Where does the h+ come from that makes atp synthase work?

The ATP Synthase

The ATP synthase (or F1F0 ATPase and also referred to as complex V) uses the free energy of an electrochemical gradient of protons (or sodium ions) generated by the respiratory chain to synthesize ATP. The ATP synthases comprise a very large group of highly conserved enzymes that are found in the bacterial cytoplasmic membranes, the thylakoid membranes of chloroplasts, and the inner membranes of mitochondria. Most members of the group use H+ as the coupling ion (the Propionigenium modestum enzyme is an example of the few ATP synthases that can use Na+ as the physiological coupling ion).

Secondary Energy Transducers

ATP synthetases (⇒) are the most important universal secondary energy transducers integrated into plasma membranes. Bacterial ATP synthetases and those of mitochondria and chloroplasts are of the F0F1-type (⇒). Functionally, the ATP synthases are reversible ion-transporting ATPases driving the reaction ADP + Pi → ATP + H2O by dissipation of a ΔμH+ or ΔμNa+ across the membrane. F1 extends to the cytosol and bears the substrate-binding sites; F0 is the membrane integral ion-conducting subcomplex. Archaea contain A0A1-type ATP synthetases that structurally resemble the eukaryotic vacuolar V-type ATPases (⇒) but, in contrast to the latter, function as ATP synthetases driven by an ion-motive ΔμH+ or ΔμNa+ according to the rotational reaction mechanism of F0F1. The peripheral substrate-binding A1 moiety has been identified from many Halobacteria, Sulfolobales, and Methanobacteria. The only integrated A0A1-complex was isolated from the methanogen Methanococcus jannashii.

Proton Pumping and ATP Synthesis

Complex I, complex III, and complex IV pump several protons into the intermembrane space for every pair of electrons that they transport to O2. A sufficient number of protons are pumped to maintain a 10:1 concentration gradient (one pH unit) between the intermembrane space and the matrix.

ATP synthase complex (FoF1-ATP synthase). This complex allows protons to flow back into the matrix and uses the free energy change from this process to synthesize ATP from ADP and inorganic phosphate (Pi). It is located in knob-shaped structures embedded in the cristae (invaginations of the inner mitochondrial membrane) and extending into matrix.

The Fo protein (the “o” in Fo refers to its sensitivity to oligomycin, a poison that blocks the flow of protons) extends through the inner mitochondrial membrane and serves as the proton channel between the intermembrane space and the matrix.

The ATP synthase (F1-ATPase) is attached to the Fo protein on the inside of the matrix. ATP synthase uses the protons flowing into the matrix to bind ADP and Pi and release ATP. The F1-ATPase is named by the reverse reaction it catalyzes when it is isolated from mitochondria and thus uncoupled from the proton gradient.

KEY POINTS ABOUT THE ELECTRON TRANSPORT CHAIN

The electron transport chain is located in the mitochondrial inner membrane and contains several different kinds of electron carriers: flavin mononucleotide, iron-sulfur proteins, coenzyme Q, heme-containing cytochromes, and copper ions.

Three large multiprotein complexes serve as proton pumps by harnessing the energy from electron flow through the ETC to oxygen; in turn, the chemiosmotic energy in the proton gradient that is created by the pumps is coupled to the synthesis of ATP by the ATP synthase complex.

ATP regulates its own synthesis and the flow of electrons through respiratory control; if ATP synthesis slows down, electron transport slows down and vice versa.

Cytosolic NADH cannot pass through the mitochondrial membrane, so it shuttles its electrons through the glycerol phosphate shuttle and the malate-aspartate shuttle.

ATP and ADP are transported in exchange for each other by the ATP/ADP translocase.

C F0F1-ATPase

The F0F1-ATPase has a fundamental role in the maintenance of pH homeostasis. Functionally, this multisubunit enzyme is organized into two distinct, but physically linked domains; the catalytic portion (F1) is cytoplasmic and incorporates the α, B, γ, δ, and ε subunits, while the integral membrane domain (F0), including the a, b, and c subunits, functions as a membranous channel for proton translocation (Sebald et al., 1982). The function of the cytoplasmic domain is to catalyze the synthesis of ATP when protons move from the outside of cells into the cytoplasm, through the membrane-bound domain, or to hydrolyze ATP when protons are moved out of the cell.

