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

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.