▲ EXPERIMENTAL FIGURE 8-27 Rotation of the y subunit of the F1 complex relative to the (ap)3 hexamer can be observed microscopically. F1 complexes were engineered that contained p subunits with an additional His6 sequence, which causes them to adhere to a glass plate coated with a metal reagent that binds histidine. The y subunit in the engineered F1 complexes was linked covalently to a fluorescently labeled actin filament. When viewed in a fluorescence microscope, the actin filaments were seen to rotate counterclockwise in discrete 120° steps in the presence of ATP, powered by ATP hydrolysis by the p subunits. [Adapted from H. Noji et al., 1997, Nature 386:299, and R. Yasuda et al., 1998, Cell 93:1117.] See also K. Nishio et al., 2002, Proc. Nat'l. Acad. Sci. 97:13448, for another way to demonstrate a and p subunit rotation relative to the c subunit ring.]

Later x-ray crystallographic analysis of the (ap)3 hexamer yielded a striking conclusion: although the three p subunits are identical in sequence and overall structure, the ADP/ATP-binding sites have different conformations in each subunit. The most reasonable conclusion was that the three p subunits cycle between three conformational states, with different nucleotide-binding sites, in an energy-dependent reaction.

In other studies, intact F0F1 complexes were treated with chemical cross-linking agents that covalently linked the y and e subunits and the c-subunit ring. The observation that such treated complexes could synthesize ATP or use ATP to power proton pumping indicates that the cross-linked proteins normally rotate together.

Finally, rotation of the y subunit relative to the fixed (ap)3 hexamer, as proposed in the binding-change mechanism, was observed directly in the clever experiment depicted in Figure 8-27. In one modification of this experiment in which tiny gold particles were attached to the y subunit, rotation rates of 134 revolutions per second were observed. Recalling that hydrolysis of 3 ATPs is thought to power one revolution (see Figure 8-26), this result is close to the experimentally determined rate of ATP hydrolysis by F0F1 complexes: about 400 ATPs per second. In a related experiment, a y subunit linked to an e subunit and a ring of c subunits was seen to rotate relative to the fixed (ap)3 hexamer. Rotation of the y subunit in these experiments was powered by ATP hydrolysis, the reverse of the normal process in which proton movement through the F0 complex drives rotation of the y subunit. Nonetheless, these observations established that the y subunit, along with the attached c ring and e subunit, does indeed rotate, thereby driving the conformational changes in the p subunits that are required for binding of ADP and P(, followed by synthesis and subsequent release of ATP.

Number of Translocated Protons Required for ATP Synthesis A simple calculation indicates that the passage of more than one proton is required to synthesize one molecule of ATP from ADP and P(. Although the AG for this reaction under standard conditions is + 7.3 kcal/mol, at the concentrations of reactants in the mitochondrion, AG is probably higher (+ 10 to + 12 kcal/mol). We can calculate the amount of free energy released by the passage of 1 mol of protons down an electrochemical gradient of 220 mV (0.22 V) from the Nernst equation, setting n = 1 and measuring AE in volts:

AG (cal/mol) = -nFAE =-(23,062 cal-V^-mol"1) AE

= (23,062 cal-V-1-mol-1)(0.22 V) = -5074 cal/mol, or -5.1 kcal/mol

Since the downhill movement of 1 mol of protons releases just over 5 kcal of free energy, the passage of at least two protons is required for synthesis of each molecule of ATP from ADP and Pi.

Proton Movement Through F0 and Rotation of the c Ring

Each copy of subunit c contains two membrane-spanning a helices that form a hairpin. An aspartate residue, Asp61, in the center of one of these helices is thought to participate in proton movement. Chemical modification of this aspartate by the poison dicyclohexylcarbodiimide or its mutation to alanine specifically blocks proton movement through F0. According to one current model, two proton half-channels lie at the interface between the a subunit and c ring (see Figure 8-24). Protons are thought to move one at a time through half-channel I from the exoplasmic medium and bind to the carboxylate side chain on Asp61 of one c subunit. Binding of a proton to this aspartate would result in a conformational change in the c subunit, causing it to move relative to the fixed a subunit, or equivalently to rotate in the membrane plane. This rotation would bring the adjacent c subunit, with its ionized aspartyl side chain, into channel I, thereby allowing it to receive a proton and subsequently move relative to the a subunit. Continued rotation of the c ring, due to binding of protons to additional c subunits, eventually would align the first c subunit containing a protonated Asp61 with the second half-channel (II), which is connected to the cy-tosol. Once this occurs, the proton on the aspartyl residue could dissociate (forming ionized aspartate) and move into the cytosolic medium.

Since the y subunit of F1 is tightly attached to the c ring of F0, rotation of the c ring associated with proton movement causes rotation of the y subunit. According to the binding-change mechanism, a 120° rotation of y powers synthesis of one ATP (see Figure 8-26). Thus complete rotation of the c ring by 360° would generate three ATPs. In E. coli, where the Fo composition is a1b2c1o, movement of 10 protons drives one complete rotation and thus synthesis of three

ATPs. This value is consistent with experimental data on proton flux during ATP synthesis, providing indirect support for the model coupling proton movement to c-ring rotation depicted in Figure 8-24. The F0 from chloroplasts contains 14 c subunits per ring, and movement of 14 protons would be needed for synthesis of three ATPs. Why these otherwise similar F0F1 complexes have evolved to have different H+:ATP ratios in not clear.

ATP-ADP Exchange Across the Inner Mitochondrial Membrane Is Powered by the Proton-Motive Force

In addition to powering ATP synthesis, the proton-motive force across the inner mitochondrial membrane also powers the exchange of ATP formed by oxidative phosphorylation inside the mitochondrion for ADP and P( in the cytosol. This exchange, which is required for oxidative phosphorylation to continue, is mediated by two proteins in the inner membrane: a phosphate transporter (HPO42"/OH" antiporter) and an ATP/ADP antiporter (Figure 8-28).

▲ FIGURE 8-28 The phosphate and ATP/ADP transport system in the inner mitochondrial membrane. The coordinated action of two antiporters (purple and green) results in the uptake of one ADP3- and one HPO42~ in exchange for one ATP4-, powered by the outward translocation of one proton during electron transport. The outer membrane is not shown here because it is permeable to molecules smaller than 5000 Da.

H concentration gradient

Membrane electric < potential

Inner mitochondrial membrane




Translocation of H during electron transport

Phosphate transporter



- ATP/ADP antiporter

Intermembrane space

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