Divalent cation binding to chloroplast membranes and its relationship to reversal of quaternary ammonium salt uncoupling of photophosphorylation

In order to elucidate the mechanism by which divalent cations such as Mn²⁺ reverse the uncoupling effects of zwitterionic buffers such as tricine and quaternary ammonium salts such as tetraethylammonium chloride (TEA), a study was made of the interaction between manganese ions and the chloroplast membrane. It was found that the chloroplast membrane binds manganese ions to the extent of 0.4–0.6 μmoles/mg chlorophyll in the dark with a dissociation constant of 6.7 μm, which corresponds very well to the concentration required for half-maximal reversal of tricine uncoupling. Manganese was displaced from the chloroplasts by the uncouplers choline and TEA as well as by other agents such as Na⁺, K⁺, Mg²⁺, Ca²⁺ or Zn²⁺ which reverse TEA uncoupling. Displacement of Mn²⁺ ions by TEA and K⁺ was noncompetitive suggesting that they do not bind to the same sites as manganese. On the other hand, displacement by Zn²⁺ was competitive suggesting that Mn²⁺ and Zn²⁺ act at the same sites. Light had no effect on manganese binding indicating that binding, not light-induced transport, is responsible for the recoupling effects.