CROSS-LINKING OF THE DELTA-SUBUNIT TO ONE OF THE 3 ALPHA-SUBUNITS HASNO EFFECT ON FUNCTIONING, AS EXPECTED IF DELTA IS A PART OF THE STATOR THAT LINKS THE F1 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 be en 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 amou nts of other cross-linked products, including alpha-alpha, delta inter nal, and alpha-alpha-delta were obtained. In ECF1F0, there was no cros s-linking between the intrinsic Cys of alpha and Cys140. Instead, the product generated between Cys(2) of alpha and Cys140 of delta was obta ined at near 90% yield. Small amounts of alpha-alpha and delta interna l were present, and under high Cu2+ concentrations, alpha-alpha-delta was also formed. The ATPase activity of ECF1 and ECF1F0 was not signif icantly affected by the presence of these cross-links, When Cys(140) o f delta was first modified with N-ethylmaleimide in ECF1F0, an alpha-d elta cross-link was still produced, although in lower yield, between C ys(64) of delta and Cys(2) of alpha. ATP hydrolysis-linked proton pump ing of inner membranes from the mutant alpha B2C was only marginally a ffected by cross-linking of the alpha to the delta subunit. These resu lts 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 su bunit near the top of the alpha-beta hexagon and not in the stalk regi on. As fixing the delta to the alpha by cross-linking does not greatly impair either the ATPase function of the enzyme, or coupled proton tr anslocation, we argue that the delta subunit forms a portion of the st ator linking F-1 to F-0.