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.     

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.     

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.     

The role of beta-Arg-182, an essential catalytic site residue in Escherichia coli F1-ATPase.

Beta-Arg-182 in Escherichia coli F1-ATPase (beta-Arg-189 in bovine mitochondrial F1) is a residue which lies close to catalytic site bound nucleotide (Abrahams et al. (1994) Nature 370, 621-628). Here we investigated the role of this residue by characterizing two mutants, betaR182Q and betaR182K. Oxidative phosphorylation and steady-state ATPase activity of purified F1 were severely impaired by both mutations. Catalytic site nucleotide-binding parameters were measured using the fluorescence quench of beta-Trp-331 that occurred upon nucleotide binding to purified F1 from betaR182Q/betaY331W and betaR182K/betaY331W double mutants. It was found that (a) beta-Arg-182 interacts with the gamma-phosphate of MgATP, particularly at catalytic sites 1 and 2, (b) beta-Arg-182 has no functional interaction with the beta-phosphate of MgADP or with the magnesium of the magnesium-nucleotide complex in the catalytic sites, and (c) beta-Arg-182 is directly involved in the stabilization of the catalytic transition state. In these features the role of beta-Arg-182 resembles that of another positively charged residue in the catalytic site, the conserved lysine of the Walker A motif, beta-Lys-155. A further role of beta-Arg-182 is suggested, namely involvement in conformational change at the catalytic site beta-alpha subunit interface that is required for multisite catalysis.     

Catalytic sites ofEscherichia coli F1-ATPase

The catalytic site ofEscherichia coli F₁-ATPase is reviewed in terms of structure and function. Structural prediction, biochemical analyses, and mutagenesis experiments suggest that the catalytic site is formed primarily by residues 137–335 of -subunit. Subdomains of the site involved in phosphate-bond cleavage/synthesis and adenine-ring binding are discussed. Ambiguities inherent in steady-state catalytic measurements due to catalytic site cooperativity are discussed, and the advantages of pre-steady-state (unisite) techniques are emphasized. The emergence of a single high-affinity catalytic site occurs as a result of F₁-oligomer assembly. Measurements of unisite catalysis rate and equilibrium constants, and their modulation by varied pH, dimethylsulfoxide, and mutations, are described and conclusions regarding the nature of the high-affinity catalytic site and mechanism of catalysis are presented.     

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.     

Structural and Functional Features of the Escherichia coli F 1 -ATPase

The structural organization and overall dimensions of the Escherichia coli F₁-ATPase in solutionhas been analyzed by synchroton X-ray scattering. Using an independent ab initio approach,the low-resolution shape of the hydrated enzyme was determined at 3.2 nm resolution. Theshape permitted unequivocal identification of the volume occupied by the _{3 3} complex ofthe atomic model of the ECF₁-ATPase. The position of the ^ and subunits were found byinteractive fitting of the solution scattering data and by cross-linking studies. Laser-inducedcovalent incorporation of 2-azido-ATP established a direct relationship between nucleotidebinding affinity and the different interactions between the stalk subunits and with the threecatalytic subunits () of the F₁-ATPase. Mutants of the ECF₁-ATPase with the introductionof Trp-for-Tyr replacement in the catalytic site of the complex made it possible to monitorthe activated state for ATP synthesis (ATP conformation) in which the and subunits arein close proximity to the subunits and the ADP conformation, with the stalk subunits arelinked to the subunit.     

Rotation of Escherichia coli F(1)-ATPase.

By applying the same method used for F(1)-ATPase (TF(1)) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302), we observed ATP-driven rotation of a fluorescent actin filament attached to the gamma subunit in Escherichia coli F(1)-ATPase. The torque value and the direction of the rotation were the same as those observed for TF(1). F(1)-ATPases seem to share common properties of rotation irrespective of the sources.     

Interaction of Escherichia coli F1-ATPase with dicyclohexylcarbodiimide-binding polypeptide

Antibody raised against the N,N'-dicyclohexylcarbodiimide (DCCD)-binding polypeptide of Escherichia coli bound to the cytoplasmic surface of the cell membrane. A weak reaction was seen with everted vesicles of the thermophile PS3. Rat-liver mitochondrial membranes did not react with the antibody. Reaction of the isolated DCCD-binding polypeptide with the antibody was prevented by oxidation of methionine residues or cleavage of the polypeptide with cyanogen bromide. Modification of the arginine residues of the DCCD-binding polypeptide did not affect interaction with the antibody. Purified F1-ATPase of E. coli bound to the isolated DCCD-binding polypeptide as shown by solid-phase radioimmune assay. Binding involved the alpha and/or beta subunits of F1 and the arginine residues of the polar central region of the DCCD-binding polypeptide. Our results are consistent with a looped arrangement of the DCCD-binding polypeptide in the membrane in which the carboxyl- and amino-terminal regions of the molecule are at the periplasmic surface and the polar central region, interacting with F1, is at the cytoplasmic surface of the cell membrane.     

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH.

