Mutations in single hairpin units of genetically fused subunit c provide support for a rotary catalytic mechanism in F(0)F(1) ATP synthase.

TitleMutations in single hairpin units of genetically fused subunit c provide support for a rotary catalytic mechanism in F(0)F(1) ATP synthase.
Publication TypeJournal Article
Year of Publication2000
AuthorsJones, PC, Hermolin, J, Fillingame, RH
JournalJ Biol Chem
Volume275
Issue15
Pagination11355-60
Date Published2000 Apr 14
ISSN0021-9258
KeywordsAdenosine Triphosphate, Artificial Gene Fusion, Catalysis, Dimerization, Mutation, Protein Conformation, Proton-Translocating ATPases
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.

DOI10.1074/jbc.275.15.11355
Alternate JournalJ. Biol. Chem.
Citation Key10.1074/jbc.275.15.11355
PubMed ID10753949
Grant ListGM23105 / GM / NIGMS NIH HHS / United States