Observations of rotation within the FoF1‐ATP synthase: deciding between rotation of the Foc subunit ring and artifact

Abstract

F_oF₁‐ATP synthase mediates coupling of proton flow in F_o and ATP synthesis/hydrolysis in F₁ through rotation of central rotor subunits. A ring structure of F_o c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F_o c subunit ring in detergent‐solubilized F_oF₁‐ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722–1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F_o inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F_oF₁‐ATP synthase into an F_o inhibitor‐insensitive state in which F₁ can hydrolyze ATP regardless of the state of the F_o. Our results raise the important issue of whether rotation of the F_o c ring in isolated F_oF₁‐ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.