The Stalk Region of the Escherichia coli ATP Synthase

Abstract

The soluble portion of the Escherichia coli F 1 F 0 ATP synthase (ECF 1 ) and E. coli F 1 F 0 ATP synthase (ECF 1 F 0 ) have been isolated from a novel mutant ␥Y205C. ECF 1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF 1 F 0 . The mutation partly disrupts the F 1 to F 0 interaction, as indicated by a reduced efficiency of proton pumping. ECF 1 containing the mutation ␥Y205C was bound to the membrane-bound portion of the E. coli F 1 F 0 ATP synthase (ECF 0 ) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes. Cu Cross-linking of ␥ to ⑀ had only a minimal effect on ATP hydrolysis. The reactivity of the Cys at ␥ 205 showed a nucleotide dependence of reactivity to maleimides in both ECF 1 and ECF 1 F 0 , which was lost in ECF 1 when the ⑀ subunit was removed. Our results show that there is close interaction of the ␥ and ⑀ subunits for the fulllength of the stalk region in ECF 1 F 0 . We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF 1 F 0 .F 1 F 0 type ATP synthases play a key role in oxidative phosphorylation and photophosphorylation. The F 1 , which can be detached from the F 0 and studied separately, is a complex of five different types of subunits called ␣, ␤, ␥, ␦, and ⑀ that are present in the molar ratio 3:3:1:1:1. The F 0 part in the Escherichia coli enzyme is composed of three different subunits: a, b, and c in the molar ratio 1:2:10 -12 (1-4). Electron microscopy first showed that the ␣ and ␤ subunits are arranged hexagonally and alternate around a central cavity in which the ␥ subunit is located (5-7). Biochemical studies place the ⑀ subunit (nomenclature for the E. coli enzyme) at the bottom of the ␣ 3 ␤ 3 ␥ core complex (4, 8) in the stalk region, which is a 40 -45-Å-long structure that links the F 1 to the F 0 part (9, 10).The recently published high resolution structure of a major part of the beef heart F 1 molecule confirms the above-described arrangement of the ␣, ␤, and ␥ subunits and adds important details (11). In particular, it shows the ␥ subunit arranged with a long C-terminal ␣-helix extending from the top of the ␣ and ␤ subunits into the stalk region. A shorter N-terminal ␣-helix is also present, running from the catalytic site region into the stalk region. These two ␣ helices form a coiled coil. A third short ␣-helix of the ␥ subunit (residues 83-99 in the E. coli sequence) is inclined at about 45°to the two larger helices at the bottom of the F 1 as it becomes the stalk. Approximately half of the ␥ subunit is unresolved in the structure, presumably because it is disordered in the crystal form.Recently, we observed cross-linking between a Cys introduced at position 44 of the polar loop of the c subunits and a site or sites on the ␥ subunit in the region between residues 202 and 230 (12). This result implies that the ␥ subunit exte...

Rotation of a γ-ε Subunit Domain in the Escherichia coli F1F0-ATP Synthase Complex

“…This is approximately the observed distribution of both ␥ and ⑀ subunits in our experiments whether enzyme activity was ended by substrate depletion or by addition of azide. In previous studies it was shown that cross-linking of ⑀ to the ␥ subunit did not block activity (7,27), indicating that these two subunits move together as a mobile domain. In electron microscopy studies (19), we have seen movements of the ␥ subunits relative to ⑀, but this is because the antibody fragments to ␣ and ⑀ used to tag specific subunits release ⑀ from ␥ so that it is fixed at one ␣-␤ subunit pair.…”

“…7 and 14, respectively) interact directly with the c subunit oligomer of the F 0 subunit. The covalent cross-linking of ␥ or ⑀ to the c subunit ring does not block ATP hydrolysis (7,14), which implies that the rotatory element in ECF 1 F 0 is a ␥-⑀-c oligomer domain moving relative to the ␣ 3 -␤ 3 -␦-a-b 2 complex.…”

“…This part is linked by a narrow stalk to the F 0 part that is composed of a, b, and c subunits in the stoichiometry 1:2:9 -12 (3)(4)(5)(6). The stalk contains a part of the ␥ and ⑀ subunits (6,7). Two other subunits, ␦ and b, are required for linkage of the F 1 and F 0 parts.…”

“…The N-terminal 10-stranded ␤ barrel region interacts with the ␥ subunit through the length of the stalk (7,13) and with the c subunit oligomer at one end (14). The helix-loop-helix C-terminal domain of ⑀ lies under the ␣ 3 ␤ 3 subunit barrel and interacts with the DELSEED region (15) of a different ␤ subunit from the one that binds to the short ␣ helix of ␥ (16).…”

Rotation of the c subunit oligomer in fully functional F1Fo ATP synthase

“…It had previously been shown by this approach that the subunit can be crosslinked to the c subunit without major effect on the coupling, while the tight relationship of the ␥ and subunits was well characterized (31,40,41). However, these experiments do not separately provide conclusive evidence that the ␥ and c subunits move together as the rotor because the function(s) of the subunit remain enigmatic.…”