The ATP synthase

The original electrochemical potential hypothesis postulated that the energy provided by the coupled proton transport is stored in the form of a transmembrane bulk electrochemical gradient of protons, and is used therefrom by the membrane-bound ATP synthase [33]. Unquestionably, the data briefly discussed above support the contention that (a) bulk electrochemical gradients are formed by coupled proton flow, and (b) such bulk electrochemical gradients can serve as the driving force for ATP synthesis. 

The ATP synthase is composed of two distinct complexes [15,16]. CFi refers to the water-soluble complex which extends from the thylakoid membrane to the stroma, and CFq to the membrane embedded complex to which it is attached. 

CFi was originally isolated as a coupling factor, that is, a protein which when removed from the thylakoid membrane leaves a membrane unable to catalyse photophosphorylation, and which when reconstituted into it restores this ability. The 

CFi-less membranes are very leaky to protons, but this leakiness is eliminated by the recombination of the membranes with the isolated CFi, or with any of several inhibitors of the CFq portion of the ATP synthase that is still membrane-bound in the CF]-less membrane. It was thus concluded that CFo is a proton channel whose function is to deliver energetic protons to CF , where ATP synthesis takes place. 

CFi is a multicomponent complex with a molecular weight of about 400000 [60,61]. It is composed of five subunits termed a, j3, y, d, e in order of decreasing molecular weight (Chapter 10). The ratio of these units per molecule has been a matter of controversy, but recent evidence would seem to support a structure composed of [60,62]. Each subunit seems to carry a specific function the 

---

The mitochondrial complex that carries out ATP synthesis is called ATP synthase or sometimes FjFo-ATPase (for the reverse reaction it catalyzes). ATP synthase was observed in early electron micrographs of submitochondrial particles (prepared by sonication of inner membrane preparations) as round, 8.5-nm-diameter projections or particles on the inner membrane (Figure 21.23). In micrographs of native mitochondria, the projections appear on the matrixfacing surface of the inner membrane. Mild agitation removes the particles from isolated membrane preparations, and the isolated spherical particles catalyze ATP hydrolysis, the reverse reaction of the ATP synthase. Stripped of these particles, the membranes can still carry out electron transfer but cannot synthesize ATP. In one of the first reconstitution experiments with membrane proteins, Efraim Racker showed that adding the particles back to stripped membranes restored electron transfer-dependent ATP synthesis. 

FIGURE 21.25 A model of the Fj and Fg components of the ATP synthase, a rotating molecnlar motor. The a, b, a, /3, and 8 snbnnits constitute the stator of the motor, and the c, y, and e subunits form the rotor. Flow of protons through the structure turns the rotor and drives the cycle of conformational changes in a and fi that synthesize ATP. 

FIGURE 21.31 Structures of several uiicouplers, molecules that dissipate the proton gradient across the inner mitochondrial membrane and thereby destroy the tight coupling between electron transport and the ATP synthase reaction. 

Uncouplers (eg, dinitrophenol) are amphipathic (Chapter 14) and increase the petmeabihty of the lipoid inner mitochondrial membrane to protons (Figure 12—8), thus teducing the electtochemical potential and shott-citcuiting the ATP synthase. In this way, oxidation can proceed without phosphotylation. 

Attention has been directed to the dechlorination of polychlorinated benzenes by strains that use them as an energy source by dehalorespiration. Investigations using Dahalococcoides sp. strain CBDBl have shown its ability to dechlorinate congeners with three or more chlorine substituents (Holscher et al. 2003). Although there are minor pathways, the major one for hexachlorobenzene was successive reductive dechlorination to pentachlorobenzene, 1,2,4,5-tetrachlorobenzene, 1,2,4-trichlorobenzene, and 1,4-dichlorobenzene (Jayachandran et al. 2003). The electron transport system has been examined by the use of specific inhibitors. lonophores had no effect on dechlorination, whereas the ATP-synthase inhibitor A,A -dicyclohexylcarbodiimide (DCCD) was strongly inhibitory (Jayachandran et al. 2004). 

The flow of electrons occurs in a similar manner from the excited pigment to cytochromes, quinones, pheophytins, ferridoxins, etc. The ATP synthase in the mitochondria of a bacterial system resembles that of the chloroplast—chloroplast proton translocating ATP synthase [37]. 

