ATP Synthesis:- Each β Subunit of ATP Synthase Can Assume Three Different Conformations
Mitochondrial F1 has nine subunits of five different types, with the composition α3β3γδε. Each of the three β subunits has one catalytic site for ATP synthesis. The crystallographic determination of the F1 structure by John E. Walker and colleagues revealed structural de tails very helpful in explaining the catalytic mechanism of the enzyme. The knoblike portion of F1is a flattened sphere, 8 nm high and 10 nm across, consisting of alternating α and β subunits arranged like the sections of an orange (Fig. 19–23a–c). The polypeptides that make up the stalk in the F1crystal structure are asymmetrically arranged, with one domain of the single γ subunit making up a central shaft that passes through F1, and another domain of associated primarily with one of the three β subunits, designated β-empty (Fig. 19–23c). Although the amino acid sequences of the three sub units are identical, their conformations differ, in part because of the association of the subunit with just one of the three. The structures of the δ and ε subunits are not revealed in these crystallographic studies. The conformational differences among β subunits extend to differences in their ATP/ADP-binding sites.
FIGURE 19–23 Mitochondrial ATP synthase complex. (a) Structure of the F1 complex, deduced from crystallographic and biochemical studies. In F1, three α and three β subunits are arranged like the segments of an orange, with alternating α (shades of gray) and (shades of purple) subunits around a central shaft, the subunit (green). (b) Crystal structure of bovine F1 (PDB ID 1BMF), viewed from the side. Two subunits and one β subunit have been omitted to reveal the central shaft (γ subunit) and the binding sites for ATP (red) and ADP (yellow) on the β subunits. The and subunits are not shown here. (c) F1 viewed from above (that is, from the N side of the membrane), showing the three β and three subunits and the central shaft (γ sub unit, green). Each β subunit, near its interface with the neighboring subunit, has a nucleotide-binding site critical to the catalytic activity. The single subunit associates primarily with one of the three pairs, forcing each of the three β subunits into slightly different con formations, with different nucleotide-binding sites. In the crystalline enzyme, one subunit (β -ADP) has ADP (yellow) in its binding site, the next (β -ATP) has ATP (red), and the third (β -empty) has no bound nu cleotide. (d) Side view of the FoF1 structure. This is a composite, in which the crystallographic coordinates of bovine mitochondrial F1 (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, δ, and were not part of the crystal structure shown here. (e) The FoF1 structure, viewed end-on in the direction P side to N side. The major structures visible in this cross section are the two trans membrane helices of each of ten c subunits arranged in concentric circles. (f) Diagram of the FoF1 complex, deduced from biochemical and crystallographic studies. The two b subunits of Fo associate firmly with the and subunits of F1, holding them fixed relative to the membrane. In Fo, the membrane-embedded cylinder of c subunits is attached to the shaft made up of F1 subunits α and β. As protons flow through the membrane from the P side to the N side through Fo, the cylinder and shaft rotate, and the β subunits of F1 change conformation as the γ subunit associates with each in turn.
When researchers crystallized the protein in the presence of ADP and App (NH)p, a close structural analog of ATP that cannot be hydrolyzed by the ATPase activity of F1, the binding site of one of the three subunits was filled with App (NH)p, the second was filled with ADP, and the third was empty. The corresponding β subunit conformations are designated β-ATP, β-ADP, and β-empty (Fig. 19–23c). This difference in nucleotide 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 Å in diameter, is made up of the carboxyl-terminal helices. The ε and γ 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.
