opposite effect on the negative cooperativity. The betaQ170 residue (M167 in beef heart) is located close to the binding site for the phosphate-Mg moiety of the nucleotide. Its replacement by tyrosine converts this site into a close state with increased affinity for the bound nucleotide and leads to an increase of negative cooperativity. In contrast, the alphaQ173L mutation (Q172 in beef heart) abolishes negative cooperativity due to the loss of two H-bonds: one stabilizing the nucleotide bound to the noncatalytic site and the other linking alphaQ173 to the adjacent betaT354, localized at the alpha(DP)-beta(TP) interface. The properties of these mutants suggest that negative cooperativity occurs through interactions between neighbor alpha- and beta-subunits. Indeed, in the beef heart enzyme, (i) the alpha(DP)-beta(TP) interface is stabilized by a vicinal alphaR171-betaD352 salt bridge (ii) betaD352 and betaT354 belong to a short peptidic stretch close to betaY345, the aromatic group of which interacts with the adenine moiety of the nucleotide bound to the catalytic site. We therefore propose that the betaY345-betaT354 stretch (beef heart numbering) constitutes a short link that drives structural modifications from a noncatalytic site to the neighbor catalytic site in which, as a result, the affinity for ADP is modulated.     

Steady-state rate of F1-ATPase turnover during ATP hydrolysis by the single catalytic site

Under the conditions of ATP regeneration and molar excess of nucleotide-depleted F1-ATPase the enzyme catalyses steady-state ATP hydrolysis by the single catalytic site. Values of Km = 10(-8) M and Vm = 0.05 s-1 for the single-site catalysis have been determined. ADP release limits single-site ATP hydrolysis under steady-state conditions. The equilibrium constant for ATP hydrolysis at the F1-ATPase catalytic site is less than or equal to 0.7.     

Complex cooperativity of ATP hydrolysis in the F(1)-ATPase molecular motor

F(1)-ATPase catalyses ATP hydrolysis and converts the cellular chemical energy into mechanical rotation. The hydrolysis reaction in F(1)-ATPase does not follow the widely believed Michaelis-Menten mechanism. Instead, the hydrolysis mechanism behaves in an ATP-dependent manner. We develop a model for enzyme kinetics and hydrolysis cooperativity of F(1)-ATPase which involves the binding-state changes to the coupling catalytic reactions. The quantitative analysis and modeling suggest the existence of complex cooperative hydrolysis between three different catalysis sites of F(1)-ATPase. This complexity may be taken into account to resolve the arguments on the binding change mechanism in F(1)-ATPase.     

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.