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

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.