The stator functions to hold F1 fixed to allow rotation of the rotor within the core of F1. The stator provides a structural support and is not involved directly in the catalytic reaction. The stator is composed of the oligomycin sensitive conferring protein OSCP, subunit 5 , subunit b, subunit d, and F6 and the X-ray structure has been solved for the peripheral stalk of bovine enzyme  .
OSCP was so named because oligomycin prevents proton flow through Fo and inhibits ATP hydrolysis only if F1 is functionally attached and coupled to proton movement. Breaking the stator uncouples ATP hydrolysis from proton translocation because the F1 core can spin instead of the rotor. The primary structure of the stator proteins shows low conservation between bovine and yeast, as evidenced by yeast subunit h and bovine F6, which have just However, the function is conserved because expression of a cDNA encoding bovine F6 complements the deletion mutation of the gene encoding subunit h in yeast .
Subunit f associates with Fo and deletion of the gene in yeast results in phenotypes that are typical for an ATP synthase that is uncoupled  see below. The role of subunit f is still ambiguous as loss of either a component of the peripheral stalk or Fo give similar phenotypes. Yeast with a null mutation in the corresponding gene is unable to grow on a nonfermentable carbon source and loses mitochondrial DNA at a high rate .
As such, subunit f is an essential component of the mitochondrial ATP synthase. A mammalian homologue for subunit i has not been identified though this does not exclude its presence. Subunit i easily dissociates from the ATP synthase complex, which explains why it had not been identified in prior studies. Deletion of the gene encoding subunit i gave different results in the two laboratories.
However, in the second laboratory, deletion of the gene encoding subunit i resulted in complete loss of the ATP synthase with absence of the mitochondrial encoded subunits . These results are not as different as they appear because, a loss of coupling of the ATP synthase will result in the loss of mitochondrial DNA .
As such, these results differ by a matter of degree, which can be caused by different growth conditions or genetic background of the yeast strains.
While the role of subunit i is uncertain, it does appear to provide a role in the efficient coupling of the yeast ATP synthase. The dimer form appears to be involved in formation of cristae of the inner mitochondrial membrane, which may be why the dimer form has not been shown for the bacterial enzyme  . Subunits e, k, and g are selectively associated but only subunits e and g are necessary for dimer formation  .
Subunit k has not been identified in the mammalian ATP synthase although this does not exclude the possibility of a mammalian homolog.
Deletion of the gene encoding subunits e or g has pleiotropic effects including a decrease of cytochrome oxidase activity  . This and an unrelated study  suggest a link, which will be discussed later, between the ATP synthase and the respiratory chain.
In yeast, there are also related molecules, Stf1p and Stf2p. Deletion of the gene encoding IF1 from yeast results in mitochondria that demonstrate uncontrolled hydrolysis of ATP in the presence of a chemical uncoupler, such as 2,4 dinitrophenol .
A number of studies have suggested that inhibitor protein has a role in preventing hydrolysis of ATP under ischemic conditions in mammalian heart      and other metabolic effects . While this is a conceptually attractive hypothesis, the energy of the hydrolysis of ATP by the ATP synthase is not lost — it is coupled to the pumping of protons across the mitochondrial membrane creating a proton gradient.
In contrast, the hydrolysis of ATP by free F1 ATPase is futile and wasteful, and it would be important for the cell to control this hydrolysis.
Stf2p appears to be important in yeast during times of stress or dehydration . Interestingly, over-expression of Stf2p in yeast results in a reduction of reactive oxygen species ROS in response to stress and thus provides a possible target to reduce ROS production in humans.
In this regard, IF1 has been shown to be important for the survival of HeLa cells when they are exposed to high concentrations of ROS, but IF1 is not important for cell survival under normal conditions . The mechanism of inhibition of IF1 was described in terms of the binding change hypothesis, but the explanation will be given in terms of the nomenclature used for the active sites of the ATP synthase. The reason as to why IF1 is unable to inhibit the reverse reaction, ATP synthesis, is still uncertain.
