title: Obstruction of transmembrane helical movements in subunit a blocks proton pumping by F1Fo ATP synthase.
abstract: Subunit a plays a key role in promoting H(+) transport-coupled rotary motion of the subunit c ring in F1Fo ATP synthase. H(+) binding and release occur at Asp-61 in the middle of the second transmembrane helix (TMH) of Fo subunit c. H(+) are thought to reach cAsp61 via aqueous half-channels formed by TMHs 2-5 of subunit a. Movements of TMH4 and TMH5 have been proposed to facilitate protonation of cAsp61 from a half channel centered in a four helix bundle at the periplasmic side of subunit a. The possible necessity of these proposed TMH movements was investigated by assaying ATP driven H(+) pumping function before and after cross-linking paired Cys substitutions at the center of TMHs within subunit a. The cross-linking of the Cys pairs aG218C/I248C in TMH4 and TMH5, and aL120C/H245C in TMH2 and TMH5, inhibited H(+) pumping by 85-90%. H(+) pumping function was largely unaffected by modification of the same Cys residues in the absence of cross-link formation. The inhibition is consistent with the proposed requirement for TMH movements during the gating of periplasmic H(+) access to cAsp61. The cytoplasmic loops of subunit a have been implicated in gating H(+) release to the cytoplasm, and previous cross-linking experiments suggest that the chemically reactive regions of the loops may pack as a single domain. Here we show that Cys substitutions in these domains can be cross-linked with retention of function and conclude that these domains need not undergo large conformational changes during enzyme function.
authors.affiliation: From the Department of Biomolecular Chemistry, School of Medicine, and Public Health, University of Wisconsin, Madison, Wisconsin 53706.
authors.forename: Kyle J
authors.initials: KJ
authors.lastname: Moore
authors.affiliation: From the Department of Biomolecular Chemistry, School of Medicine, and Public Health, University of Wisconsin, Madison, Wisconsin 53706. Electronic address: rhfillin@wisc.edu.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Fillingame
mesh_terms: D019943:Amino Acid Substitution; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D029968:Escherichia coli Proteins; D025243:Bacterial Proton-Translocating ATPases; C557221:Escherichia coli Proton-Translocating ATPase
keywords: ATP Synthase; ATPases; Cysteine-mediated Cross-linking; F1Fo ATPase; Proton Transport; Subunit a; Transmembrane Helices