Conformational Changes in the Escherichia coli ATP Synthase (ECF1F0) Monitored by Nucleotide-dependent Differences in the Reactivity of Cys-87 of the γ Subunit in the Mutant βGlu-381 → Ala

Abstract

Cys-87, one of two intrinsic cysteines of the ␥ subunit of the Escherichia coli ATP synthase (ECF 1 F 0 ), is in a short segment of this subunit that binds to the bottom domain of a ␤ subunit close to a glutamate (Glu-381). Cys-87 was unreactive to maleimides under all conditions in wild-type ECF 1 and ECF 1 F 0 but became reactive when Glu-381 of ␤ was replaced by a cysteine or alanine. The reactivity of Cys-87 with maleimides was nucleotidedependent, occurring with ATP or ADP ؉ EDTA in catalytic sites, in the presence of AMP⅐PNP ؉ Mg 2؉ but not with ADP ؉ Mg 2؉ bound, whether P i was present or not, and not when nucleotide binding sites were empty. Binding of N-ethylmaleimide had no effect, whereas 7-diethyl-amino-3-(4-maleimidylphenyl)-4-methylcoumarin increased the ATPase activity of ECF 1 more than 2-fold by reaction with Cys-87. In ECF 1 F 0 , these reagents inhibited activity. The nucleotide dependence of the reaction of Cys-87 of the ␥ subunit depended on the presence of the ⑀ subunit. In ⑀ subunit-free ECF 1 , maleimides reacted with Cys-87 under all nucleotide conditions, including when catalytic sites were empty. These results are discussed in terms of nucleotide-dependent movements of the ␥ subunit during functioning of the F 1 F 0 -type ATPase.

The Stalk Region of the Escherichia coli ATP Synthase

“…As with the conformational changes detected at other parts of the ␥ subunit (30,31), the nucleotide dependence of the reaction of Cys-205 was lost on removal of the ⑀ subunit. ECF 1 from which the ⑀ subunit has been removed is a highly active ATPase, and therefore, as we have discussed before (30,31), the conformational changes observed at residues 8, 87, 106, and now 205 cannot be necessary for ATP hydrolysis.…”

“…ECF 1 from which the ⑀ subunit has been removed is a highly active ATPase, and therefore, as we have discussed before (30,31), the conformational changes observed at residues 8, 87, 106, and now 205 cannot be necessary for ATP hydrolysis. Rather, they likely represent structural changes that are part of the energy coupling within the complex that are in addition to rotation of the ␥ and ⑀ subunits that occurs during the cooperative functioning of catalytic sites (32)(33)(34).…”

“…This site reacted significantly faster with maleimides in both isolated ECF 1 and in ECF 1 F 0 in the ATP state (with Mg AMP-PNP bound) than in the ADP (Mg 2ϩ ) state. We have previously observed nucleotidedependent conformational changes in the ␥ subunit near catalytic sites (by cross-linking and fluorescence changes of probes attached at Cys introduced at position 8 (30)) at Cys-87 (by changes in reactivity to maleimides (31)) and at position 106 (from fluorescence changes of CM bound to a Cys at this site (30)). The results for ␥ Cys-205 now establish that the conformational change occurring with ATP hydrolysis is propagated by the ␥ subunit from the catalytic sites to the interface of the F 1 with F 0 .…”

Cross‐Linking of Chloroplast F0F1‐ATPase Subunit ɛ to γ Without Effect on Activity ɛ and γ are Parts of the Rotor

“…In E. coli subunit c is indispensable for binding F, to Fo [61, 621 whereas in chloroplasts F,(-E) binds well to F,, [63]. Cross-linking yields in the E. coli enzyme were dependent on the nucleotide load [lo, 23,26,27,351, whereas the chloroplast enzyme behaved much more slackly in this respect (this work).…”

The vacuolar H+-ATPase: a universal proton pump of eukaryotes

“…The crucial linking subunit is the γ polypeptide, which forms a spindle of extended α-helices at the centre of the pseudo-hexameric arrangement of alternating α and β subunits in the F " sector [6]. There is now experimental evidence to show that, during the catalytic cycle, the γ subunit rotates about its long axis with respect to each α\β dimer in the F " sector, in a socalled ' entropic motor ' [173,[214][215][216]. The γ subunit also interfaces with the ε subunit [217], and both of these subunits in turn contact both the β subunit in F " [218] and subunit c in the F o sector [143,219,220].…”