First, haploinsufficiency is the situation where loss of one of the wild type alleles in a diploid organism causes a disease. Second is the situation where a null mutation causes a dominant negative or gain of dysfunctional activity. Both of these cases appear to be applicable in the yeast model system of null mutations in genes encoding subunits of the ATP synthase as well as for the yeast vacuolar ATPase  .
A priori, it would be predicted that deletion of any one of the five genes encoding the subunits of F1 would result in similar, if not identical, phenotypes. However, the situation is much more complicated than predicted . With loss of the rotor, one would predict that the F1Fo subcomplex would allow protons to flow through Fo without Fo being coupled to the rotation of the rotor.
Since Fo subunits are encoded in the mitochondrial DNA, it is impossible to assess defects in coupling when the strain becomes entirely petite. Two different approaches helped resolve this problem. Analysis of mitochondria isolated from these strains indicated that the deletion mutations caused an uncoupling of the ATP synthase presumably due to a leakage of protons into the mitochondria via the uncoupled ATP synthase subcomplex . While this has not been conclusively answered, there is a reasonable explanation that is consistent with what is known about mitochondrion biogenesis .
The biogenesis of the mitochondrion is dependent on the import and processing of proteins that are encoded in the nucleus and made in the cytoplasm. The import of the mitochondrial precursor proteins from the cytoplasm into the mitochondrion is dependent on proton potential across the mitochondrial membrane  .
As such, uncoupling of the mitochondrial membrane will prevent the import of newly synthesized proteins and thus inhibit mitochondrion biogenesis. While yeast is a facultative anaerobe and able to survive without oxidative phosphorylation, all eukaryotic cells require mitochondrion and thus inhibiting the biogenesis is lethal. If the ATP synthase is uncoupled, the cell can only survive if the proton leak is blocked and that is most easily achieved in yeast by eliminating the mitochondrial DNA, which encodes subunits a, c and 8 which comprise the proton pathway.
Thus, there is a correlation between the degree of uncoupling of the ATP synthase and the propensity of the cells to become cytoplasmic petite. Mutations that inhibit activity of the ATP synthase can act either by inhibiting the enzymatic reaction, as in an active site mutation, or by uncoupling proton movement with the reaction cycle.
The coupling of rotation of the rotor with flow of proton and the synthesis of ATP is an intricate mechanism caused by numerous inter-subunit contacts. As such, mutations exist that alter these interactions and affect coupling. A genetic selection scheme, seemingly unrelated to the ATP synthase, selected cells with mutations in genes encoding the ATP synthase and these mutations cause an uncoupling of the ATP synthase.
The yeast, Kluyveromyces lactis, is a facultative anaerobe like Saccharomyces cerevisiae, but differs in that it requires mitochondrial DNA even when grown on a fermentable carbon source. This is in contrast to S. Mutations in K. The yme1 mutation converts S. A number of mutations were isolated from a yme1 S. These mutations also mapped to the genes encoding the ATP synthase with some of them being identical to mgi mutations identified in K.
In a manner analogous to K. These diverse systems converging on common mutations, suggest a shared mechanism linking the ATP synthase with loss or retention of mitochondrial DNA. A variety of biochemical studies on the isolated mitochondria indicated that the mgi mutations caused an uncoupling of the ATP synthase presumably caused by proton leakage through Fo.
X-ray crystallography of some of the mutant structures provided a structural basis for the mechanism of uncoupling rotation of the rotor from ATP hydrolysis or synthesis . The mgi residues are as labeled. The nucleotide binding P-loop domain is colored red and labeled. While the structural and biochemical studies gave an insight into the effect of mgi mutations on the biochemistry of the ATP synthase, it is still not clear how the mutations convert a petite negative strain into a petite positive strain.
There is a correlation between the percentage of petite mutations formed and the severity of the mutation on the coupling of the ATP synthase, but this is distinct from how uncoupling allows the petite negative cells to lose their mitochondrial DNA. The rotation is believed to proceed with minimal resistance but maintains critical interactions to cause catalysis. When disulfide bridges were introduced in the E. The location of the cross-links apparently provides an explanation to resolve this paradox.