The role of the F0F1-ATPase in organisms capable of oxidative phosphorylation is to synthesize ATP aerobically as a result of protons passing into the cell, and to generate a proton motive force (PMF) anaerobically, via the expulsion of protons. As a consequence of the latter mechanism, it is thought that the F0F1-ATPase can increase the intracellular pH in situations where it becomes acidified. For many bacteria, activity data demonstrate that a lowering of the intracellular pH is accompanied by induction of the F0F1-ATPase (Koebmann et al., 2000; Kuhnert and Quivey, 2003; Kullen and Klaenhammer, 1999), while in a number of bacterial species, including Lactobacillus acidophilus (Kullen and Klaenhammer, 1999), S. mutans (Kuhnert et al., 2004), and S. pneumoniae (Martin-Galiano et al., 2001) transcription of the F0F1 operon is induced by exposure to low pH. The inherent acid tolerance of various bacteria is related both to the levels of F0F1-ATPase, as well as to the pH optima of the enzymes. It is clear that naturally acid tolerant organisms possess F0F1-ATPases with lower pH optima (Sturr and Marquis, 1992; Martin-Galiano et al., 2001).

Analysis of the role of the F0F1-ATPase in acid tolerance is limited by the fact that the system is essential for life in many bacterial species (Koebmann et al., 2000). We have been unable to create clean deletion of the genes encoding the system in L. monocytogenes (unpubished data). However, 2-dimensional gel electrophoresis studies have shown that the listerial F0F1-ATPase subunit is induced as a consequence of exposure to mild acid treatment (Phan-Thanh and Mahouin, 1999). Administration of DCCD, an ATPase inhibitor demonstrated that the ATPase is involved in acid tolerance and the ATR (Datta and Benjamin, 1997). In addition, we have created a partial insertion mutant in the F0F1-ATPase in L. monocytogenes that is not compromised in fitness. This mutant demonstrated that the F0F1-ATPase of L. monocytogenes is involved in the induction of the ATR in this bacterium (Cotter et al., 2000).

9.13 The ATP Synthase Inhibitor Protein IF1

Further reading: Campanella et al. (2009), Faccenda and Campanella (2012)

The ATP synthase is freely reversible, and its direction depends on the thermodynamic balance between Δp and the matrix ΔGp. Damage to the electron transport chain, increased proton leakage, or severe hypoxia can lower Δp such that the ATP synthase reverses in the cell and starts to hydrolyse cytoplasmic ATP generated by glycolysis. Experimentally, this reversal can be detected as a decrease in Δψm upon addition of the ATP synthase inhibitor oligomycin (see Figure 12.2). Under these conditions, glycolysis is called on to service not only the entire ATP demand of the cell but also the synthase reversal. One result of this is that cells may deplete their cytoplasmic ATP to the extent that glycolysis and fatty acid oxidation, both of which require ATP, cannot proceed and the cell dies. This condition is also approached in many published experiments in which protonophores are added to cells, in which case mitochondrial ATP hydrolysis can be extremely rapid, being no longer limited by the low inner membrane proton permeability. Although the ATP depletion can be alleviated in vitro by the addition of oligomycin, a more subtle physiological mechanism exists in many cells, mediated by the 10-kDa inhibitor protein (IF1; Section 7.6).

IF1 can bind to the F1-ATP synthase under conditions of acidic matrix pH, partially inhibiting its catalytic activity. At the molecular level, studies with Escherichia coli ATP synthase suggest that IF1 acts as a ‘ratchet’ preventing reversal of the enzyme (Section 7.6). Because an acidic matrix is normally only seen under hypoxic conditions, when the electron transport pathway is inhibited, or in the presence of a protonophore, this essentially means that IF1 can inhibit ATP synthase reversal but is without effect on ATP synthesis (when the matrix is alkaline). The inhibition is not complete but depends on the ratio of IF1 to the ATP synthase complex and may play an important role in limiting ATP depletion in hypoxia (Figure 9.18). Neurons generally possess higher ratios of IF1 to F1 than astrocytes, with the result that electron chain inhibition causes a more profound depolarisation of the former while slowing cytoplasmic ATP depletion.

Disease Association

Circulating ASA of the IgM class have been first described in seven patients with nephrotic syndrome; some of these patients also presented a combination of ASA and anti-actin (three cases) and anti-nuclear (three cases) autoantibodies. Therefore, ASA represents the most reproducible marker of autoimmunity in these patients but a clear clinical and pathogenetic relationship with the disease cannot be defined. Overall, patients with circulating ASA could not be readily differentiated from other patients with primary NS with respect to clinical and pathologic characteristics. As detailed in Table 67.1, all patients of our series had an early onset of proteinuria (between 1 and 8 years) and had strict resistance or dependence to steroids; 6 had been treated with cyclosporine and only 4 were sensitive. Sensitivity to cyclosporine was associated with good long-term outcome in all cases. High plasma levels of IgM was a constant finding in all patients and in one reached the relevant limit of 1.000 mg%, in the absence of monoclonal band. The histological background was focal segmental glomerulosclerosis (FSGS) in 3 children while 2 presented diffuse mesangial proliferation with IgM deposition and 1 had an unspecific pattern. Three patients developed during the follow-up positiveness for antinuclear antibodies (ANA).

