On the role of Arg-210 and Glu-219 of subunit α in proton translocation by the Escherichia coli F0F1-ATP synthase

Francis Valiyaveetil, Robert H. Fillingame

Research output: Contribution to journalArticle

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Abstract

A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F0F1-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F0 subunits b and c inserted normally into the membrane in the ΔuncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The ΔuncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit α, residues previously postulated to be essential in proton translocation. The αE219G and αE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80% of wild type membranes). We conclude that the αGlu-219 residue cannot play a critical role in proton translocation. The αR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F1 from membrane, the αR210A mutant F0 was active in passive proton translocation, i.e. in dissipating the ΔpH normally established by NADH oxidation with these membrane vesicles. αR210A membranes with F1 bound were also proton permeable. Arg-210 of subunit α may play a critical role in active H+ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.

Original languageEnglish (US)
Pages (from-to)32635-32641
Number of pages7
JournalJournal of Biological Chemistry
Volume272
Issue number51
DOIs
StatePublished - Dec 19 1997
Externally publishedYes

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Escherichia coli
Protons
Adenosine Triphosphate
Membranes
Genes
Succinic Acid
Adenosine Triphosphatases
Kanamycin Resistance
Gene encoding
Kanamycin
Active Biological Transport
Oxidative Phosphorylation
Bacilli
Bacillus subtilis
NAD
Quenching
Hydrolysis
Carbon
Oxidation
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

On the role of Arg-210 and Glu-219 of subunit α in proton translocation by the Escherichia coli F0F1-ATP synthase. / Valiyaveetil, Francis; Fillingame, Robert H.

In: Journal of Biological Chemistry, Vol. 272, No. 51, 19.12.1997, p. 32635-32641.

Research output: Contribution to journalArticle

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abstract = "A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F0F1-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F0 subunits b and c inserted normally into the membrane in the ΔuncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The ΔuncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit α, residues previously postulated to be essential in proton translocation. The αE219G and αE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80{\%} of wild type membranes). We conclude that the αGlu-219 residue cannot play a critical role in proton translocation. The αR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F1 from membrane, the αR210A mutant F0 was active in passive proton translocation, i.e. in dissipating the ΔpH normally established by NADH oxidation with these membrane vesicles. αR210A membranes with F1 bound were also proton permeable. Arg-210 of subunit α may play a critical role in active H+ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.",
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