The isoforms differ by a single amino acid; the liver isoform has an additional residue, an aspartate, at the C-terminus. The isoforms are formed by alternative splicing, which is conserved and highly regulated    . The heart isoform is expressed in heart, skeletal muscle, and intercostal muscle, diaphragm, all containing tissues with a high and variable energy demand. The liver isoform is expressed in brain, thyroid, spleen, pancreas, kidney, testis and liver.
Of these, all but the brain consume relatively low amount of ATP and have a steady energy demand . The expectation is that these isoforms have biochemical significance and are important for the physiology of the tissues. However, comparison of the ATPase activity of the two isoforms by both bulk ATPase activity measurements and analysis of rotation of single molecules did not demonstrate any significant difference in kinetics of ATPase reaction cycle  .
This suggests that if the isoforms serve as a regulatory mechanism, that the regulation must occur in conjunction with another molecule. An early mutation isolated in E. The resultant bacteria were lacking membrane bound F1Fo and exhibited a reduced ability to accumulate sugars and amino acids but this could be restored by the addition DCCD, a covalent inhibitor of Fo.
DCCD is apparently acting to prevent proton leakage through Fo. As such, this mutation exhibited qualities that are predicted for mutations that uncouple the ATP synthase, which allow free movement of protons. However, the rotation rate was greatly reduced and mutants with large deletions exhibited irregular motion . The patient was a 22 year old that presented with symptoms typical of a patient with a mitochondrial disease. This disease is not lethal and thus consistent with the impairment, but not elimination, of activity of the ATP synthase.
The mutation likely alters the assembly of the intact ATP synthase. In this case, the patient had symptoms that were typical of a severe mitochondrial disease resulting in death of the child at age 3.
The patient had one sister with similar symptoms who died at 15 months. Both patients paradoxically had combined respiratory chain deficiency.
Despite more severe symptoms and outcome, the TyrCys mutation had a much less apparent effect on the function of the ATP synthase. The most pronounced effect of the mutations was observed in both the muscle and liver of the patient, where there was a moderate depletion of the mitochondrial DNA.
A similar finding was observed when the comparable mutation was modeled in yeast . Tyr is within the region where the mgi mutations were clustered in the yeast F1 ATPase suggesting that the mutation might affect the coupling capacity. The increased loss of mitochondrial DNA is also consistent with the mgi phenotype and thereby suggesting that the mutation might decrease the coupling of the ATP synthase.
However, while there was some indication that the corresponding mutation when made in yeast uncoupled the mitochondrial ATP synthase, the effect was not large. One possible explanation for this paradox is that even mild uncoupling of the ATP synthase causes loss of mitochondrial DNA over many generations, acting analogous to a degenerative disease, and the loss in the mitochondrial DNA is ultimately the reason for the disease symptoms.
Both children died in the first weeks of life due to severe encephalopathy with associated extensive cerebral damage. In addition, there was damage to the lungs, kidney, and skeletal muscle. The mutation caused a dramatic decrease in the cellular level of the ATP synthase as measured by activity. Coupling of oxidation and phosphorylation can also be demonstrated using oligomycin or venturicidin, toxic antibiotics that bind to the ATP synthase in mitochondria.
These compounds are potent inhibitors of both ATP synthesis and the transfer of electrons through the chain of carriers to O 2 Fig. Because oligomycin is known not to interact directly with the electron carriers but only with ATP synthase, it follows that electron transfer and ATP synthesis are obligatorily coupled; neither reaction occurs without the other.
There are, however, certain conditions and reagents that uncouple oxidation from phosphorylation. When intact mitochondria are disrupted by treatment with detergent or physical shear, the resulting memfbrane fragments are still capable of catalyzing electron transfer from succinate or NADH to O 2 , but no ATP synthesis is coupled to this respiration.
Certain chemical compounds also cause uncoupling Fig. The chemical uncouplers Table include 2,4-dinitrophenol DNP and a group of compounds related to carbonylcyanide phenylhydrazone Fig. All of these uncouplers are weak acids with hydrophobic properties. Ionophores p. These agents bind to inorganic ions and surround them with hydrophobic moieties; the ionophore-metal ion complexes pass easily through membranes.
We shall see later how the chemiosmotic theory accounts for the action of uncouplers. Figure Two chemical uncouplers of oxidative protons across the inner mitochondrial membrane, phosphorylation.
