6120 Biochemistry, Vol. 49, No. 29, 2010 suggests that reorientation of LacY prior to sugar binding does not correspond to the minor electrogenic reaction. The same argument also applies for periplasmic proton binding, the other step in the reaction cycle that takes place before sugar binding. In this case, the mutant E325A which is permanently proto- nated (23, 24, 27, 35) should not be able to generate an electrical response, again in opposition to the experimental results. Alter- natively, electrogenic sugar binding to LacY may be responsible for this electrogenic reaction. In this case, however, the observed rate constant would be linearly dependent on the substrate concentration, also opposed to the experimental results. Thus, we propose that sugar binding to LacY triggers an electrogenic partial reaction that takes place in the reaction cycle after sugar binding (eq 1). In this scenario, the observed rate constants hyperbolically increase with substrate concentration (see eq 2), as observed experimentally. To further define the sugar-induced electrogenic reaction, consideration of the LacY C154G mutant is helpful. In contrast to E325A LacY, mutant C154G is strongly impaired in equilib- rium lactose exchange (36, 37). However, C154G LacY binds sugar substrates in a two-step process (26), which suggests that after sugar binding C154G LacY forms an intermediate that is unable to release sugar to the cytoplasm, possibly an occluded state. Interestingly, the C154G mutant also displays a sugar- induced electrogenic conformational transition similar to that observed with wild-type LacY at pH 5.2 (Figure 2) and E325A LacY (17). Therefore, it seems reasonable to propose that sugar binding also triggers an electrogenic partial reaction in wild-type and E325A LacY leading to formation of an occluded state as proposed previously (13). A reaction cycle for lactose/Hþ symport is shown in Figure 6. It has been suggested that in the absence of sugar LacY is oriented in the membrane in an inward-facing conformation and deprotonated (17). From the kinetic analysis presented, it is apparent that LacY binds sugar with a rate constant of at least 400 s-1 (see the model in Figure 5C, inset, and the assignment of rate constants at the beginning of the discussion) followed by a weakly electrogenic step (CoutHþLac f CoccHþLac). We pro- pose that this corresponds to the formation of an intermediate “occluded” state from the outward-facing tertiary complex. In this reaction, only 6% of the total translocated charge is displaced per turnover. The rate constants (kþ ∼ 180 s-1, and k- ∼ 30 s-1) are taken from the analysis of the transient currents shown in Figure 2. Release of sugar (CoccHþLac f CinHþ) is electro- neutral and also rapid, proceeding with a rate constant of ∼250 s-1 (see the parameters of the kinetic model in Figure 5). The rate- limiting process is Hþ release (CinHþ f Cin), which is also the major electrogenic step with 94% of the total translocated charge. The rate constant varies with pH; at alkaline pH, assuming a value of 30 s-1 at alkaline pH. The process has an apparent pK of 7.5. In Figure 6, minor and major electrogenic steps are illustrated by a shallow periplasmic and by a deep cytoplasmic Hþ binding site. However, Hþ displacement through the protein dielectric is only one of several alternative sources of charge displacement such as tilting of helices or movement of charged residues during the transport reaction. It is possible that the sugar-induced minor charge trans- location (green arrows in Figure 6) is due to an electrogenic conformational transition not involving the transported cation which was suggested for the melibiose permease (MelB) (38). Indeed, MelB appears to be related to LacY (39), although they belong to different families (melB to the glycoside-pento- side-hexuronide:cation symporter and LacY to the MFS family). Garcia-Celma et al. FIGURE 6: Schematic representation of two proposed electrogenic steps in the reaction cycle of wild-type LacY. Inward-facing, out- ward-facing, and occluded conformations are marked with subscripts in, out, and occ, respectively. L in yellow squares represents the sugar (lactose or melibiose), and red circles with a white cross represent Hþ. Addition of sugar leads to a rapid sugar binding (CoutHþLac) with an estimated lower limit for the rate of ∼400 s-1 (see the text). Sugar binding is followed by a formation of the occluded state (CoccHþLac) and is accompanied by a minor electrogenic reaction (green) with forward and reverse rate constants of 181 and 33 s-1, respectively. De-occlusion and release of the sugar are electroneutral and proceed with an estimated rate of ∼250 s-1 (see the text). Hþ release on the cytoplasmic side (red) is the major electrogenic step in the overall transport cycle and characterized by a pH-dependent rate constant (0.3-30 s-1). The observation that sugar occlusion is only weakly electrogenic (6% of the total charge translocated per turnover) is graphically represented by a “shallow” Hþ binding site in CoccHþLac, while strongly electrogenic Hþ release (94% of the total charge trans- located per turnover) is represented by a “deep” Hþ release site in Cin. 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