Kinetic Analysis of Cooperative Interactions Induced by Mn²⁺ Binding to the Chloroplast H⁺-ATPase

The kinetics of Mn²⁺ binding to three cooperatively interacting sites in chloroplast H⁺-ATPase (CF₁) were measured by EPR following rapid mixing of the enzyme with MnCl₂ with a time resolution of 8 ms. Mixing of the enzyme-bound Mn²⁺ with MgCl₂ gave a measure of the rate of exchange. The data could be best fitted to a kinetic model assuming three sequential, positively cooperative binding sites. (1) In the latent CF₁ the binding to all three sites had a similar on-rate constants of (1.1 ± 0.04) × 10⁴ M⁻¹ s⁻¹. (2) Site segregation was found in the release of ions with off-rate constants of 0.69 ± 0.04 s⁻¹ for the first two and 0.055 ± 0.003 s⁻¹ for the third. (3) Addition of one ADP per CF₁ caused a decrease in the off-rate constants to 0.31 ± 0.02 and 0.033 ± 0.008 s⁻¹ for the first two and the third sites, respectively. (4) Heat activation of CF₁ increased the on-rate constant to (4.2 ± 0.92) × 10⁴ M⁻¹ s⁻¹ and the off-rate constants of the first two and the third site to 1.34 ± 0.08 and 0.16 ± 0.07 s⁻¹, respectively. (5) The calculated thermodynamic dissociation constants were similar to those previously obtained from equilibrium binding studies. These findings were correlated to the rate constants obtained from studies of the catalysis and regulation of the H⁺-ATPase. The data support the suggestion that regulation induces sequential progress of catalysis through the three active sites of the enzyme.