Rotatory catalysis by F-ATPase: Real-time recording of intersubunit rotation

Abstract

ATP synthase (F-ATPase) is an ubiquitous enzyme in photosynthesis and respiration of prokaryotic and eukaryotic organisms. It couples proton translocation through its membrane portion, F-0, to the synthesis of the `'energy carrier molecule'' ATP at the peripheral portion, F-1 [1]. Three cooperative reaction sites are distributed with trigonal symmetry over the hexagonal array of (alpha beta)(3) in F-1 [2, 3]. It has been proposed that the endergonic release of spontaneously formed ATP [4, 5] might involve mechanical energy transduction [6, 7] through the proton driven rotation of subunit gamma within (alpha beta)(3) [2, 8, 9]. We recorded the putative intersubunit rotation in real rime [10]. Applying polarized absorption relaxation after photobleaching (PARAP) to immobilized F-1 with eosinlabeled gamma, we observed the rotational motion of gamma relative to immobilized (alpha beta)(3) in the time range of 100 ms, compatible with the rate of ATP hydrolysis by immobilized F-1. Its angular domain of at least 200 degrees favours a rotatory tri-site mechanism of catalysis with gamma acting as a crankshaft in the center of (alpha beta)(3).