Introduction of a Sodium-Binding Motif into Subunits a and c of Bacillus sp. PS3 Proton F-ATPase Does Not Result in Sodium Specificity of the Enzyme

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Abstract

In bacteria F-type ATPase (F-ATPase) plays a key role in bioenergetics and couples ATP synthesis/hydrolysis with the transport of ions (H+ or Na+) across the membrane. The ion specificity of the enzyme is determined by the amino acid sequence of subunits c and а. Here, we introduced several mutations (7 in subunit c and 6 in subunit a) into F-ATPase of thermophilic bacterium Bacillus sp. PS3 in order to change the ion specificity of the enzyme from proton to sodium. The mutations did not affect the ATPase activity of the enzyme, but led to loss of proton conductivity and impaired the binding of subunit a to the c-subunit oligomer, rather than changed the ion specificity.

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About the authors

S. M. Bruman

Lomonosov Moscow State University; Winogradsky Institute of Microbiology, Federal Research Center of Biotechnology, Russian Academy of Sciences

Email: feniouk@belozersky.msu.ru
Russian Federation, Moscow; Moscow

A. V. Litvin

Winogradsky Institute of Microbiology, Federal Research Center of Biotechnology, Russian Academy of Sciences; Skolkovo Institute of Science and Technology

Email: feniouk@belozersky.msu.ru
Russian Federation, Moscow; Moscow

A. S. Lapashina

Lomonosov Moscow State University; Winogradsky Institute of Microbiology, Federal Research Center of Biotechnology, Russian Academy of Sciences

Email: feniouk@belozersky.msu.ru
Russian Federation, Moscow; Moscow

B. A. Fenyuk

Lomonosov Moscow State University

Author for correspondence.
Email: feniouk@belozersky.msu.ru
Russian Federation, Moscow

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2. Fig. 1. Normalised fluorescence of AFMA in the presence of membrane particles containing TF0F1-WT (continuous curve) or TF0F1-Tr-12 (discontinuous curve). a - sodium succinate was added to 3 mM followed by sodium malonate to 30 mM; b - ATP was added to 500 μM followed by a mixture of valinomycin and nigericin (uncoupler) to 500 nM of each in the cuvette

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3. Fig. 2. a - Effect of incubation with DCCD on ATPase activity of VLS with TF0F1-WT or TF0F1-Tr-12. The ratios of the ATPase activity values in the sample with DCCD to those in similar control samples without DCCD are shown. Points for which the standard deviation is shown were replicated in three repeats, the others are averages calculated from two repeats. b - Subunit composition of purified TF0F1-WT and TF0F1-Tr-12 complexes (electrophoresis under denaturing conditions in 12% polyacrylamide gel). Letters on the right indicate individual subunits

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