How release of phosphate from mammalian F1-ATPase generates a rotary substep

The rotation of the central stalk of F-1-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F-1-ATPase, each 360 degrees rotation consists of three 120 degrees steps composed of substeps of about 65 degrees, 25 degrees, and 30 degrees, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F-1-ATPase inhibitor protein, IF1, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F-1-ATPase inhibited by IF1 represents the catalytic dwell state. Another structure, described here, of bovine F-1-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the alpha(E)beta(E)-catalytic interface, whereas in F-1-ATPase inhibited with IF1, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the beta(E)-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the alpha(E)-subunit disrupts interactions in the alpha(E)beta(E)-catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the gamma-subunit and the C-terminal domain of the alpha(E)-subunit. To restore most of these interactions, and to make compensatory new ones, the gamma-subunit rotates through 25 degrees- 30 degrees.