Trapping of conformations of the Escherichia coli F₁ ATPase by disulfide bond formation: A state of the enzyme with all three catalytic sites of equal and low affinity for nucleotides

A mutant of Escherichia coli F₁F₀-ATPase, αS411C/βY331W/βE381C/γC87S, has been generated. CuCl₂ treatment of this mutant led to cross-linking between α and β subunits in yields of up to 90%. This cross-linking across non-catalytic site interfaces inhibited ATP hydrolysis activity. In the absence of cross-linking, MgATP bound in catalytic sites of the mutant with three different affinities of 0.1 μM, 6 μM and 60 μM, respectively, values that are comparable to wild-type. For MgADP, there was one tight site (0.34 μM) and two sites of lower affinity (each 27 μM), again comparable to wild-type enzyme. After cross-linking all three catalytic sites bound MgATP or MgADP with the same relatively low affinity (~ 60 μM). Thus cross-linking fixed all three catalytic sites in the same conformation. Trypsin cleavage experiments showed that cross- linking fixed the ε subunit in the ATP+EDTA conformation.