Rotation of subunits during catalysis by Escherichia coli F1-ATPase.

Abstract

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

Exploiting the Nitrilotriacetic Acid Moiety for Biolabeling with Ultrastable Perylene Dyes

“…First, a PCR-based method 4 was used to create a unique NsiI restriction site (ATGCAT) at the 5'-end of the atpD gene for β in plasmid p3U 5 . A cassette of complementary, 5'-phosphorylated primers was then ligated into the cleaved, dephosphorylated NsiI site of p3U.…”

“…The modified atpD gene encodes β with N-terminal sequence MHHHHHHMHTGK (underlined residues replace wild-type Ala). F O F 1 was expressed from p3UβH 6 in strain LE392Δ(atpI-C) 6 , and βHis 6 -tagged F 1 was purified by standard methods 5 . The βHis 6 tags had minimal effect on the specific ATPase activity of F 1 .…”

Biological application of multi-component nanowires in hybrid devices powered by F1-ATPase motors

“…However, not until the first atomic-level (0.28 • A) resolution structure of bovine mitochondria F 1 was obtained (Abrahams et al, 1994), as well as a great deal of related Fig. 1 Schematic of F 1 -ATPase motor, whose propeller is nickel nanowire fabricated by electrochemical deposition studies (Duncan et al, 1995;Ogilvie et al, 1997;Engelbrecht and Junge, 1997;Groth and Walker, 1997), the understanding of the mechanism of F 1 motor was turned from the 'cartoon' stage to detailed calculations (Noji et al, 1997;Oster and Wang, 2003). Now, the prevailing and accepted mechanism for isolated F 1 -ATPase is that the central γ subunit rotates against the surrounding α 3 β 3 subunits during the hydrolysis of ATP in the three catalytic β subunits of the F 1 -ATPase.…”

Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site

“…The catalytic domain, whose structure was solved by X-ray crystallography [5], consists of α $ β $ γ with one active site in each β subunit with some contribution from the neighbouring α subunit. Rotation of the γ subunit relative to the α $ β $ hexamer has been shown to have an integral role in steady-state turnover [6][7][8][9].…”

“…Furthermore, Stock et al [11] obtained a 3.9 A / resolution map of the partial mitochondrial complex from yeast, which included a decamer of c subunits arranged in the predicted ring [6,[12][13][14] with the C-terminal helix on the perimeter. Although the resolution did not permit the assignment of side chains, the electron densities of the yeast map were similar to the hairpin structure solved by NMR.…”