Modification of subunit b of the F o complex from Escherichia coli ATP synthase by a hydrophobic maleimide and its effects on F o functions

Purified F 0 from Escherichia coli ATP synthase was labelled with N ‐(7‐dimethylamino‐4‐methyl‐coumarinyl)‐maleimide (DACM), a hydrophobic reagent which forms a stable, strongly fluorescent adduct with SH groups. Sodium dodecyl sulfate gel electrophoresis clearly demonstrated that subunit b was exclusively labelled, most likely at Cys‐21, the only cysteine residue in E. coli F 0 . The amount of two molecules of DACM bound per F 0 , which was calculated from the absorption spectrum at 380 nm, is in full agreement with the postulated stoichiometry of two copies of subunit b/F 0 complex. Thus the label provides a useful tool for simply detecting subunit b in protein chemical studies. DACM‐labelled F 0 was incorporated into liposomes and assayed for H + translocating activity and its capacity to bind purified F 1 . Whereas the initial rate of H + uptake was inhibited about 40% the reconstitution of a dicyclohexylcarbodiimide‐sensitive F 1 F 0 ATPase activity was completely unaffected. In a second set of experiments we reconstituted an F 0 complex from DACM‐labelled purified subunit b and an ac complex. In contrast to the results obtained with intact, DACM‐labelled F 0 , both H + translocating activity and F 1 binding capacity were greatly reduced. Our data indicate that cysteine‐21, probably together with other amino acids, is involved in maintaining a proper interaction of the hydrophobic N‐terminal region of subunit b with the ac complex. This interplay seems to be a prerequisite for at least the in vitro assembly of a functional F 0 complex.