Using manual rapid-mixing procedures in which small, equal volumes of Escherichia coli F1-ATPase and [gamma-32P]ATP were combined at final concentrations of 2 and 0.2 microM, respectively (i.e., unisite catalysis conditions), it was shown that greater than or equal to 66% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi equal to 0.4 and the rate of dissociation of bound [32P]Pi equal to 3.5 x 10(-3) s-1, similar to previously published values. Azide is known to inhibit cooperative but not unisite catalysis in F1-ATPase [Noumi, T., Maeda, M., & Futai, M. (1987) FEBS Lett. 213, 381-384]. In the presence of 1 mM sodium azide, 99% of the 32P became bound to the enzyme, with the ratio of bound ATP/bound Pi being 0.57. These experiments demonstrated that when conditions are used which minimize cooperative catalysis, most or all of the F1 molecules bind substoichiometric ATP tightly, hydrolyze it with retention of bound ATP and Pi, and release the products slowly. The data justify the validity of previously published rate constants for unisite catalysis. Unisite catalysis in E. coli F1-ATPase was studied at varied pH from 5.5 to 9.5 using buffers devoid of phosphate. Rate constants for ATP binding/release, ATP hydrolysis/resynthesis, Pi release, and ADP binding/release were measured; the Pi binding rate constant was inferred from the delta G for ATP hydrolysis. ATP binding was pH-independent; ATP release accelerated at higher pH. The highest KaATP (4.4 x 10(9) M-1) was seen at physiological pH 7.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of F1-ATPase residue alpha-Arg-376 for catalytic transition state stabilization

The functional role of essential residue alpha-Arg-376 in the catalytic site of F1-ATPase was studied. The mutants alpha R376C, alpha R376Q, and alpha R376K were constructed, and combined with the mutation beta Y331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis. Despite the apparent proximity of alpha-Arg-376 to bound nucleoside di- and triphosphate in published X-ray structures, the mutations had little effect on MgADP or MgATP binding affinities, particularly at the highest affinity catalytic site, site 1. Both Cys and Gln mutants abolished transition state formation, demonstrating that alpha-Arg-376 is normally involved at this step of catalysis. A model of the F1-ATPase catalytic transition state structure is presented and discussed. The Lys mutant, although severely impaired, supported transition state formation, suggesting that an additional essential role for the alpha-Arg-376 guanidinium group exists, likely in alpha/beta conformational signal transmission required for steady-state catalysis. Parallels between alpha-Arg-376 and GAP/G-protein "arginine finger" residues are evident.     

On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase.

1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F1-ATPase. The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F1, with Kd1 = 0.20 microM and Kd2,3 = 5.5 microM. lin-Benzo-ATP was a good substrate for hydrolysis. 2) The mutants investigated were beta Y331F, L, A and E. kcat/KM for ATP hydrolysis in purified F1 was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen. 3) Fluorescence properties of lin-benzo-ADP bound to F1 differed widely, depending on the residue present at position beta 331. Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E. There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E. 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same. 5) F1 from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal. Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.     

Directed mutagenesis of the beta-subunit of F1-ATPase from Escherichia coli

Oligonucleotide-directed mutagenesis was used to generate six mutant strains of Escherichia coli which had the following specific amino acid substitutions in the beta-subunit of F1-ATPase: (i) Lys-155----Gln; (ii) Lys-155----Glu; (iii) Gly-149----Ile; (iv) Gly-154----Ile; (v) Tyr-297----Phe;(vi) Tyr-354----Phe. The effects of each mutation on growth of cells on succinate plates or limiting (3 mM) glucose and on cell membrane ATPase activity and ATP-driven pH gradient formation were studied. The results showed Lys-155 to be essential for catalysis, as has been predicted previously from sequence homology and structural considerations; however, the results appear to contradict the hypothesis that Lys-155 interacts with one of the substrate phosphate groups because the Lys-155----Glu mutation was less detrimental than Lys-155----Gln. Gly-149 and Gly-154 have been predicted to be involved in essential conformational changes in F1-ATPase by virtue of their position in a putative glycine-rich flexible loop structure. The mutation of Gly-154----Ile caused strong impairment of catalysis, but the Gly-149----Ile mutation produced only moderate impairment. The two tyrosine residues chosen for mutation were residues which have previously received much attention due to their being the sites of reaction of the inactivating chemical modification reagents 4-chloro-7-nitrobenzofurazan (Tyr-297) and p-fluorosulfonylbenzoyl-5'-adenosine (Tyr-354). We found that mutation of Tyr-297----Phe caused only minor impairment of catalysis, and mutation of Tyr-354----Phe produced no impairment. Therefore, a direct role for either of these tyrosine residues in catalysis is unlikely.     

