Uni-site ATP synthesis in thylakoids

Uni-site ATP synthesis was measured with thylakoids. The membrane-bound ATP-synthase, CF₀F₁ was brought into the active, reduced state by illumination in the presence of thioredoxin, dithiothreitol and phosphate. This enzyme contains two tightly bound ATP per CF_oF₁. ATP was released from the enzyme when ADP was added in substoichiometric amounts during illumination. Experiments with [¹⁴C]ADP indicated that after binding the same nucleotide was phosphorylated and released as [¹⁴C]ATP, i.e. only one site is involved in ATP-synthesis (uni-site ATP-synthesis'). The two tightly bound ATP are not involved in the catalytic turnover. The rate constant for ADP binding was (4 ± 2) × 10⁶ M^-1s^-1. Compared to deenergized conditions the rate constant for ADP binding and that for ATP-release were drastically increased, i.e. membrane energization increased the rate constants for the ATP-synthesis direction.