Concentration gradient effects of sodium and lithium ions and deuterium isotope effects on the activities of H+-ATP synthase from chloroplasts

Abstract

We explored the concentration gradient effects of the sodium and lithium ions and the deuterium isotope's effects on the activities of H(+)-ATP synthase from chloroplasts (CF(0)F(1)). We found that the sodium concentration gradient can drive the ATP synthesis reaction of CF(0)F(1). In contrast, the lithium ion can be an efficient enzyme-inhibitor by blocking the entrance channel of the ion translocation pathway in CF(0). In the presence of sodium or lithium ions and with the application of a membrane potential, unexpected enzyme behaviors of CF(0)F(1) were evident. To account for these observations, we propose that both of the sodium and lithium ions could undergo localized hydrolysis reactions in the chemical environment of the ion channel of CF(0). The protons generated locally could proceed to complete the ion translocation process in the ATP synthesis reaction of CF(0)F(1). Experimental and theoretical deuterium isotope effects of the localized hydrolysis on the activities of CF(0)F(1), and the energetics of these related reactions, support this proposed mechanism. Our experimental observations could be understood in the framework of the well-established ion translocation models for the H(+)-ATP synthase from Escherichia coli, and the Na(+)-ATP synthase from Propionigenium modestum and Ilyobacter tartaricus.

Figure 1

Schematic diagram of CF₀F₁ proteoliposome in experimental sample cell. An example of ionic concentrations inside and outside the proteoliposome in a specific experimental run is indicated. Notation conventions shown here were used throughout this report.

Figure 2

Effects of sodium ions inside proteoliposomes on ATP synthesis activities of CF₀F₁. In five experiments, concentrations of [Na⁺]_in/[Na⁺]_out = 150 mM/8 mM, and pH_in = pH_put = 8.0, remained the same. Curve a, ATP yield with Δϕ = 0 mV. The fitted initial rate is 3.5 s⁻¹. Curve b, adding proton ionophore FCCP. Curve c, adding sodium ionophore. Curve d, applying a potassium membrane potential of 30 mV ([K⁺]_out/[K⁺]_in= 130 mM/50 mM). The fitted initial rate is 14.7 s⁻¹. Curve e, under conditions of curve d, plus addition of FCCP. The fitted initial rate is 9.5 s⁻¹. Each signal trace represents a single experimental measurement of ATP concentration.

Figure 3

Competition of Na⁺ inside proteoliposome on driving ATP synthesis reaction under conditions of ΔpH = 3.5 (pH_out/pH_in = 8.8/5.3), [Na⁺]_out = 1mM, and [Na⁺]_in at a range of 1–100 mM. Smooth curve represents simulation with Eq. (1), where a = 0.341 ± 0.035, d = 0.146 ± 0.051, and e = 0.108 ± 0.032 mM⁻¹. Each experimental data point represents the result of two independent experimental measurements. The same batch of CF₀F₁ sample was used throughout the experiments.

Figure 4

Effects of lithium ions inside proteoliposomes on ATP synthesis activites of CF₀F₁. Curve a, ATP yield with [Li⁺]_in/[Li⁺]_out =150 mM/5 mM and Δϕ = 0 mV. Curve b, [Li⁺]_in/[Li⁺]_out = 100 mM/5 mM and Δϕ = 140 mV. The fitted initial rate is 13.2 s⁻¹. Curve c, [Li⁺]_in/[Li⁺]_out = 10 mM/5 mM and Δ ϕ =140 mV. The initial rate is 8.4 s⁻¹. Curve d, under conditions of curve b, plus addition of FCCP. Each signal trace represents a single experimental measurement of ATP concentration.

Figure 5

Deuterium isotope's effects on relative initial ATP synthesis rates of CF₀F₁. Membrane potentials were all set at 140 mV. Experimental conditions are: (a) pH_out/pH_in = 8.0/5.0, (b) pH_out/pH_in = 8.0/6.3, and (c) pH_out/pH_in = 8.0/8.0, and [Li⁺]_in/[Li⁺]_out =100 mM/5 mM. Each experimental data point represents the result of four independent experimental measurements. The same batch of CF₀F₁ sample was used throughout the experiments.

Figure 6

Blocking effects of Li⁺ inside proteoliposome under conditions of ΔpH = 3.5 (pH_out/pH_in = 8.8/5.3), [Li⁺]_out = 1 mM, and [Li⁺]_in at a range of 1–75 mM. Two fitted curves were results of Hill coefficient n set at 4 and 14, respectively. Original experimental data were corrected with the residual synthesis rates originating from the localized hydrolysis of lithium ions. A strong cooperative blocking effect on the ATP synthesis rate was observed. Each experimental data point represents the result of two independent experimental measurements. The same batch of CF₀F₁ sample was used throughout the experiments.

Figure 7

Blocking effects of Li⁺ outside proteoliposome under conditions of ΔpH = 3.3 (pH_out/pH_in = 8.0/4.7), [Li⁺]_in = 3 mM, and [Li⁺]_out at a range of 3–300 mM. The fitted Hill coefficient n is 1.1. A weak cooperative blocking effect on ATP synthesis rates was observed. Each experimental data point represents the result of four independent experimental measurements. The same batch of CF₀F₁ sample was used throughout the experiments.