Amino acid substitutions in the epsilon-subunit of the F1F0-ATPase of Escherichia coli

A mutant strain of Escherichia coli was isolated in which Gly-48 of the mature epsilon-subunit of the energy-transducing adenosine triphosphatase was replaced by Asp. This amino acid substitution caused inhibition of ATPase activity (about 70%), loss of ATP-dependent proton translocation and lowered oxidative phosphorylation, but did not affect proton translocation through the F0. Purified F1-ATPase from the mutant strain bound to stripped membranes with the same affinity as the normal F1-ATPase. Partial revertant strains were isolated in which Pro-47 of the epsilon-subunit was replaced by Ser or Thr. Pro-47 and Gly-48 are predicted to be residues 2 and 3 in a Type II beta-turn and the Gly-48 to Asp substitution is predicted to cause a change from a Type II to a Type I or III beta-turn. Space-filling models of the beta-turn (residues 46-49) in the normal, mutant and partial revertant epsilon-subunits indicate that the peptide oxygen between Pro-47 and Gly-48 is in a different position to the peptide oxygen between Pro-47 and Asp-48 and that the substitution of Pro-47 by either Ser or Thr restores an oxygen close to the original position. It is suggested that the peptide oxygen between Pro-47 and Gly-48 of the epsilon-subunit is involved either structurally in inter-subunit H-bonding or directly in proton movements through the F1-ATPase.     

NMR study of the phosphate-binding elements of Escherichia coli elongation factor Tu catalytic domain

The phosphoryl-binding elements in the GDP-binding domain of elongation factor Tu were studied by heteronuclear proton observe methods. Five proton resonances were found below 10.5 ppm. Two of these were assigned to the amide groups of Lys 24 and Gly 83. These are conserved residues in each of the consensus sequences. Their uncharacteristic downfield proton shifts are attributed to strong hydrogen bonds to phosphate oxygens as for resonances in N-ras-p21 [Redfield, A. G., & Papastavros, M. Z. (1990) Biochemistry 29, 3509-3514]. The Lys 24 of the EF-Tu G-domain has nearly the same proton and nitrogen shifts as the corresponding Lys 16 in p21. These results suggest that this conserved lysine has a similar structural role in proteins in this class. The tentative Gly 83 resonance has no spectral analogue in p21. A mutant protein with His 84 changed to glycine was fully 15N-labeled and the proton resonance assigned to Gly 83 shifted downfield by 0.3 ppm, thereby supporting the assignment.     

Structure of the nucleotide-binding domain in the beta-subunit of Escherichia coli F1-ATPase

We propose a working model for the tertiary structure of the nucleotide-binding domain of the beta-subunit of E. coli F1-ATPase, derived from secondary structure prediction and from comparison of the amino acid sequence with the sequences of other nucleotide-binding proteins of known three-dimensional structure. The model is consistent with previously published results of specific chemical modification studies and of analyses of mutations in the beta-subunit and its implications for subunit interactions and catalytic mechanism in F1-ATPases are discussed.     

Purification of F1-ATPase with impaired catalytic activity from partial revertants of Escherichia coli uncA mutant strains

It is shown that F1-ATPase preparations having impaired catalytic rates may be purified from partial revertants of uncA mutant strains of Escherichia coli. Recovery of catalytic activity in the partial revertant F1 was accompanied by recovery of alpha in equilibrium beta intersubunit conformational interaction, supporting the hypothesis that such interaction is required for normal catalysis in F1. The specific ATPase activities of the partial revertant F1 preparations were in the range 1-29% of normal, and some of the preparations showed unusual insensitivity to inhibitors. The properties of a new uncA mutant F1 preparation (uncA498) which has approximately half of normal catalytic rate are also briefly described.     

Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli.

beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively. The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes. The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding. These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.     

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.     

Thermodynamic analyses of the catalytic pathway of F1-ATPase from Escherichia coli. Implications regarding the nature of energy coupling by F1-ATPases

Thermodynamic properties of 12 different F1-ATPase enzymes were analyzed in order to gain insights into the catalytic mechanism and the nature of energy coupling to delta mu H+. The enzymes were normal soluble Escherichia coli F1, a group of nine beta-subunit mutant soluble E. coli F1 enzymes (G142S, K155Q, K155E, E181Q, E192Q, M209I, D242N, D242V, R246C), and both soluble and membrane-bound bovine heart mitochondrial F1. Unisite activity was studied by use of Gibbs free energy diagrams, difference energy diagrams, and derivation of linear free energy relationships. This allowed construction of binding energy diagrams for both the unisite ATP hydrolysis and ATP synthesis reaction pathways, which were in agreement. The binding energy diagrams showed that the step of Pi binding is a major energy-requiring step in ATP synthesis, as is the step of ATP release. It is suggested that there are two major catalytic enzyme conformations, and ATP- and an ADP-binding conformation. The effects of the mutations on the rate-limiting steps of multisite as compared to unisite activity were correlated, suggesting a direct link between the rate-limiting steps of the two types of activity. Multisite activity was analyzed by Arrhenius plots and by study of relative promotion from unisite to multisite rate. Changes in binding energy due to mutation were seen to have direct effects on multisite catalysis. From all the data, a model is derived to describe the mechanism of ATP synthesis. ATP hydrolysis, and energy coupling to delta mu H+ in F1F0-ATPases.