The inhibitor protein IF1 from mammalian mitochondria inhibits ATP hydrolysis but not ATP synthesis by the ATP synthase complex.

The hydrolytic activity of the ATP synthase in bovine mitochondria is inhibited by a protein called IF1, but bovine IF1 has no effect on the synthetic activity of the bovine enzyme in mitochondrial vesicles in the presence of a proton motive force. In contrast, it has been suggested based on indirect observations that human IFI inhibits both the hydrolytic and synthetic activities of the human ATP synthase and that the activity of human IF1 is regulated by the phosphorylation of Ser-14 of mature IF1. Here, we have made both human and bovine IF1 which are 81 and 84 amino acids long, respectively, and identical in 71.4% of their amino acids and have investigated their inhibitory effects on the hydrolytic and synthetic activities of ATP synthase in bovine submitochondrial particles. Over a wide range of conditions, including physiological conditions, both human and bovine IF1 are potent inhibitors of ATP hydrolysis, with no effect on ATP synthesis. Also, substitution of Ser-14 with phosphomimetic aspartic and glutamic acids had no effect on inhibitory properties, and Ser-14 is not conserved throughout mammals. Therefore, it is unlikely that the inhibitory activity of mammalian IF1 is regulated by phosphorylation of this residue.

Interface mobility between monomers in dimeric bovine ATP synthase participates in the ultrastructure of inner mitochondrial membranes.

The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle, and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. Disruption of the interface between dimers by mutation affects the morphology of the cristae severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76 and 95°. These particles represent active dimeric ATP synthase. Some angular variations arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer-monomer interaction is mediated mainly by j subunits attached to the surface of wedge-shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other interwedge contacts, molding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae.

TMEM70 and TMEM242 help to assemble the rotor ring of human ATP synthase and interact with assembly factors for complex I.

Human mitochondrial ATP synthase is a molecular machine with a rotary action bound in the inner organellar membranes. Turning of the rotor, driven by a proton motive force, provides energy to make ATP from ADP and phosphate. Among the 29 component proteins of 18 kinds, ATP6 and ATP8 are mitochondrial gene products, and the rest are nuclear gene products that are imported into the organelle. The ATP synthase is assembled from them via intermediate modules representing the main structural elements of the enzyme. One such module is the c8-ring, which provides the membrane sector of the enzyme's rotor, and its assembly is influenced by another transmembrane (TMEM) protein, TMEM70. We have shown that subunit c interacts with TMEM70 and another hitherto unidentified mitochondrial transmembrane protein, TMEM242. Deletion of TMEM242, similar to deletion of TMEM70, affects but does not completely eliminate the assembly of ATP synthase, and to a lesser degree the assembly of respiratory enzyme complexes I, III, and IV. Deletion of TMEM70 and TMEM242 together prevents assembly of ATP synthase and the impact on complex I is enhanced. Removal of TMEM242, but not of TMEM70, also affects the introduction of subunits ATP6, ATP8, j, and k into the enzyme. TMEM70 and TMEM242 interact with the mitochondrial complex I assembly (the MCIA) complex that supports assembly of the membrane arm of complex I. The interactions of TMEM70 and TMEM242 with MCIA could be part of either the assembly of ATP synthase and complex I or the regulation of their levels.

Structure of the ATP synthase from Mycobacterium smegmatis provides targets for treating tuberculosis.

The structure has been determined by electron cryomicroscopy of the adenosine triphosphate (ATP) synthase from Mycobacterium smegmatis This analysis confirms features in a prior description of the structure of the enzyme, but it also describes other highly significant attributes not recognized before that are crucial for understanding the mechanism and regulation of the mycobacterial enzyme. First, we resolved not only the three main states in the catalytic cycle described before but also eight substates that portray structural and mechanistic changes occurring during a 360° catalytic cycle. Second, a mechanism of auto-inhibition of ATP hydrolysis involves not only the engagement of the C-terminal region of an α-subunit in a loop in the γ-subunit, as proposed before, but also a "fail-safe" mechanism involving the b'-subunit in the peripheral stalk that enhances engagement. A third unreported characteristic is that the fused bδ-subunit contains a duplicated domain in its N-terminal region where the two copies of the domain participate in similar modes of attachment of the two of three N-terminal regions of the α-subunits. The auto-inhibitory plus the associated "fail-safe" mechanisms and the modes of attachment of the α-subunits provide targets for development of innovative antitubercular drugs. The structure also provides support for an observation made in the bovine ATP synthase that the transmembrane proton-motive force that provides the energy to drive the rotary mechanism is delivered directly and tangentially to the rotor via a Grotthuss water chain in a polar L-shaped tunnel.

