Biochemistry 2010, 49, 6115–6121 6115 DOI: 10.1021/bi100492p Delineating Electrogenic Reactions during Lactose/Hþ Symport† Juan J. Garcia-Celma,‡ Julian Ploch,‡ Irina Smirnova,§ H. Ronald Kaback,*,§, ,)^ and Klaus Fendler*,‡ ‡Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, D-60438 Frankfurt/M, Germany, §Department of Physiology, D)epartment of Microbiology and Immunology, and ^Department of Molecular Genetics, University of California, Los Angeles, California 90095-7327 Received April 1, 2010; Revised Manuscript Received June 17, 2010 ABSTRACT: Electrogenic reactions accompanying downhill lactose/Hþ symport catalyzed by the lactose permease of Escherichia coli (LacY) have been assessed using solid-supported membrane-based electro- physiology 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 Hþ translocation is abolished either by acidic pH or by a Glu325 f Ala mutation in the Hþ 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 Hþ 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. The lactose permease of Escherichia coli (LacY)1 is a galacto- pyranoside/Hþ symporter that belongs to the major facilitator superfamily (MFS) of membrane transport proteins (1, 2). LacY is arguably the most thoroughly studied ion-coupled membrane transport protein (3-7). X-ray crystal structures exhibit an inward-open conformation with a tightly packed, closed peri- plasmic side and a large hydrophilic cavity open only to the cytoplasm with sugar- and Hþ-binding sites in the middle of the molecule at the apex of the hydrophilic cavity. Although an outward-open conformation has not yet been obtained by X-ray crystallography, it is well documented that LacY undergoes conformational changes upon sugar binding that lead to closing of the cytoplasmic cavity and opening of a relatively large hydrophilic cavity on the periplasmic side (the alternating access model). Therefore, estimates of distance from cross-linking measurements in the inward-facing structure (3), site-directed alkylation (8-11), single-molecule Fo€rster resonance energy transfer (12), double electron-electron resonance (13), site- directed thiol cross-linking (14), and site-directed quenching and unquenching of Trp fluorescence (15) all provide strong, independent support for the alternating access model. For a detailed investigation of the alternating access model, resolution of the partial reactions in the transport cycle is essential. This requires a method with sufficient time resolution †This work was funded by the Deutsche Forschungsgemeinschaft SFB 807 (to K.F.) and by National Institutes of Health Grants DK051131, DK069463, GM073210, and GM074929 and National Science Foundation Grant 0450970 to H.R.K. *To whom correspondence should be addressed. K.F.: e-mail, klaus. fendler@biophys.mpg.de; phone, þ49-69-63032035; fax, þ49-69- 63032222. H.R.K.: e-mail, rkaback@mednet.ucla.edu; phone, (310) 206-5053; fax, (310) 206-8623. 1Abbreviations: LacY, lactose permease; MelB, melibiose permease; SSM, solid-supported membrane; DTT, dithiothreitol; MFS, major facilitator superfamily; SE, standard error. such as solid-supported membrane (SSM)-based electrophysio- logy (16, 17), which allows concentration jumps as rapid as 4.5 ms (17). This fast solution exchange in combination with quantita- tive temporal information concerning the surface substrate concentration rise time is utilized to detect rapid charge displace- ments in the reaction cycle of LacY and to determine rate constants. A kinetic model for sugar transport in LacY is pre- sented, and a transport mechanism including an occluded state is proposed. EXPERIMENTAL PROCEDURES Construction of Mutants and LacY Purification. Con- struction of mutants and purification of the His-tagged proteins were conducted as described previously (13). Purified proteins (10-15 mg/mL) in a 50 mM sodium phosphate/0.01% DDM (pH 7.5) mixture were frozen in liquid nitrogen and stored at -80 C until they were used. Reconstitution of Proteoliposomes. Reconstitution of the purified wild type or E325A mutant was conducted with E. coli total phospholipids (Avanti Polar Lipids, Alabaster, AL) by using octyl glucoside dilution, followed by one cycle of freeze- thaw sonication (18, 19). Purified wild-type LacY or the E325A mutant and liposomes were mixed at a lipid:protein ratio of 5 (weight/weight), as indicated. Prior to use, the samples were thawed on ice and gently sonicated for 2-5 s. SSM-Based Electrophysiology. SSM measurements were performed as described previously (16, 17, 20-22). Briefly, 40 μL of proteoliposomes at a protein concentration of 1 mg/mL was allowed to adsorb for 1 h to an octadecanethiol/diphytanoylpho- sphatidylcholine hybrid bilayer on a gold surface of the sensor. The solution exchange protocol consisted of three phases: (1) nonactivating solution (2 s), (2) activating solution (2 s), and (3) nonactivating solution (2 s). A valveless diverter fluidic geometry r 2010 American Chemical Society Published on Web 06/22/2010 pubs.acs.org/Biochemistry