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bioRxiv · 10.1101/2024.12.06.627301

Rapid kinetics of H+ transport by membrane pyrophosphatase: evidence for a direct-coupling mechanism

Anashkin, V. A.·Bogachev, A. V.·Serebryakova, M. V.·Zavyalova, E. G.·Bertsova, Y. V.·Baykov, A. A.
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Abstract

Stress resistance-conferring membrane pyrophosphatase (mPPase) found in microbes and plants couples pyrophosphate hydrolysis with H+ transport out of the cytoplasm. There are two opposing views on the energy-coupling mechanism in this transporter: the pumping is associated with either pyrophosphate binding to mPPase or the hydrolysis step. We used our recently developed stopped-flow pyranine assay to measure H+ transport into mPPase-containing inverted membrane vesicles on the timescale of a single turnover. The vesicles were prepared from Escherichia coli overproducing the H+-translocating mPPase of Desulfitobacterium hafniense. Pyrophosphate induced linear accumulation of H+ in the vesicles, without evident lag or burst. In contrast, the binding of three nonhydrolyzable pyrophosphate analogs essentially induced no H+ accumulation. These findings are inconsistent with the "pumping-before-hydrolysis" model of mPPase functioning and support the alternative model positing the hydrolysis reaction as the source of the transported H+ ions. mPPase is thus a first "directly-coupled" proton pump.

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Anashkin, V. A., Bogachev, A. V., Serebryakova, M. V., Zavyalova, E. G., Bertsova, Y. V., Baykov, A. A.. 2024-12-10. Rapid kinetics of H+ transport by membrane pyrophosphatase: evidence for a direct-coupling mechanism. https://doi.org/10.1101/2024.12.06.627301

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