However, the membrane of A. woodii lacks cytochromes or menaquinone. The chemiosmotic energy is most likely generated by the activities of a membrane-bound methyl transferase in this bacterium (Muller and Gottschalk 1994). The primary ion for the chemiosmotic energy in M. thermoacetica is H+ (Ivey and Ljungdahl 1986) and that in A. woodii is Na+ (Heise et al. 1992). The difference in the ionic specificity is reflected in the properties of their ATP synthases. Thus the ATP synthase from M. thermoacetica and M. thermoautotrophica pump protons (Das et al. 1997) while that from A. woodii is a Na+-pump (Heise et al. 1992 Redlinger and Muller 1994). 

Transport of protons from the intermembrane space into the matrix across the inner membrane of the mitochondria occurs via the ATP synthase complex (Fo-Ei), which generates ATP. Somewhat surprisingly, it was discovered that proteins that transport protons back into the matrix, but without generating ATP, do, in fact, exist and are... 

The activity of the ATP synthase is low in comparison with that in mitochondria from other tissues i.e. mitochondria in brown adipose tissue can generate very little ATP. 

The ATP synthase (EC3.6.1.34, complex V) that transports H"" is a complex molecular machine. The enzyme consists of two parts—a proton channel (Fq, for oligomycin-sensitive ) that is integrated into the membrane and a catalytic unit (Fi) that protrudes into the matrix. The Fo part consists of 12 membrane-spanning c-peptides and one a-subunit. The head of the Fi part is composed of three a and three p subunits, between which there are three active centers. The stem between Fo and Fi consists of one y and one e subunit. Two more polypeptides, b and 8, form a kind of stator, fixing the a and p subunits relative to the Fo part. 

As protons pass through a channel in the ATP synthase complex, ADP and P, are joined to form ATP. 

B. The ATP synthase inhibitor oligomycin binds directly to the enzyme complex and plugs up the H channel, which blocks ATP formation. 

Fig. 7. Extension of the sequence of events at a single p subunit to the ATP synthase enzyme as a whole during ATP synthesis, as proposed by the torsional mechanism. The diagram is drawn at 60° intervals of the movement of the e subunit. Binding of substrate converts the two-nucleotide state (the resting or ground state) to the three-nucleotide state. Catalysis takes place in the three-nucleotide state, which converts back to the two-nucleotide state with release of product [18]...

For the continued synthesis of ATP, the enzyme must cycle between a form that binds ATP very tightly and a form that releases ATP. Chemical and crystallographic studies of the ATP synthase have revealed the structural basis for this alternation in function. 

A second membrane transport system essential to oxidative phosphorylation is the phosphate translocase, which promotes symport of one H2PO4 and one H+ into the matrix. This transport process, too, is favored by the transmembrane proton gradient (Fig. 19-26). Notice that the process requires movement of one proton from the P to the N side of the inner membrane, consuming some of the energy of electron transfer. A complex of the ATP synthase and both translocases, the ATP synthasome, can be isolated from... 

In ischemic (oxygen-deprived) cells, a protein inhibitor blocks ATP hydrolysis by the ATP synthase operating in reverse, preventing a drastic drop in [ATP]. 

Electron microscopy of sectioned chloroplasts shows ATP synthase complexes as knoblike projections on the outside (stromal or N) surface of thylalcoid membranes these complexes correspond to the ATP synthase complexes seen to project on the inside (matrix or N) surface of the inner mitochondrial membrane. Thus the relationship between the orientation of the ATP synthase and the direction of proton pumping is the same in chloroplasts and mitochondria. In both cases, the Fl portion of ATP synthase is located on the more alkaline (N) side of the membrane through which protons flow down their concentration gradient the direction of proton flow relative to Fi is the same in both cases P to N (Fig. 19-58). 

The mechanism of chloroplast ATP synthase is also believed to be essentially identical to that of its mitochondrial analog ADP and P, readily condense to form ATP on the enzyme surface, and the release of this enzyme-bound ATP requires a proton-motive force. Rotational catalysis sequentially engages each of the three JS subunits of the ATP synthase in ATP synthesis, ATP release, and ADP + Pj binding (Figs 19-24, 19-25). 

Chloroplasts, like mitochondria, evolved from bacteria living endosymbiotically within early eukaryotic cells. The ATP synthases of eubacteria, cyanobacteria, mitochondria, and chloroplasts share a common evolutionary precursor and a common enzymatic mechanism. 

Boyer, P.D. (1997) The ATP synthase—a splendid molecular machine. Annu. Rev. Biochem. 66, 717-749. 

An advanced review of kinetic, structural, and biochemical evidence for the ATP synthase mechanism. 

---