Distances between the b-subunits in the tether domain of F0F1-ATP synthase from E. coli

The arrangement of the b-subunits in the holo-enzyme F0F1-ATP synthase from E. coli is investigated by site-directed mutagenesis spin-label EPR. F0F1-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The hydrophilic F-1-part and the hydrophobic membrane-integrated F-0-part are connected by a central and a peripheral stalk. The peripheral stalk consists of two b-subunits. Cysteine mutations are introduced in the tether domain of the b-subunit at b-40, b-51, b-53, b-62 or b-64 and labeled with a nitroxide spin label. Conventional (9 GHz), high-field (95 GHz) and pulsed EPR spectroscopy reveal: All residues are in a relatively polar environment, with mobilities consistent with helix sites. The distance between the spin labels at each b-subunit is 2.9 nm in each mutant, revealing a parallel arrangement of the two helices. They can be in-register but separated by a large distance (1.9 nm), or at close contact and displaced along the helix axes by maximally 2.7 nm, which excludes an in-register coiled-coil model suggested previously for the b-subunit. Binding of the non-hydrolysable nucleotide AMPPNP to the spin-labeled enzyme had no significant influence on the distances compared to that in the absence of nucleotides. (c) 2005 Elsevier B.V. All rights reserved.