TABLE 67.1. Clinical Features of 7 Patients with Nephrotic Syndrome who Presented Anti-podocyte Antibodies

Abbreviations: IF, immunofluorescence; ESRF, end stage renal failure; FSGS, focal segmental glomerulosclerosis; TX, renal transplant.

Abstract

F1-ATPase (F1) is the water-soluble portion of ATP synthase and a rotary molecular motor in which the rotary shaft, the γ subunit, rotates with 120° steps against the α3β3 stator ring upon ATP hydrolysis. While the crystal structures of F1 exhibit essentially one stable conformational state of F1, single-molecule rotation studies revealed that there are two stable conformations of F1 in each 120° step: the ATP-binding dwell state and the catalytic dwell state. This chapter provides the experimental procedure for the determination of which catalytic state the crystal structures of F1 represent, by the use of a cross-linking technique in the single-molecule rotation assay. The β and γ subunits are cross-linked through a disulfide bond between two cysteine residues genetically introduced at the positions where the β and γ subunits have a specific contact in the crystal structures of the ADP-bound form. In the single-molecule rotation assay, the cross-linked F1 shows a pause at the catalytic dwell state that corresponds to the dwell angle in one turn where the β subunit undergoes ATP hydrolysis. Thus, this experiment reveals not only that the crystal structure represents the catalytic dwell state but also that the ADP-bound β subunit represents the catalytically active state. A protocol for inhibition of the wild-type F1 with chemical inhibitors such as adenosine-5′-(β,γ-imino)-triphosphate (AMP-PNP) or/and N3− under crystallization conditions is also provided.

B Rotary Catalysis of Rotorless F1-ATPase

F1-ATPase, a water-soluble portion of ATP synthase, is a rotary motor protein. It is composed of a hexameric headpiece assembled from alternating α and β subunits, denoted (α3β3). The nucleotide-binding sites lie in the interface clefts between the subunits, but they are not all identical. Three catalytic sites are located mostly in the β subunits and alternate with three noncatalytic sites that lie mostly in the α subunits. The (α3β3) hexamer surrounds an eccentric coiled-coil “shaft” (γ subunit), the rotation of which is driven by sequential ATP hydrolysis at the three catalytic sites. Some biochemical studies suggested that the α3β3 ring alone does not possess intrinsic cooperativity and that the γ subunit mediates the interplay among the β subunits (Kaibara et al., 1996; Garcıa and Capaldi, 1998). In contrast, recent studies evidenced that F1 retains catalytic power to rotate γ unidirectionally, even when most interaction sites between β and γ are abolished (Aloise et al., 1991; Furuike et al., 2008).

Recently, a detailed real time analysis was performed to clarify this issue (Uchihashi et al., 2011). The α3β3 subcomplex was covalently immobilized on a mica surface and imaged using HS-AFM with a frame capture time of 80 ms (Fig. 15). In the absence of nucleotides, α3β3 exhibited a pseudo sixfold symmetric ring in which three alternately arranged subunits were elevated relative to the other three (Fig. 15A, left). The simulated (Fig. 15A, right) and observed AFM images confirmed that the N-terminal side was selectively attached to the mica. The addition of the nonhydrolyzable ATP analog AMPPNP rendered the ring triangular and obscured the central hole (Fig. 15B, left). Although the three α subunits with lower protrusions retained the same conformation as those observed under nucleotide-free conditions, two of the three β subunits retracted toward the center and simultaneously lowered their protrusions. Consequently, the ring showed a single high protrusion. The simulated image (Fig. 15B, right) also showed an asymmetric ring similar to that of the observed image. This indicates that only two β subunits can assume the closed conformation, even in saturating AMPPNP conditions.

Next, the conformational changes of β subunits were observed in the presence of ATP at an imaging speed of 12.5 frames/s. After the addition of ATP, β subunits exhibited distinct conformational dynamics; each β subunit underwent a conformational transition between the outwardly extended high state (open) and the retracted low state (closed) (Fig. 15C). The most notable features were that only a single β subunit assumed the open state, as in the presence of AMPPNP, and that when the open-to-closed transition occurred at one β subunit, the opposite closed-to-open transition occurred simultaneously at its counterclockwise neighboring β subunit in most cases. Thus, the high and outwardly extended conformation propagates in the counterclockwise direction. These results proved that the stator α3β3 ring alone possesses high cooperativity for sequential power stroking among the three catalytic β subunits. However, the ATP-binding rate and the efficiency of unidirectionality of the α3β3 subcomplex were distinctly lower than those of F1. Thus, the interaction with γ is dispensable but important for rapid and precise rotary catalysis.

