Rotation of the Ft 7 Subunit Driven by Proton Movement Through F0 Powers ATP Synthesis

Each of the three p subunits in the complete F0F1 complex can bind ADP and P( and catalyze ATP synthesis. However, the coupling between proton flow and ATP synthesis must be indirect, since the nucleotide-binding sites on the p subunits of F1, where ATP synthesis occurs, are 9-10 nm from the surface of the mitochondrial membrane. The most widely accepted model for ATP synthesis by the F0F1 complex—the binding-change mechanism—posits just such an indirect coupling (Figure 8-26).

According to this mechanism, energy released by the "downhill" movement of protons through F0 directly powers rotation of the c-subunit ring together with its attached 7 and e subunits (see Figure 8-24). The 7 subunit acts as a cam, or rotating shaft, whose movement within F1 causes cyclical changes in the conformations of the p subunits. As schematically depicted in Figure 8-26, rotation of the 7 subunit relative to the fixed (ap)3 hexamer causes the nucleotide-binding site of each p subunit to cycle through three conformational states in the following order:

1. An O state that binds ATP very poorly and ADP and Pj weakly

2. An L state that binds ADP and P( more strongly

3. A T state that binds ADP and P( so tightly that they spontaneously form ATP and that binds ATP very strongly

A final rotation of 7 returns the p subunit to the O state, thereby releasing ATP and beginning the cycle again. ATP or ADP also binds to regulatory or allosteric sites on the three a subunits; this binding modifies the rate of ATP synthesis according to the level of ATP and ADP in the matrix, but is not directly involved in synthesis of ATP from ADP and Pi.

Several types of evidence support the binding-change mechanism, which is now generally accepted. First, biochemical studies showed that one of the three p subunits on isolated F1 particles can tightly bind ADP and Pi and then form ATP, which remains tightly bound. The measured AG for this reaction is near zero, indicating that once ADP and Pi are bound to what is now called the T state of a p subunit, they spontaneously form ATP. Importantly, dissociation of the bound ATP from the p subunit on isolated F1 particles occurs extremely slowly. This finding suggested that dissociation of ATP would have to be powered by a conformational change in the p subunit, which, in turn, would be caused by proton movement.

Energy—120° rotation of y

Energy—120° rotation of y

Ann i n

ATP formation P2\ in T site

Ann i n

ATP formation P2\ in T site

Energy—120 P2\ rotation of y

Energy—120 P2\ rotation of y

▲ FIGURE 8-26 The binding-change mechanism of ATP synthesis from ADP and Pi by the F0F1 complex. This view is looking up at F1 from the membrane surface (see Figure 8-24). Each of the F1 p subunits alternate between three conformational states that differ in their binding affinities for ATP ADP and P,. Step □: After ADP and P, bind to one of the three p subunits (here, arbitrarily designated p1) whose nucleotide-binding site is in the O (open) conformation, proton flux powers a 120° rotation of the y subunit (relative to the fixed p subunits). This causes an increase in the binding affinity of the p1 subunit for ADR and P, to L (low), an increase in the binding affinity of the p3 subunit for ADR

and Pi from L to T (tight), and a decrease in the binding affinity of the p2 subunit for ATP from T to O, causing release of the bound ATP StepB: The ADP and P, in the T site (here the p3 subunit) form ATP a reaction that does not require an input of energy, and ADP and P, bind to the p2 subunit, which is in the O state. This generates an F1 complex identical with that which started the process (left) except that it is rotated 120°. Step 13: Another 120° rotation of y again causes the O n L n T n O conformational changes in the p subunits described above. Repetition of steps |1 and |2| leads to formation of three ATP molecules for every 360° rotation of y. [Adapted from P Boyer, 1989, FASEB J. 3:2164, and Y Zhou et al., 1997, Proc. Nat'l. Acad. Sci. USA 94:10583.]

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