Na⁺ Translocation Dominates over H⁺-Translocation in the Membrane Pyrophosphatase with Dual Transport Specificity

Cation-pumping membrane pyrophosphatases (mPPases; EC 7.1.3.1) vary in their transport specificity from obligatory H⁺ transporters found in all kingdoms of life, to Na⁺/H⁺-co-transporters found in many prokaryotes. The available data suggest a unique "direct-coupling" mechanism of H⁺ transport, in which the transported proton is generated from nucleophilic water molecule. Na⁺ transport is best rationalized by assuming that the water-borne proton propels a prebound Na⁺ ion through the ion conductance channel ("billiard" mechanism). However, the "billiard" mechanism, in its simple form, is not applicable to the mPPases that simultaneously transport Na⁺ and H⁺ without evident competition between the cations (Na⁺,H⁺-PPases). In this study, we used a pyranine-based fluorescent assay to explore the relationship between the cation transport reactions catalyzed by recombinant Bacteroides vulgatus Na⁺,H⁺-PPase in membrane vesicles. Under appropriately chosen conditions, including the addition of an H⁺ ionophore to convert Na⁺ influx into equivalent H⁺ efflux, the pyranine signal measures either H⁺ or Na⁺ translocation. Using a stopped-flow version of this assay, we demonstrate that H⁺ and Na⁺ are transported by Na⁺,H⁺-PPase in a ratio of approximately 1:8, which is independent of Na⁺ concentration. These findings were rationalized using an "extended billiard" model, whose most likely variant predicts the kinetic limitation of Na⁺ delivery to the pump-loading site.