Delineating Electrogenic Reactions during Lactose/H(+) Symport

[Image: see text] Electrogenic reactions accompanying downhill lactose/H(+) symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electrophysiology with improved time resolution. Rates of charge translocation generated by purified LacY reconstituted into proteoliposomes were analyzed over a pH range from 5.2 to 8.5, which allows characterization of two electrogenic steps in the transport mechanism: (i) a weak electrogenic reaction triggered by sugar binding and observed under conditions where H(+) translocation is abolished either by acidic pH or by a Glu325 → Ala mutation in the H(+) binding site (this step with a rate constant of ∼200 s(−1) for wild-type LacY leads to an intermediate proposed to represent an “occluded” state) and (ii) a major electrogenic reaction corresponding to 94% of the total charge translocated at pH 8, which is pH-dependent with a maximum rate of ∼30 s(−1) and a pK of 7.5. This partial reaction is assigned to rate-limiting H(+) release on the cytoplasmic side of LacY during turnover. These findings together with previous electrophysiological results and biochemical−biophysical studies are included in an overall kinetic mechanism that allows delineation of the electrogenic steps in the reaction pathway.