title: Mutations in single hairpin units of genetically fused subunit c provide support for a rotary catalytic mechanism in F(0)F(1) ATP synthase.
abstract: Previously, we generated genetically fused dimers and trimers of subunit c of the Escherichia coli ATP synthase based upon the precedent of naturally occurring dimers in V-type H(+)-transporting ATPases. The c(2) and c(3) oligomers have proven useful in testing hypothesis regarding the mechanism of energy coupling. In the first part of this paper, the uncoupling Q42E substitution has been introduced into the second loop of the c(2) dimer or the third loop of the c(3) trimer. Both mutant proteins proved to be as functional as the wild type c(2) dimer or wild type c(3) trimer. The results argue against an obligatory movement of the epsilon subunit between loops of monomeric subunit c in the c(12) oligomer during rotary catalysis. Rather, the results support the hypothesis that the c-epsilon connection remains fixed as the c-oligomer rotates. In the second section of this paper, we report on the effect of substitution of the proton translocating Asp(61) in every second helical hairpin of the c(2) dimer, or in every third hairpin of the c(3) trimer. Based upon the precedent of V-type ATPases, where the c(2) dimer occurs naturally with a single proton translocating carboxyl in every second hairpin, these modified versions of the E. coli c(2) and c(3) fused proteins were predicted to have a functional H(+)-transporting ATPase activity, with a reduced H(+)/ATP stoichiometry, but to be inactive as ATP synthases. A variety of Asp(61)-substituted proteins proved to lack either activity indicating that the switch in function in V-type ATPases is a consequence of more than a single substitution.
authors.affiliation: Medical Research Council, Dunn Human Nutritional Unit, Cambridge CB2 2XY, United Kingdom.
authors.forename: P C
authors.initials: PC
authors.lastname: Jones
authors.forename: J
authors.initials: J
authors.lastname: Hermolin
authors.forename: R H
authors.initials: RH
authors.lastname: Fillingame
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019677:Artificial Gene Fusion; D002384:Catalysis; D019281:Dimerization; D009154:Mutation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases