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
Members of the F o F 1 , A o A 1 and V o V 1 family of ATP synthases and ATPases have undergone at least two reversals in primary function. The first was from a progenitor protonpumping ATPase to a proton-driven ATP synthase. The second involved transforming the synthase back into a proton-pumping ATPase. As proposed earlier [FEBS Lett. 259 (1990) 227], these reversals required changes in the H þ /ATP coupling ratio from an optimal value of about 2 for an ATPase function to about 4 for an ATP synthase function. The doubling of the ratio that occurred at the ATPase-to-Synthase transition was accomplished by duplicating the gene that encodes the nucleotidebinding catalytic subunits followed by loss of function in one of the genes. The halving of the ratio that occurred at the Synthaseto-ATPase transition was achieved by a duplication/fusion of the gene that encodes the proton-binding transporter subunits, followed by a loss of function in one half of the double-sized protein. These events allowed conservation of quaternary structure, while maintaining a sufficient driving force to sustain an adequate phosphorylation potential or electrochemical gradient. Here, we describe intermediate evolutionary steps and a finetuning of the H þ /ATP coupling ratio to optimize synthase function in response to different environments. In addition, we propose a third reversal of function, from an ATPase back to an ATP synthase. In contrast to the first two reversals which required a partial loss in function, the change in coupling ratio required for the third reversal is explained by a gain in function.
The Pi z HOH exchange reaction of oxidative phosphorylation is considerably less sensitive to uncouplers than the Pi T ATP and ATP ;± HOH exchanges. The uncoupler-insensitive Pi 2. HOH Previous findings have shown that the Pi T HOH exchange is less sensitive to 2,4-dinitrophenol than the Pi T. ATP exchange or the capacity for net oxidative l)hosphorylation (10)(11)(12). However, the significance or the source of this exchange has not been known. The possibility exists that it might reflect activities of enzymes such as alkaline phosphatase or pyrophosphatase known to catalyze a Pi ± HOH exchange (7), perhaps activated in some manner by 2,4-dinitrophenol. In addition, whether such behavior is limited to 2,4-dinitrol)henol or might be shown by more potent uncouplers of oxidative phosphorylation has not been shown. Results with other uncouplers and with oligomycin inhibition reported here cover these points.The effects of increasing concentrations of the potent uncoupler 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13) (13) on the exchanges catalyzed by mitochondria are shown ill Fig. 1. In the absence of uncoupler, the relative rates of the reactions Pi 2. HOH, ATP ¢± HOH, and Pi T ATP are about 12:6: 1, respectively, under the conditions used. At low concentrations of uncoupler, the Pi ± ATP and the ATP z HOH exchanges are much more sensitive to the uncoupler than the Pi z HOH exchange. At a concentration of S-13 sufficient to inhibit the Pi T ATP and ATP 2 HOH exchange by about 50%, the Pi T HOH exchange is inhibited by less than 5%. At a concentration S-13 that gives a near zero value for the Pi T ATP and ATP T HOH exchanges and a maximum value for the uncoupler-stimulated ATPase activity, the Pi T HOH exchange is still rapid and inhibited by only 35%. Responses similar to those reported in Fig. 1 for S-13 with mitochondria are also observed with 2,4-dinitrophenol and m-chlorocarbonvlcvanide phenylhydrazone.Important for the present considerations are the demonstrations, not given in detail here, of the effects of oligomycin.This antibiotic is a potent inhibitor of oxidative phosphorylation and inhibits the Pi T HOH exchange (14). In the absence of uncouplers and under conditions like those described with Fig. 1 Abbreviation: S-13, 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.