Article FIGURE 5: Simulation of the transient currents with a four-state kinetic model. (A) Experimental data. For a better comparison between the experimental transient currents and the four-state kinetic model, the experimental transients of Figure 1A (100 mM lactose concentration jumps at different pH values) are represented on an expanded time scale. (B) Numerical solution of the four-state model. The color code for different pH values is given in panel A and applies to both panels. (C) pH dependence of k40 (rate constant k4 at potential 0). The kinetic model with rate constants is shown in the inset. Partial reactions colored black are electroneutral. The minor electrogenic reaction (6% of the total translocated charge per turn- over) is colored green, and the major electrogenic partial reaction (94% of the total translocated charge per turnover) is colored red. The rate constant k40 of the major electrogenic partial reaction takes the values given in the figure at the individual traces (B). A detailed description of the model is given in the text. nent electrogenicity, the voltage dependence of k4 is taken into account (note that the voltage dependence of the weakly electro- genic B f C reaction is neglected). In response to the potential generated by the continuous transport activity of LacY, the voltage dependence of k4 leads to a decay of the currents at >20 ms (Figure 5A), which is taken into account in the model. The kinetic parameter characterizing this partial reaction is its rate constant at zero voltage (k40) which was obtained by manual adjustment of the model to the experimental current traces. As discussed below, this partial reaction corresponds to cytoplasmic Hþ release and is pH-dependent. For each of the current traces measured at different pH values, a given rate constant (k40) was determined (Figure 5B). The model reproduces the electrogenic response of the sym- porter from pH 5.2 to 8.5 remarkably well. Notably, all para- meters were kept constant, and only k40 was varied. Moreover, the model explains the major features of electrogenic transport at different pH values. (i) At alkaline pH (7.6 and 8.5), the transients decay with a τ1/2 of >20 ms and the magnitude of the peak current decreases at the lower pH value. (ii) At pH 7.0, two Biochemistry, Vol. 49, No. 29, 2010 6119 maxima are apparent. (iii) At pH 6.3, biphasic behavior is obtained, and the initial charge displacement is observed. The agreement between the model and the experimental findings gives further support to the proposal that two electrogenic steps with very different electrogenicities are involved in the turnover of wild-type LacY. Using the rate constants k40 at different pH values, a binding curve for a single titratable acidic group with a pK of 7.5 is obtained (Figure 5C). This agrees well with the pH dependence of lactose efflux measured with proteoliposomes reconstituted with purified LacY, where a functional group or proton binding site with a pK of 8.3 must be deprotonated for LacY to catalyze efflux at maximum rates (31). In conclusion, the four-state kinetic model is able to make correct predictions for the measured currents under a variety of different conditions. But most importantly, the kinetic model is minimal in the sense that it contains the fewest partial reactions needed to reproduce the experimental findings. Therefore, the model delineates a unique sequence of partial reactions with the individual rate constants and electrogenicities. Wild-Type and Mutant E325A. The mutant E325A is specifically defective in all steps involving Hþ release but cata- lyzes exchange and counterflow at least as well as the wild type (23, 24). Moreover, both the wild type and the mutant have a similar KD of ∼20 mM for lactose binding. Kinetic differences are found for the second reaction of the two-step binding model (eq 1). Sugar binding is approximately twice as fast in the mutant (kþ ∼ 400 s-1) as in the wild type (kþ ∼ 200 s-1). We can further characterize the ES f ES* step with a stability constant (Ks = kþ/k-), which is 50 for the mutant and only 5.5 for the wild type, indicating an ∼10-fold shift in the distribution toward ES* in the mutant. On the other hand, the rate constant for another transport impaired mutant, C154G LacY, was considerably smaller (53 ( 5 s-1) than that of the wild type (17). The four-state kinetic model can also be used for the mutant E325A. As the D f A step in the kinetic model represents deprotonation of LacY, a k4 of 0 s-1 should yield the transient currents for mutant E325A. Indeed, the transient current pre- dicted by the model, assuming k4 = 0 s-1, consists of an initial charge displacement followed by a slow negative component, in agreement with the measured transients (Figure 3A,B). As the only pH-dependent step in the four-state model is the fourth partial reaction, the model predicts no variation of the transient currents with pH for E325A LacY, which also agrees with the experimental findings (Figure 3A,B). LacY Transport Mechanism. On the basis of the previous discussion, it is apparent that two electrogenic steps in the LacY reaction cycle can be detected: a rapid sugar-induced initial charge translocation with low electrogenicity and a major electro- genic transition late in the reaction cycle. The latter is also the rate-limiting step in downhill sugar/Hþ symport (31) and has been assigned to cytoplasmic Hþ release (29), but what is responsible for the minor initial charge displacement? As LacY before addition of the sugar is in a conformation facing the inside of the proteoliposomes (3, 32-34), the question of whether reorientation of the transporter from the inward- facing conformation to the outward-facing conformation can cause the minor charge displacement arises. In this case, if reorientation of LacY is an electrogenic equilibrium followed by fast electroneutral sugar binding, the observed rate would decrease hyperbolically with substrate concentration, in opposi- tion to the experimental results (Figure 2B). This observation