Cross-linking of the delta subunit to one of the three alpha subunits has no effect on functioning, as expected if delta is a part of the stator that links the F-1 and F-0 parts of the Escherichia coli ATP synthase

A mutant of the Escherichia coli F1F0-ATPase has been generated (alpha Q2C) in which the glutamine at position 2 of the alpha subunit has been replaced with a cysteine residue. Cu2+ treatment of ECF1 from this mutant crosslinked an alpha subunit to the delta subunit in high yield. Two different sites of disulfide bond formation were involved, i.e. between Cys(90) (or the closely spaced Cys(47)) of alpha with Cys(140) of delta, and between Cys(2) of alpha and Cys140 of delta. Small amounts of other cross-linked products, including alpha-alpha, delta internal, and alpha-alpha-delta were obtained. In ECF1F0, there was no cross-linking between the intrinsic Cys of alpha and Cys140. Instead, the product generated between Cys(2) of alpha and Cys140 of delta was obtained at near 90% yield. Small amounts of alpha-alpha and delta internal were present, and under high Cu2+ concentrations, alpha-alpha-delta was also formed. The ATPase activity of ECF1 and ECF1F0 was not significantly affected by the presence of these cross-links, When Cys(140) of delta was first modified with N-ethylmaleimide in ECF1F0, an alpha-delta cross-link was still produced, although in lower yield, between Cys(64) of delta and Cys(2) of alpha. ATP hydrolysis-linked proton pumping of inner membranes from the mutant alpha B2C was only marginally affected by cross-linking of the alpha to the delta subunit. These results indicate that Cys(140) and Cys(64) of the delta subunit and Cys(2) of the alpha subunit are in close proximity. This places the delta subunit near the top of the alpha-beta hexagon and not in the stalk region. As fixing the delta to the alpha by cross-linking does not greatly impair either the ATPase function of the enzyme, or coupled proton translocation, we argue that the delta subunit forms a portion of the stator linking F-1 to F-0.