Structure of the dimeric ATP synthase from bovine mitochondria.

The structure of the dimeric ATP synthase from bovine mitochondria determined in three rotational states by electron cryo-microscopy provides evidence that the proton uptake from the mitochondrial matrix via the proton inlet half channel proceeds via a Grotthus mechanism, and a similar mechanism may operate in the exit half channel. The structure has given information about the architecture and mechanical constitution and properties of the peripheral stalk, part of the membrane extrinsic region of the stator, and how the action of the peripheral stalk damps the side-to-side rocking motions that occur in the enzyme complex during the catalytic cycle. It also describes wedge structures in the membrane domains of each monomer, where the skeleton of each wedge is provided by three α-helices in the membrane domains of the b-subunit to which the supernumerary subunits e, f, and g and the membrane domain of subunit A6L are bound. Protein voids in the wedge are filled by three specifically bound cardiolipin molecules and two other phospholipids. The external surfaces of the wedges link the monomeric complexes together into the dimeric structures and provide a pivot to allow the monomer-monomer interfaces to change during catalysis and to accommodate other changes not related directly to catalysis in the monomer-monomer interface that occur in mitochondrial cristae. The structure of the bovine dimer also demonstrates that the structures of dimeric ATP synthases in a tetrameric porcine enzyme have been seriously misinterpreted in the membrane domains.

Assembly of the peripheral stalk of ATP synthase in human mitochondria.

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.

Correlates of high phosphatidylethanol (PEth) levels and their concordance with self-reported heavy alcohol consumption among men who have sex with men who binge drink alcohol.

BACKGROUND: Heavy alcohol use, including binge drinking, is associated with high morbidity and mortality among men who have sex with men (MSM). Self-reported alcohol measures may lead to inaccurate estimates due to recall and social desirability biases. Objective alcohol biomarkers like phosphatidylethanol (PEth) can be used to corroborate self-report and could help to inform treatment approaches and research strategies for alcohol using MSM. METHODS: From 2015 to 2020, alcohol using MSM ≥18 years were enrolled in a randomized controlled trial evaluating the efficacy of naltrexone in reducing binge drinking. Using this trial's baseline data, we applied multivariable logistic regression to identify the correlates of high PEth levels (i.e., ≥87 ng/ml) and concordance between PEth levels and self-reported heavy drinking. RESULTS: Of 118 MSM, 64% had PEth levels ≥87 ng/ml and 72% had PEth levels that were concordant with self-reported heavy alcohol use. Factors significantly associated in separate models with elevated PEth levels were income ≥$60,000 (adjusted odds ratio [aOR] = 4.09; 95% CI = 1.13 to 14.82), being employed (aOR = 4.04; 95% CI = 1.45 to 11.32), episodic cannabis use (aOR = 4.63; 95% CI = 1.27 to 16.92), and any alcohol/substance use prior to or during anal intercourse (aOR = 2.52; 95% CI = 1.08 to 5.90). Living with HIV was associated with significantly lower odds of elevated PEth levels (aOR = 0.23; 95% CI = 0.09 to 0.61). Factors associated with significantly higher concordance between PEth levels and self-reported heavy alcohol use included at least weekly use of poppers (aOR = 6.41; 95% CI = 1.27 to 32.28) and polysubstance use (aOR = 2.53; 95% CI = 1.02 to 6.27). Living with HIV was associated with lower odds of concordance (aOR = 0.36; 95% CI = 0.14 to 0.97). CONCLUSIONS: PEth may enhance the detection of heavy drinking among MSM, including the identification of subpopulations that may benefit from targeted alcohol reduction interventions. However, PEth values for MSM living with HIV showed modest concordance with self-reported alcohol use and may need to be supplemented with additional biomarkers or evaluated against a different cutoff.

Persistence of the permeability transition pore in human mitochondria devoid of an assembled ATP synthase.

The opening of the permeability transition pore, a nonspecific channel in inner mitochondrial membranes, is triggered by an elevated total concentration of calcium ions in the mitochondrial matrix, leading to disruption of the inner membrane and necrotic cell death. Cyclosporin A inhibits pore opening by binding to cyclophilin D, which interacts with the pore. It has been proposed that the pore is associated with the ATP synthase complex. Previously, we confirmed an earlier observation that the pore survives in cells lacking membrane subunits ATP6 and ATP8 of ATP synthase, and in other cells lacking the enzyme's c8 rotor ring or, separately, its peripheral stalk subunits b and oligomycin sensitive conferral protein. Here, we investigated whether the pore is associated with the remaining membrane subunits of the enzyme. Individual deletion of subunits e, f, g, and 6.8-kDa proteolipid disrupts dimerization of the complex, and deletion of DAPIT (diabetes-associated protein in insulin sensitive tissue) possibly influences oligomerization of dimers, but removal of each subunit had no effect on the pore. Also, we removed together the enzyme's membrane bound c8 ring and the δ-subunit from the catalytic domain. The resulting cells assemble only a subcomplex derived from the peripheral stalk and membrane-associated proteins. Despite diminished levels of respiratory complexes, these cells generate a membrane potential to support uptake of calcium into the mitochondria, leading to pore opening, and retention of its characteristic properties. It is most unlikely that the ATP synthase, dimer or monomer, or any component, provides the permeability transition pore.

