During oxidative and photo-phosphorylation, F0Fj-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis ofATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within FoF1. Guided by a recent, high-resolution structure for (Fig. la). The first is that the major energy-requiring step is not synthesis of ATP at the catalytic site but rather its release from the site (8). Second, the tight binding of substrates and release of product occur simultaneously at separate but interacting sites (9). A third premise has been more speculative: that the required binding changes are coupled to proton transport by rotation of a complex of subunits extending through FoF1 (10). Rotation of the y subunit in F1 is thought to deform the catalytic sites to give the binding changes ( Fig. la), whereas rotation within Fo is believed to be required for completion of the proton pathway (Fig. lb). Evidence both for (5, 10-15) and against (16-19) subunit rotation has been presented, but no critical test has been reported. However, such a test now seems possible in light of a recent, highresolution structure for bovine F1 (14). Specific points of contact between -y and the three catalytic 13 subunits that encircle it include positioning of the bovine homolog of Escherichia coli 'y-subunit C87 (-yC87) close to one of the 1-subunit 380DELSEED386 sequences. In preliminary studies, we identified a Cys mutation within this X3-subunit sequence, ,BD380C, that allowed rapid and specific crosslinking of 13 to y subunit on membrane-bound FoF1 and concomitant inactivation of ATP hydrolysis and ATP-driven proton pumping (20).In this study, we induced a specific disulfide between PD380C on one 13 subunit and yC87 in soluble F1 (Fig. lc, step 1). Then, by subunit dissociation/reconstitution, we incorporated radiolabeled 13 subunits specifically into the two noncrosslinked 13-subunit positions of reconstituted hybrid F1 (Fig. lc, step 2).After reduction of the initial nonradioactive P-y crosslink, this allowed us to test the effects of ligand binding and catalysis on the ability of y subunit to reposition itself relative to specific 13 subunits (Fig. lc, step 3). Our results show that, after catalytic turnover of reduced hybrid F1, unlabeled and radiolabeled P subunits show a similar capacity to form a disulfide with yC87, as expected for a rotary mechanism. MATERIALS AND METHODSPurification of Soluble E. coli Fl. E. coli FoF1 was overexpressed from wild-type or mutant forms of plasmid p3U (20) and membranes were isolated as described (21,22

We report evidence for proton-driven subunit rotation in membrane-bound F o F 1 -ATP synthase during oxidative phosphorylation. A ␤D380C͞␥C87 crosslinked hybrid F 1 having epitope-tagged ␤D380C subunits (␤ f lag ) exclusively in the two noncrosslinked positions was bound to F o in F 1 -depleted membranes. After reduction of the ␤-␥ crosslink, a brief exposure to conditions for ATP synthesis followed by reoxidation resulted in a significant amount of ␤ f lag appearing in the ␤-␥ crosslinked product. Such a reorientation of ␥C87 relative to the three ␤ subunits can only occur through subunit rotation. Rotation was inhibited when proton transport through F o was blocked or when ADP and P i were omitted. These results establish F o F 1 as the second example in nature where proton transport is coupled to subunit rotation.

We recently demonstrated that the gamma subunit in soluble F1-ATPase from Escherichia coli rotates relative to surrounding beta subunits during catalytic turnover (Duncan et al. (1995) Proc. Natl. Acad. Sci. USA 92, 10964-10968). Here, we extend our studies to the more physiologically relevant membrane-bound F0F1 complex. It is shown that beta D380C-F1, containing a beta-gamma intersubunit disulfide bond, can bind to F1-depleted membranes and can restore coupled membrane activities upon reduction of the disulfide. Using a dissociation/reconstitution approach with crosslinked beta D380C-F1, beta subunits containing an N-terminal Flag epitope (beta flag) were incorporated into the two non-crosslinked beta positions and the hybrid F1 was reconstituted with membrane-bound F0. Following reduction and ATP hydrolysis, reoxidation resulted in a significant amount of crosslinking of beta flag to the gamma subunit. This demonstrates that gamma rotates within F1 during catalytic turnover by membrane-bound F0-F1. Furthermore, the rotation of gamma is functionally coupled to F0, since preincubation with DCCD to modify F0 blocked rotation.

As indicated above, there was a time in the pioneering days of the 1960s that many coupling factors, e.g. F2, F3, F4 and F5, which are not known today, were being considered as serious candidates for key roles in oxidative phosphorylation. Beechey was not convinced as to the validity of many of these claims and argued [13-151 that many of the factors were distinguished only by a differential enrichment of one particular component, e.g. the OSCP. He did much to clarify what was, and what was not, a discrete coupling factor.Brian Beechey's work in the ATPase field (and by no means all of it can be summarized here: see for example Lowe and Beechey [16] for a later contribution), and others in which he has worked such as investigating the claim that iodohistidine was an intermediate in oxidative phosphorylation [ 171 , is characterized by its careful and thorough execution. This has meant that it has stood the test of time and has, therefore, contributed very significantly indeed to what we know today about the ATP synthase.

No abstract