Charge displacements during ATP-hydrolysis and synthesis of the Na+-transporting FoF1-ATPase of Ilyobacter tartaricus

Abstract

Transient electrical currents generated by the Na(+)-transporting F(o)F(1)-ATPase of Ilyobacter tartaricus were observed in the hydrolytic and synthetic mode of the enzyme. Two techniques were applied: a photochemical ATP concentration jump on a planar lipid membrane and a rapid solution exchange on a solid supported membrane. We have identified an electrogenic reaction in the reaction cycle of the F(o)F(1)-ATPase that is related to the translocation of the cation through the membrane bound F(o) subcomplex of the ATPase. In addition, we have determined rate constants for the process: For ATP hydrolysis this reaction has a rate constant of 15-30 s(-1) if H(+) is transported and 30-60 s(-1) if Na(+) is transported. For ATP synthesis the rate constant is 50-70 s(-1).

FIGURE 1

BLM and adsorbed proteoliposomes. (A) ATPase in the hydrolytic mode. The ATPase is activated by caged ATP. The current I(t) is measured via the electrodes E. This circuit is omitted in B. (B) ATPase in the synthetic mode. “Background ATP” generates the driving force for ATP synthesis, which is activated by caged ADP.

FIGURE 2

(A) Transient current after activation with caged ATP at the BLM. Conditions: 50 mM HEPES, pH 7,0 (TRIS); 100 mM KCl; 2 mM NaCl; 1 mM MgCl₂;1 mM DTT (dithiothreitole); 10 μM monensin; 360 μM caged ATP (η = 0,22). The solid line is a fit using a triexponential function. (B and C) Transient current in the absence (B) and presence (C) of 1 mM NaN₃. Conditions as in A. Traces B and C are from a separate experiment.

FIGURE 3

Transient currents after activation with caged ATP at the BLM. (A) The peak currents are presented as a function of released ATP concentrations. (B) Relaxation rates of the transient currents as a function of released ATP concentrations. Conditions: 50 mM H₃PO₄/KOH, pH 7,0; 2 mM NaCl; 1 mM MgCl₂; 1 mM DTT; 10 μM monensin. Filled circles: 0–2 mM caged ATP, η = 0.23. Open circles: 2 mM caged ATP, η = 0.23–0.

FIGURE 4

Transient currents after activation with caged ATP at the BLM at different Na⁺ concentrations. (A) Peak currents. (B) Relaxation rates. The curved solid lines in A and B represent fits using hyperbolic functions with an offset. Conditions: 50 mM HEPES, pH 7,0 (TRIS); 100 mM KCl; various concentrations of NaCl, 1 mM MgCl₂; 1 mM DTT; 10 μM monensin; 500 μM caged ATP (η = 0,35).

FIGURE 5

Relaxation rates of the transient currents after activation with caged ATP at the BLM at different pH. The dashed line is the rate of ATP released from caged ATP in solution calculated according to Walker et al. (1988). Conditions: 50 mM HEPES, pH 7,0 (TRIS); 100 mM KCl; 1 mM MgCl₂; 2 mM NaCl; 1 mM DTT; 10 μM monensin; 400 μM caged ATP (η = 0,26).

FIGURE 6

Transient currents after activation with caged ADP at the BLM. Conditions: 50 mM HEPES, pH 7,0 (TRIS); 100 mM NaCl; 1 mM MgCl₂; 1 mM DTT. After adsorption of the proteoliposomes the cuvette was perfused with 2 ml protein free solution. The transient currents were consecutively recorded after addition of (a) 500 μM caged ADP, (b) 50 μM “background” ATP, and (c) 0.5 mM H₃PO₄(KOH).

FIGURE 7

Transient currents after activation with a rapid concentration jump at the SSM. (A) SSM signal after an ATP jump (1 mM ATP) in the absence of NaCl. (B) SSM signal after an ATP jump (0,5 mM ATP) in the presence of 2 mM NaCl. (C) Inhibition of A by 60 μM DCCD. Activating solution for A–C: 50 mM HEPES, pH 7,0 (TRIS); 300 mM KCl; 0 or 2 mM NaCl; 3 mM MgCl₂; 0.5 or 1 mM ATP. Nonactivating solution: as activating solution but no ATP. (D) SSM signal after an ADP jump (0.3 mM ADP) in the presence of NaCl, P_i, and “background ATP.” (E) Without “background ATP”. Activating solution for D and E: 50 mM HEPES, pH 7,0 (TRIS); 300 mM KCl; 2 mM NaCl; 3 mM MgCl₂; 1 mM H₃PO₄(KOH); 0/30 μM external ATP; 0,3 mM ADP. Nonactivating solution: as activating solution but no ADP.

FIGURE 8

(A) Kinetic model describing the activation of the F_oF₁-ATPase using a photolytic concentration jump with caged ATP. (B) Structure of F_oF₁-ATPase and rotary mechanism of Na⁺ transport during the process characterized by the rate constant k₂. For clarity the α-, β-, δ-, and ɛ-subunits of F₁ have been omitted. L, T, and O refer to the loose, tight, and open states, respectively, of the ATP binding site.