The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs.

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 α3ß3-component of the catalytic domain is similar to those in active F1-ATPases in Escherichia coli and Geobacillus stearothermophilus However, its ε-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 α3ß3-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 ßE-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.

Extrinsic conditions influence the self-association and structure of IF1, the regulatory protein of mitochondrial ATP synthase.

The endogenous inhibitor of ATP synthase in mitochondria, called IF1, conserves cellular energy when the proton-motive force collapses by inhibiting ATP hydrolysis. Around neutrality, the 84-amino-acid bovine IF1 is thought to self-assemble into active dimers and, under alkaline conditions, into inactive tetramers and higher oligomers. Dimerization is mediated by formation of an antiparallel α-helical coiled-coil involving residues 44-84. The inhibitory region of each monomer from residues 1-46 is largely α-helical in crystals, but disordered in solution. The formation of the inhibited enzyme complex requires the hydrolysis of two ATP molecules, and in the complex the disordered region from residues 8-13 is extended and is followed by an α-helix from residues 14-18 and a longer α-helix from residue 21, which continues unbroken into the coiled-coil region. From residues 21-46, the long α-helix binds to other α-helices in the C-terminal region of predominantly one of the ß-subunits in the most closed of the three catalytic interfaces. The definition of the factors that influence the self-association of IF1 is a key to understanding the regulation of its inhibitory properties. Therefore, we investigated the influence of pH and salt-types on the self-association of bovine IF1 and the folding of its unfolded region. We identified the equilibrium between dimers and tetramers as a potential central factor in the in vivo modulation of the inhibitory activity and suggest that the intrinsically disordered region makes its inhibitory potency exquisitely sensitive and responsive to physiological changes that influence the capability of mitochondria to make ATP.

ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites.

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 α3ß3-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 ß-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.

Assembly of the membrane domain of ATP synthase in human mitochondria.

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F1-c8 complex inhibited by the ATPase inhibitor protein IF1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast Fo membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase.

The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O, had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.

Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase.

The permeability transition in human mitochondria refers to the opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane, and ATP synthesis, followed by cell death. Recent proposals suggest that the pore is associated with the ATP synthase complex and specifically with the ring of c-subunits that constitute the membrane domain of the enzyme's rotor. The c-subunit is produced from three nuclear genes, ATP5G1, ATP5G2, and ATP5G3, encoding identical copies of the mature protein with different mitochondrial-targeting sequences that are removed during their import into the organelle. To investigate the involvement of the c-subunit in the PTP, we generated a clonal cell, HAP1-A12, from near-haploid human cells, in which ATP5G1, ATP5G2, and ATP5G3 were disrupted. The HAP1-A12 cells are incapable of producing the c-subunit, but they preserve the characteristic properties of the PTP. Therefore, the c-subunit does not provide the PTP. The mitochondria in HAP1-A12 cells assemble a vestigial ATP synthase, with intact F1-catalytic and peripheral stalk domains and the supernumerary subunits e, f, and g, but lacking membrane subunits ATP6 and ATP8. The same vestigial complex plus associated c-subunits was characterized from human 143B ρ0 cells, which cannot make the subunits ATP6 and ATP8, but retain the PTP. Therefore, none of the membrane subunits of the ATP synthase that are involved directly in transmembrane proton translocation is involved in forming the PTP.

Structure of the mitochondrial ATP synthase from Pichia angusta determined by electron cryo-microscopy.

The structure of the intact monomeric ATP synthase from the fungus, Pichia angusta, has been solved by electron cryo-microscopy. The structure provides insights into the mechanical coupling of the transmembrane proton motive force across mitochondrial membranes in the synthesis of ATP. This mechanism requires a strong and integral stator, consisting of the catalytic α3ß3-domain, peripheral stalk, and, in the membrane domain, subunit a and associated supernumerary subunits, kept in contact with the rotor turning at speeds up to 350 Hz. The stator's integrity is ensured by robust attachment of both the oligomycin sensitivity conferral protein (OSCP) to the catalytic domain and the membrane domain of subunit b to subunit a. The ATP8 subunit provides an additional brace between the peripheral stalk and subunit a. At the junction between the OSCP and the apparently stiff, elongated α-helical b-subunit and associated d- and h-subunits, an elbow or joint allows the stator to bend to accommodate lateral movements during the activity of the catalytic domain. The stator may also apply lateral force to help keep the static a-subunit and rotating c10-ring together. The interface between the c10-ring and the a-subunit contains the transmembrane pathway for protons, and their passage across the membrane generates the turning of the rotor. The pathway has two half-channels containing conserved polar residues provided by a bundle of four α-helices inclined at ∼30° to the plane of the membrane, similar to those described in other species. The structure provides more insights into the workings of this amazing machine.

