Nonhydrolyzable ß-y bond OH OH

App(NH)p (ß,y-imidoadenosine 5'-triphosphate)



FIGURE 19-23 Mitochondrial ATP synthase complex. (a) Structure of the Ft complex, deduced from crystallographic and biochemical studies. In Ft, three a and three 3 subunits are arranged like the segments of an orange, with alternating a (shades of gray) and 3 (shades of purple) subunits around a central shaft, the y subunit (green). (b) Crystal structure of bovine Ft (PDB ID 1BMF), viewed from the side. Two a subunits and one 3 subunit have been omitted to reveal the central shaft (y subunit) and the binding sites for ATP (red) and ADP (yellow) on the 3 subunits. The 8 and e subunits are not shown here. (c) Ft viewed from above (that is, from the N side of the membrane), showing the three 3 and three a subunits and the central shaft (y subunit, green). Each 3 subunit, near its interface with the neighboring a subunit, has a nucleotide-binding site critical to the catalytic activity. The single y subunit associates primarily with one of the three a3 pairs, forcing each of the three 3 subunits into slightly different conformations, with different nucleotide-binding sites. In the crystalline enzyme, one subunit (3-ADP) has ADP (yellow) in its binding site, the next (3-ATP) has ATP (red), and the third (3-empty) has no bound nucleotide. (d) Side view of the FoFT structure. This is a composite, in which the crystallographic coordinates of bovine mitochondrial Ft (shades of purple and gray) have been combined with those of yeast mitochondrial Fo (shades of yellow and orange) (PDB ID 1QO1). Sub-units a, b, 8, and e were not part of the crystal structure shown here. (e)The FoFT structure, viewed end-on in the direction P side to N side. The major structures visible in this cross section are the two transmembrane helices of each of ten c subunits arranged in concentric circles. (f) Diagram of the FoF-i complex, deduced from biochemical and crystallographic studies. The two b subunits of Fo associate firmly with the a and 3 subunits of Fi, holding them fixed relative to the membrane. In Fo, the membrane-embedded cylinder of c subunits is attached to the shaft made up of Ft subunits y and e. As protons flow through the membrane from the P side to the N side through Fo, the cylinder and shaft rotate, and the 3 subunits of Ft change conformation as the y subunit associates with each in turn.

ADP, and the third was empty. The corresponding ¡ subunit conformations are designated ¡-ATP, ¡-ADP, and ¡-empty (Fig. 19-23c). This difference in nucleo-tide binding among the three subunits is critical to the mechanism of the complex.

The Fo complex making up the proton pore is composed of three subunits, a, b, and c, in the proportion ab2c10-12. Subunit c is a small (Mr 8,000), very hydrophobic polypeptide, consisting almost entirely of two transmembrane helices, with a small loop extending from the matrix side of the membrane. The crystal structure of the yeast FoF1, solved in 1999, shows the arrangement of the c subunits. The yeast complex has ten c subunits, each with two transmembrane helices roughly perpendicular to the plane of the membrane and arranged in two concentric circles (Fig. 19-23d, e). The

inner circle is made up of the amino-terminal helices of each c subunit; the outer circle, about 55 A in diameter, is made up of the carboxyl-terminal helices. The e and y subunits of F1 form a leg-and-foot that projects from the bottom (membrane) side of F1 and stands firmly on the ring of c subunits. The schematic drawing in Figure 19-23f combines the structural information from studies of bovine F1 and yeast FoF1.

Rotational Catalysis Is Key to the Binding-Change Mechanism for ATP Synthesis

On the basis of detailed kinetic and binding studies of the reactions catalyzed by FoF1, Paul Boyer proposed a rotational catalysis mechanism in which the three active sites of F1 take turns catalyzing ATP synthesis

(Fig. 19-24). A given p subunit starts in the p-ADP conformation, which binds ADP and Pi from the surrounding medium. The subunit now changes conformation, assuming the p-ATP form that tightly binds and stabilizes ATP, bringing about the ready equilibration of ADP + Pi with ATP on the enzyme surface. Finally, the subunit changes to the p-empty conformation, which has very low affinity for ATP, and the newly synthesized ATP leaves the enzyme surface. Another round of catalysis begins when

FIGURE 19-24 Binding-change model for ATP synthase. The F1 complex has three nonequivalent adenine nucleotide-binding sites, one for each pair of a and p subunits. At any given moment, one of these sites is in the p-ATP conformation (which binds ATP tightly), a second is in the p-ADP (loose-binding) conformation, and a third is in the p-empty (very-loose-binding) conformation. The proton-motive force causes rotation of the central shaft—the y subunit, shown as a green arrowhead—which comes into contact with each ap subunit pair in succession. This produces a cooperative conformational change in which the p-ATP site is converted to the p-empty conformation, and ATP dissociates; the p-ADP site is converted to the p-ATP conformation, which promotes condensation of bound ADP + P| to form ATP; and the p-empty site becomes a p-ADP site, which loosely binds ADP + Pi entering from the solvent. This model, based on experimental findings, requires that at least two of the three catalytic sites alternate in activity; ATP cannot be released from one site unless and until ADP and P| are bound at the other.

this subunit again assumes the p-ADP form and binds ADP and Pi.

The conformational changes central to this mechanism are driven by the passage of protons through the Fo portion of ATP synthase. The streaming of protons through the Fo "pore" causes the cylinder of c subunits and the attached y subunit to rotate about the long axis of y, which is perpendicular to the plane of the membrane. The y subunit passes through the center of the a3p3 spheroid, which is held stationary relative to the membrane surface by the b2 and 8 subunits (Fig. 19-23f). With each rotation of 120°, y comes into contact with a different p subunit, and the contact forces that p subunit into the p-empty conformation.

The three p subunits interact in such a way that when one assumes the p-empty conformation, its neighbor to one side must assume the p-ADP form, and the other neighbor the p-ATP form. Thus one complete rotation of the y subunit causes each p subunit to cycle through all three of its possible conformations, and for each rotation, three ATP are synthesized and released from the enzyme surface.

One strong prediction of this binding-change model is that the y subunit should rotate in one direction when FoF1 is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP. This prediction was confirmed in elegant experiments in the laboratories of Masasuke Yoshida and Kazuhiko Kinosita, Jr. The rotation of y in a single F1 molecule was observed microscopically by attaching a long, thin, fluorescent actin polymer to y and watching it move relative to a3p3 immobilized on a microscope slide, as ATP was hydrolyzed. When the entire FoF1 complex (not just F1) was used in a similar experiment, the entire ring of c subunits rotated with y (Fig. 19-25). The "shaft" rotated in the predicted direction through 360°. The rotation was not smooth, but occurred in three discrete steps of 120°. As calculated from the known rate of ATP hydrolysis by one F1 molecule and from the frictional drag on the long actin polymer, the efficiency of this mechanism in converting chemical energy into motion is close to 100%. It is, in Boyer's words, "a splendid molecular machine!"

Chemiosmotic Coupling Allows Nonintegral Stoichiometries of O2 Consumption and ATP Synthesis

Before the general acceptance of the chemiosmotic model for oxidative phosphorylation, the assumption was that the overall reaction equation would take the following form:

with the value of x—sometimes called the P/O ratio or the P/2e_ ratio—always an integer. When intact mito

Actin filament

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