Pause and rotation of F(1)-ATPase during catalysis.

F(1)-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120 degrees rotation of the central gamma subunit relative to the surrounding alpha(3)beta(3) ring. Here, we show that the rotation of F(1)-ATPase spontaneously lapses into long (approximately 30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F(1)-ATPase previously known as the ADP-Mg inhibited form in which F(1)-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the gamma subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90 degrees position.

Redox regulation of the rotation of F(1)-ATP synthase.

In F(1)-ATPase, the smallest known motor enzyme, unidirectional rotation of the central axis subunit gamma is coupled to ATP hydrolysis. In the present study, we report the redox switching of the rotation of this enzyme. For this purpose, the switch region from the gamma subunit of the redox-sensitive chloroplast F(1)-ATPase was introduced into the bacterial F(1)-ATPase. The ATPase activity of the obtained complex was increased up to 3-fold upon reduction (Bald, D., Noji, H., Stumpp, M. T., Yoshida, M. & Hisabori, T. (2000) J. Biol. Chem. 275, 12757-12762). Here, we successfully observed the modulation of rotation of gamma in this chimeric complex by changes in the redox conditions. In addition we revealed that the suppressed enzymatic activity of the oxidized F(1)-ATPase complex was characterized by more frequent long pauses in the rotation of the gamma subunit. These findings obtained by the single molecule analysis therefore provide new insights into the mechanisms of enzyme regulation.