Both have a dissociable proton dissipating the proton gradient. Both also uncouple and are very hydrophobic. They act by carrying photophosphorylation p. The subscript letter 0 in F 0 denotes that it is the portion of the ATP synthase that confers sensitivity to oligomycin, a potent inhibitor of this enzyme complex and thus of oxidative phosphorylation Table Figure The ATP synthase complex from mitochondria.
F 1 was first extracted from the mitochondrial inner membrane and purified by Efraim Racker and his colleagues in the early s. When F 1 is carefully extracted from inside-out vesicles prepared from the inner mitochondrial membrane Fig.
When a preparation of isolated F 1 is added back to such depleted vesicles, their capacity to couple electron transfer and ATP synthesis is restored. Membrane reconstitution experiments of this kind, pioneered by Racker, opened new doors to research on membrane structure and function.
It is a peripheral membrane protein complex, held to the membrane by its interaction with Fo, an integral membrane protein complex of four different polypeptides that forms a transmembrane channel through which protons can cross the membrane. Highresolution electron micrographs of the isolated FoFI complex show the knoblike F 1 head, a stalk, and a base piece Fo , which normally extends across the inner membrane Fig.
The complete F.
There is very little cross-linking data positioning Helix 1 of subunit-a and thus the position is inferred from the absence, rather than presence, of cross-linking. Also shown is a schematic of the orientation of the transmembrane helices TMH for subunits a, b, and c in the membrane. This rotation will change the subunit in the T conformation into the O conformation, allowing the subunit to release the ATP that has been formed within it.
Both have a dissociable proton dissipating the proton gradient. The proton turbine is powered by the flow of protons down a potential gradient across the mitochondrial membrane created by the electron transport chain during respiration. Of these, all but the brain consume relatively low amount of ATP and have a steady energy demand . In the second cycle, these 50 molecules of CoQH2 will transport protons into the intermembrane space and generate 50 cycling electrons, which will in turn generate 25 new molecules of reduced CoQH2. The absence of a catalytic base and the inability to transition between different catalytic conformations, as evidenced by the absence of open conformation, are major determinants in the catalytic incapacity of the non-catalytic sites.
We describe also the membrane transport systems that move substrates, products, and reducing equivalents between the cytosol and the mitochondrial matrix. Mitochondria are suspended in a buffered medium, and an O 2 electrode is used to monitor O 2 consumption. Certain poisons, called uncouplers , render the inner mitochondrial membrane permeable to protons. The patient had one sister with similar symptoms who died at 15 months. In the first passage through the cycle, protons will be transported across the membrane into the intermembrane space and electrons will be released. Of these, all but the brain consume relatively low amount of ATP and have a steady energy demand .
When physically separated from the membrane by mechanical agitation, F1 is capable only of catalyzing ATP hydrolysis.
F1 forms the knobs that protrude from the matrix side of the inner membrane. When a preparation of isolated F 1 is added back to such depleted vesicles, their capacity to couple electron transfer and ATP synthesis is restored. We describe also the membrane transport systems that move substrates, products, and reducing equivalents between the cytosol and the mitochondrial matrix. Subunit-a was shown to be necessary for the activity, but not for the assembly of the ATP synthase, and subcomplexes of — the enzyme can be formed devoid of subunit-a. The stator is composed of the oligomycin sensitive conferring protein OSCP, subunit 5 , subunit b, subunit d, and F6 and the X-ray structure has been solved for the peripheral stalk of bovine enzyme  .
A composite representation of the ATP synthase is shown on the right with the F1, subunits of the stator, and the cring shown. The import of the mitochondrial precursor proteins from the cytoplasm into the mitochondrion is dependent on proton potential across the mitochondrial membrane  . The c subunits form a donut-shaped ring in the plane of the membrane. The complexes are present in nonequal amounts: for each NADH-CoQ reductase complex, there are about three CoQH2 — cytochrome c reductase complexes and seven cytochrome c oxidase complexes. The half-channels are thought to be comprised of subunit-a, but may also include subunit b and other components of Fo. This mechanism fits nicely with the biochemical and structural properties of the enzyme.