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The adenosine triphosphate (ATP) synthase in human mitochondria is a membrane bound assembly of 29 proteins of 18 kinds organized into F1-catalytic, peripheral stalk (PS), and c8-rotor ring modules. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, imported into the mitochondrial matrix, and assembled into the complex with the mitochondrial gene products ATP6 and ATP8. Intermediate vestigial ATPase complexes formed by disruption of nuclear genes for individual subunits provide a description of how the various domains are introduced into the enzyme. From this approach, it is evident that three alternative pathways operate to introduce the PS module (including associated membrane subunits e, f, and g). In one pathway, the PS is built up by addition to the core subunit b of membrane subunits e and g together, followed by membrane subunit f. Then this b-e-g-f complex is bound to the preformed F1-c8 module by subunits OSCP and F6. The final component of the PS, subunit d, is added subsequently to form a key intermediate that accepts the two mitochondrially encoded subunits. In another route to this key intermediate, first e and g together and then f are added to a preformed F1-c8-OSCP-F6-b-d complex. A third route involves the addition of the c8-ring module to the complete F1-PS complex. The key intermediate then accepts the two mitochondrially encoded subunits, stabilized by the addition of subunit j, leading to an ATP synthase complex that is coupled to the proton motive force and capable of making ATP. [ABSTRACT FROM AUTHOR]

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined from Mycobacterium smegmatis which hydrolyzes ATP very poorly. The structure of the α3β3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus. However, its e-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. In E. coli and G. stearothermophilus, the α-helices adopt an "up" state where the α-helices enter the α3β3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase from Caldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The βE-subunits in both enzymes are in the usual "open" conformation but appear to be occupied uniquely by the combination of an adenosine 5'-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1 domain in Mycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen. [ABSTRACT FROM AUTHOR]

The structures and functions of the components of ATP synthases, especially those subunits involved directly in the catalytic formation of ATP, are widely conserved in metazoans, fungi, eubacteria, and plant chloroplasts. On the basis of a map at 32.5-Å resolution determined in situ in the mitochondria of Trypanosoma brucei by electron cryotomography, it has been proposed that the ATP synthase in this species has a noncanonical structure and different catalytic sites in which the catalytically essential arginine finger is provided not by the α-subunit adjacent to the catalytic nucleotide-binding site as in all species investigated to date, but rather by a protein, p18, found only in the euglenozoa. A crystal structure at 3.2-Å resolution of the catalytic domain of the same enzyme demonstrates that this proposal is incorrect. In many respects, the structure is similar to the structures of F1-ATPases determined previously. The α3β3-spherical portion of the catalytic domain in which the three catalytic sites are found, plus the central stalk, are highly conserved, and the arginine finger is provided conventionally by the α-subunits adjacent to each of the three catalytic sites found in the β-subunits. Thus, the enzyme has a conventional catalytic mechanism. The structure differs from previous described structures by the presence of a p18 subunit, identified only in the euglenozoa, associated with the external surface of each of the three α-subunits, thereby elaborating the F1-domain. Subunit p18 is a pentatricopeptide repeat (PPR) protein with three PPRs and appears to have no function in the catalytic mechanism of the enzyme. [ABSTRACT FROM AUTHOR]

The crystal structure has been determined of the F1-catalytic domain of the F-ATPase from Caldalkalibacillus thermarum, which hydrolyzes adenosine triphosphate (ATP) poorly. It is very similar to those of active mitochondrial and bacterial F1-ATPases. In the F-ATPase from Geobacillus stearothermophilus, conformational changes in the ε-subunit are influenced by intracellular ATP concentration and membrane potential. When ATP is plentiful, the ε-subunit assumes a "down" state, with an ATP molecule bound to its two C-terminal a-helices; when ATP is scarce, the a-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the a-helices, devoid of ATP, enter the a3β3-catalytic region. However, in the Escherichia coli enzyme, there is no evidence that such ATP binding to the ε-subunit is mechanistically important for modulating the enzyme's hydrolytic activity. In the structure of the F1-ATPase from C. thermarum, ATP and a magnesium ion are bound to the a-helices in the down state. In a form with a mutated ε-subunit unable to bind ATP, the enzyme remains inactive and the ε-subunit is down. Therefore, neither the ?-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the a3β3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the βE-catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesiumion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis. [ABSTRACT FROM AUTHOR]

The structures of F-ATPases have predominantly been determined from mitochondrial enzymes, and those of the enzymes in eubacteria have been less studied. Paracoccus denitrificans is a member of the α-proteobacteria and is related to the extinct protomitochondrion that became engulfed by the ancestor of eukaryotic cells. The P. denitrificans F-ATPase is an example of a eubacterial F-ATPase that can carry out ATP synthesis only, whereas many others can catalyse both the synthesis and the hydrolysis of ATP. Inhibition of the ATP hydrolytic activity of the P. denitrificans F-ATPase involves the ζ inhibitor protein, an α-helical protein that binds to the catalytic F1 domain of the enzyme. This domain is a complex of three α-subunits and three β-subunits, and one copy of each of the γ-, δ- and ɛ-subunits. Attempts to crystallize the F1-ζ inhibitor complex yielded crystals of a subcomplex of the catalytic domain containing the α- and β-subunits only. Its structure was determined to 2.3 Å resolution and consists of a heterodimer of one α-subunit and one β-subunit. It has no bound nucleotides, and it corresponds to the `open' or `empty' catalytic interface found in other F-ATPases. The main significance of this structure is that it aids in the determination of the structure of the intact membrane-bound F-ATPase, which has been crystallized. [ABSTRACT FROM AUTHOR]

The rotation of the central stalk of F1-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F1-ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F1-ATPase inhibitor protein, IF1, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F1-ATPase inhibited by IF1 represents the catalytic dwell state. Another structure, described here, of bovine F1-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the αEβE-catalytic interface, whereas in F1-ATPase inhibited with IF1, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the βE-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the βE-subunit disrupts interactions in the αEβE-catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the αE-subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°-30°. [ABSTRACT FROM AUTHOR]