Title: Rotation of a γ-ε Subunit Domain in the Escherichia coli F1F0-ATP Synthase Complex
Abstract: A triple mutant of <i>Escherichia coli</i>F<sub>1</sub>F<sub>0</sub>-ATP synthase, αQ2C/αS411C/εS108C, has been generated for studying movements of the γ and ε subunits during functioning of the enzyme. It includes mutations that allow disulfide bond formation between the Cys at α411 and both Cys-87 of γ and Cys-108 of ε, two covalent cross-links that block enzyme function (Aggeler, R., and Capaldi, R. A. (1996) <i>J. Biol. Chem.</i> 271, 13888–13891). A cross-link is also generated between the Cys at α2 and Cys-140 of the δ subunit, which has no effect on functioning (Ogilvie, I., Aggeler, R., and Capaldi, R. A. (1997) <i>J. Biol. Chem.</i> 272, 16652–16656). CuCl<sub>2</sub> treatment of the mutant αQ2C/αS411C/εS108C generated five major cross-linked products. These are α-γ-δ, α-γ, α-δ-ε, α-δ, and α-ε. The ratio of α-γ-δ to the α-γ product was close to 1:2, <i>i.e.</i> in one-third of the ECF<sub>1</sub>F<sub>0</sub> molecules the γ subunit was attached to the α subunit at which the δ subunit is bound. Also, 20% of the ε subunit was present as a α-δ-ε product. With regard to the δ subunit, 30% was in the α-γ-δ, 20% in the α-δ-ε, and 50% in the α-δ products when the cross-linking was done after incubation in ATP + MgCl<sub>2</sub>. The amounts of these three products were 40, 22, and 38%, respectively, in experiments where Cu<sup>2+</sup> was added after preincubation in ATP + Mg<sup>2+</sup> + azide. The δ subunit is fixed to, and therefore identifies, one specific α subunit (α<sub>δ</sub>). A distribution of the γ and ε subunits, which is essentially random with respect to the α subunits, can only be explained by rotation of γ-ε relative to the α<sub>3</sub>β<sub>3</sub> domain in ECF<sub>1</sub>F<sub>0</sub>.