4.6 ATP Synthase Reversal

The ATP synthase is reversible and is only constrained to run in the direction of net ATP synthesis by the continual regeneration of Δp and the use of ATP by the cell. If the respiratory chain is inhibited and ATP is supplied to the mitochondrion, or if sufficient Ca2+ is added to depress Δp below that for thermodynamic equilibrium with the ATP synthase reaction, the enzyme complex functions as an ATPase, generating a Δp comparable to that produced by the respiratory chain. The proton circuit generated by ATP hydrolysis must be completed by a means of proton re-entry into the matrix. Proton translocators therefore accelerate the rate of ATP hydrolysis, just as they accelerate the rate of respiration; this is the ‘uncoupler-stimulated ATPase activity,’ which is of particular importance in the cellular context, when mitochondrial dysfunction can cause ATP synthase reversal and drain glycolytically generated ATP. In some circumstances, an ‘inhibitor protein,’ IF1, can limit this reversal (Sections 7.7 and 9.13Section 7.7Section 9.13).

The classic means of discriminating whether a mitochondrial energy-dependent process is driven directly by Δp or indirectly via ATP is to investigate the sensitivity of the process to the ATP synthase inhibitor oligomycin. A Δp-driven event would be insensitive to oligomycin when the potential was generated by respiration, but it would be sensitive when Δp was produced by ATP hydrolysis. The converse would be true of an ATP-dependent event. If Δp or Δψ is being monitored, mitochondria (isolated or in situ), which are net generators of ATP, will hyperpolarise (i.e., Δp will increase) on addition of oligomycin, whereas those whose Δp is supported by ATP hydrolysis will depolarise. This ‘null-point’ assay is a simple way of monitoring mitochondrial function within cells (Figure 12.2).

Under physiological conditions, the mitochondrial ATP synthase will not normally be called upon to act as a proton-translocating ATPase, except possibly during periods of anoxia when glycolytic ATP could be utilised to maintain the mitochondrial Δp. However, some bacteria, such as Streptococcus faecalis when grown on glucose, lack a functional respiratory chain and rely entirely on hydrolysis of glycolytic ATP to generate a Δp across their membrane and enable them to transport metabolites.

Processing and Assembly of Atp6

The F1FO ATP synthase converts the proton gradient across the mitochondrial inner membrane into chemical energy in the form of ATP [4]. It consists of mitochondrial as well as nuclear encoded subunits, the latter being synthesized in the cytosol and imported into mitochondria via conserved protein translocases [5]. The FO subunit Atp6 is mitochondrial encoded and integrated into the F1FO ATP synthase at a late step of the assembly process [6]. While it has been recognized already in 1988 that Atp6 in S. cerevisiae is synthesized as a precursor protein with an N-terminal extension of 10 amino acid residues that are cleaved off [7], only recently Atp23 has been identified as the peptidase that mediates N-terminal Atp6 processing [1,2]. Replacement of the catalytically active glutamate residue at position 168 (Glu168Gln) within the metallopeptidase domain of Atp23 inactivates the peptidase and results in the accumulation of the precursor form of Atp6. Surprisingly, Δatp23 cells expressing Atp23E168Q remain respiration competent indicating that Atp6 maturation is not required for its assembly into the F1FO ATP synthase. In contrast, deletion of ATP23 impaired the respiratory competence of the yeast cells. Atp6 accumulates at drastically reduced steady state levels in these cells and the assembly of the F1FO ATP synthase is impaired [1,2]. Co-immunoprecipitation experiments revealed direct binding of Atp23 to newly synthesized, mature Atp6 [2]. Therefore, Atp23 exerts dual activities during the biogenesis of the F1FO ATP synthase in yeast mitochondria: it mediates the processing of newly synthesized Atp6 and ensures the assembly of mature Atp6 into the FO-moiety of the F1FO ATP synthase. Similar-sized assembly intermediates accumulate in the absence of Atp23 or of the assembly factor Atp10, which is localized in the matrix space and facilitates the interaction of Atp6 with the Atp9 ring complex within the FO-particle [7,8]. Overexpression of Atp23 was found to be an effective suppressor of an atp10 null mutant [1]. These findings suggest a sequential binding of newly synthesized Atp6 to Atp10 and to Atp23, which mediates Atp6 maturation in the intermembrane space and its association with Atp9 ring complexes.