Cardiolipin binds selectively but transiently to conserved lysine residues in the rotor of metazoan ATP synthases.

The anionic lipid cardiolipin is an essential component of active ATP synthases. In metazoans, their rotors contain a ring of eight c-subunits consisting of inner and outer circles of N- and C-terminal α-helices, respectively. The beginning of the C-terminal α-helix contains a strictly conserved and fully trimethylated lysine residue in the lipid head-group region of the membrane. Larger rings of known structure, from c9-c15 in eubacteria and chloroplasts, conserve either a lysine or an arginine residue in the equivalent position. In computer simulations of hydrated membranes containing trimethylated or unmethylated bovine c8-rings and bacterial c10- or c11-rings, the head-groups of cardiolipin molecules became associated selectively with these modified and unmodified lysine residues and with adjacent polar amino acids and with a second conserved lysine on the opposite side of the membrane, whereas phosphatidyl lipids were attracted little to these sites. However, the residence times of cardiolipin molecules with the ring were brief and sufficient for the rotor to turn only a fraction of a degree in the active enzyme. With the demethylated c8-ring and with c10- and c11-rings, the density of bound cardiolipin molecules at this site increased, but residence times were not changed greatly. These highly specific but brief interactions with the rotating c-ring are consistent with functional roles for cardiolipin in stabilizing and lubricating the rotor, and, by interacting with the enzyme at the inlet and exit of the transmembrane proton channel, in participation in proton translocation through the membrane domain of the enzyme.

Regulation of the thermoalkaliphilic F1-ATPase from Caldalkalibacillus thermarum.

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 α-helices; when ATP is scarce, the α-helices are proposed to inhibit ATP hydrolysis by assuming an "up" state, where the α-helices, devoid of ATP, enter the α3ß3-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 α-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 γ-subunit nor the regulatory ATP bound to the ε-subunit is involved in the inhibitory mechanism of this particular enzyme. The structure of the α3ß3-catalytic domain is likewise closely similar to those of active F1-ATPases. However, although the ßE-catalytic site is in the usual "open" conformation, it is occupied by the unique combination of an ADP molecule with no magnesium ion and a phosphate ion. These bound hydrolytic products are likely to be the basis of inhibition of ATP hydrolysis.

Structure of ATP synthase from Paracoccus denitrificans determined by X-ray crystallography at 4.0 Å resolution.

The structure of the intact ATP synthase from the α-proteobacterium Paracoccus denitrificans, inhibited by its natural regulatory ζ-protein, has been solved by X-ray crystallography at 4.0 Å resolution. The ζ-protein is bound via its N-terminal α-helix in a catalytic interface in the F1 domain. The bacterial F1 domain is attached to the membrane domain by peripheral and central stalks. The δ-subunit component of the peripheral stalk binds to the N-terminal regions of two α-subunits. The stalk extends via two parallel long α-helices, one in each of the related b and b' subunits, down a noncatalytic interface of the F1 domain and interacts in an unspecified way with the a-subunit in the membrane domain. The a-subunit lies close to a ring of 12 c-subunits attached to the central stalk in the F1 domain, and, together, the central stalk and c-ring form the enzyme's rotor. Rotation is driven by the transmembrane proton-motive force, by a mechanism where protons pass through the interface between the a-subunit and c-ring via two half-channels in the a-subunit. These half-channels are probably located in a bundle of four α-helices in the a-subunit that are tilted at ∼30° to the plane of the membrane. Conserved polar residues in the two α-helices closest to the c-ring probably line the proton inlet path to an essential carboxyl group in the c-subunit in the proton uptake site and a proton exit path from the proton release site. The structure has provided deep insights into the workings of this extraordinary molecular machine.

How release of phosphate from mammalian F1-ATPase generates a rotary substep.

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 α(E)ß(E)-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 ß(E)-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 ß(E)-subunit disrupts interactions in the α(E)ß(E-)catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the γ-subunit and the C-terminal domain of the α(E)-subunit. To restore most of these interactions, and to make compensatory new ones, the γ-subunit rotates through 25°-30°.