Subunit organization of the stator part of the F-0 complex from Escherichia coli ATP synthase

Abstract

Membrane-bound ATP synthases (F1F0) catalyze the synthesis of ATP via a rotary catalytic mechanism utilizing the energy of an electrochemical ion gradient. The transmembrane potential is supposed to propel rotation of a subunit c ring of F-0 together with subunits gamma and epsilon of F-1, thereby forming the rotor part of the enzyme, whereas the remainder of the F1F0 complex functions as a stator for compensation of the torque generated during rotation. This review focuses on our recent work on the stator part of the F-o complex, e.g., subunits a and b. Using epitope insertion and antibody binding, subunit a was shown to comprise six transmembrane helixes with both the N- and C-terminus oriented toward the cytoplasm. By use of circular dichroism (CD) spectroscopy, the secondary structure of subunit b incorporated into proteoliposomes was determined to be 80% alpha -helical together with 14% beta turn conformation, providing flexibility to the second stalk. Reconstituted subunit b together with isolated ac subcomplex was shown to be active in proton translocation and functional F-1 binding revealing the native conformation of the polypeptide chain. Chemical crosslinking in everted membrane vesicles led to the formation of subunit b homodimers around residues bQ37 to bL65, whereas bA32C could be crosslinked to subunit a, indicating a close proximity of subunits a and b near the membrane. Further evidence for the proposed direct interaction between subunits a and b was obtained by purification of a stable ab(2) subcomplex via affinity chromatography using His tags fused to subunit a or b. This ab(2) subcomplex was shown to be active in proton translocation and F-1 binding, when coreconstituted with subunit c. Consequences of crosslink formation and subunit interaction within the F1F0 complex are discussed.