ATP synthesis by an uncoupler-resistant mutant of Bacillus megaterium

A. A. Guffanti, H. Blumenfeld, T. A. Krulwich

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Abstract

ATP synthesis has been studied in starved whole cells of B. megaterium and its uncoupler-resistant mutant derivative, C8. In the present studies, ATP synthesis was examined as a function of the transmembrane electrical potential under conditions in which this potential was the sole component of the electrochemical proton gradient (λμ̄H+). Energization by respiration, with L-malate as substrate, was compared to energization by a valinomycin-induced potassium diffusion potential. Both strains synthesized ATP only at respiration-generated λμ̄H+ values above -30 mV. At -120 mV the ratio of the phosphorylation potential to the λμ̄H+ was 3-4 for both strains. As the λμ̄H+ was dissipated with low, increasing concentrations of uncoupler, ATP synthesis was inhibited in the wild type more profoundly than in C8. By contrast, when energization of ATP synthesis was mediated by a potassium diffusion potential, both strains exhibited little synthesis at λμ̄H+ values below -150 mV; C8 showed consistently poorer ATP synthesis than the wild type; there was no resistance of either strain to low concentrations of uncoupler; and the phosphorylation potential/λμ̄H+ ratios were consistently lower than observed during respiration. The mutation in C8 appeared to be a single mutation, with a reversion frequency of 1 in 8 x 105. C8 exhibited lower ATPase activity and higher respiratory activity than the wild type. Taken together, the results are consistent with a model in which the energy form that is directly coupled to ATP synthesis is a relatively uncoupler-insensitive microscopic gradient that is produced during natural proton pumping. During establishment of artificial gradients, the uncoupler-sensitive bulk transmembrane gradient might predominate. The mutant strain C8 could possess an alteration in the TPase so that it is even more selectively coupled than the wild type to the microscopic gradient.

Original languageEnglish
Pages (from-to)8416-8421
Number of pages6
JournalJournal of Biological Chemistry
Volume256
Issue number16
StatePublished - 1981
Externally publishedYes

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