Standard experiments

The so-called standard experiment, a basic approach of the steady-state technique (2.5.2a.), can be used to characterize the electrogenic ion-binding properties of the KdpFABC in both conformational states, E1 and P-E2. In this set of experiments, the fluorescence response of the RH421 dye was monitored, at various pH of the buffer and in the presence of different substrates that stabilize specific stationary states of the enzyme throughout the reaction cycle. In the absence of ATP, the KdpFABC is supposed to be stabilized in its E₁ conformation, and binding of K⁺ and possibly H⁺, is assumed to occur at the cytoplasmic side. Upon addition of saturating 1 mM Mg-ATP, the enzyme proceeds to the P-E₂ state, in which the ion binding is assumed to take place at the extracellular side.

Another possibility to trap the enzyme in E₂-P state is via the so-called backdoor phosphorylation, triggered by addition of inorganic phosphate, P_i, which reverses the direction of dephosphorylation step in the transport cycle, E₁ + P_i → E₂-P (158).

Corresponding experiments were performed to study the electrogenicity of ion binding in the unphosphorylated state, after phosphorylation by 1 mM Mg-ATP and by 1 mM Tris-phosphate (Pi).

An important observation necessary to mention before presenting obtained results is the requirement of pH jumps from pH 8 down to pH 6 and backward for stabilization of the fluorescence signal before any subsequent experimental studies. First results involving pH changes of the buffer were misleading, suggesting strong proton binding by solubilized KdpFABC complex. An example of proton titration experiment, in the absence of K⁺, ATP and Pi, shown in Figure 29A, reveals a fluorescence decrease of ~20 % as a result of binding of H⁺, whereas a maximal fluorescence decrease was ~5 % in case of the K⁺ binding (see text further below). Since the fluorescence change is a measure of the positive charge bound to the membrane domain of KdpFABC, this observation would indicate binding of four times as many protons as K⁺ ions. This assumption is not in agreement with any known behavior described for P-type ATPases so far, and it is highly unlikely to be the case for the KdpFABC mode of work. A possible explanation for this observation, however, could be the conformational rearrangement of the dye/lipid system induced by forward and backward pH jumps that leads to a new equilibrium state, and finally produces a stable fluorescence

65 signal. Therefore, all RH421 experiments were carried out with initial pH decrease from 8 to 6 (by adding HCl) and successive, instantaneous increase back to pH 8 (by adding NaOH).

After these pH jumps, successive H⁺ titration resulted in florescence decrease of ~5 % (Figure 29B) that are comparable to the steps obtained for addition of saturating K⁺ concentrations.

Figure 29. Effect of pH on the fluorescence signal of RH421, most likely induced by conformational rearrangement in the RH421/lipid system induced by sudden pH jumps. A: H⁺ titration in the absence of K⁺, showing ~20 % fluorescence decrease upon reaching pH 6. B: Forward and backward pH jumps followed by decreasing the pH back to 6 (by adding HCl), only now producing a fluorescence decrease of ~5 % due to proton binding.

According to the described protocol, a cuvette with 2 mL buffer of 50 mM Tris-HCl (pH 8) and 2 mM MgCl₂ was thermally equilibrated, before 200 nM RH421 dye and 9 µg/mL detergent-solubilized KdpFABC were added. After achieving a stable fluorescence signal

A

B

66 (10-15 minutes), aliquots of HCl were added to adjust the desired pH value of the buffer, followed by the addition of saturating concentrations of 1 mM ATP (pH adjusted) and 250 µM KCl. Addition of ATP did not alter the pH of the buffer more than ± 0.1. An example of the single fluorescence signal trace is shown in Figure 30A, whereas the corresponding set of HCl, ATP and KCl additions is given is Figure 30B.

Figure 30. RH421 standard experiment to reveal the electrogenic contributions of substrate additions to the KdpFABC complex. A: Fluorescence trace induced by the addition of HCl to obtain pH 6.5 (6.5 µl HCl 4 M), 1 mM ATP and 250 µM KCl. B: Schematic representation of the fluorescence changes detected after addition of various amounts of HCl to obtain the indicated pH, followed by the addition of ATP and KCl.

C: Changing the order of substrate addition revealed no significant difference in the substrate-induced fluorescence decreases.

6.0 6.5 7.0 7.5 8.0

pH

-0.1 -0.08 -0.06 -0.04

fluorescence (norm.)

K⁺/ATP full line

ATP/K⁺ dashed line C

67 Additions of both H⁺ and K⁺, and to a minor extent ATP, generated a fluorescence decrease.

To check whether there is a mutual effect on the binding behavior, in the next set of experiments, the order of ATP and HCl additions was reversed (Figure 30C). There was no significant difference in the substrate-induced fluorescence decreases.

The further evaluation of the results obtained from standard experiments is given in Figure 31. The substrate-induced decreases of the RH421 fluorescence indicate that binding of both ion species, K⁺ and H⁺, is an electrogenic process in the unphosphorylated as well as in the phosphorylated states of the KdpFABC. This implies that the ions are bound to sites located in a protein domain embedded in the membrane dielectric.

Figure 31. Analysis of the electrogenicity of substrate binding to the KdpFABC complex. A: pH dependence of the fluorescence changes induced by the additions of aliquots of HCl, 1 mM ATP or P_i, and 250 µM KCl. Solid symbols: experiments with addition of ATP; open symbols: experiments with addition of P_i, grey squares: K⁺ titration experiments in E₁. B: pH dependence of the fluorescence changes induced by the additions of 1 mM ATP or P_i first, then appropriate aliquots of HCl, and finally 250 µM KCl. The lines were drawn to guide the eye.

68 Results presented in Figure 31A show that only a very small fluorescence change (<0.5 %) is produced during the phosphorylation step both by ATP and Pi, indicating that no significant charge movements occur during the corresponding reaction steps. Further on, it was demonstrated that the H⁺ concentration affects the amount of electrochemically bound K⁺, and that the H⁺-binding kinetics differs in the unphosphorylated and phosphorylated state of the KdpFABC, produced both by ATP and Pi. On the other hand, binding of K⁺ was not significantly dependent neither on the pH of the buffer nor the specific state of the enzyme, E1 or E2-P. The addition of saturating KCl and HCl concentrations produced a fluorescence decrease of up to ~5 % (Figure 31B).