Removal of Mn from Spinach Chloroplasts by Sodium Cyanide and the Binding of Mn 2+ to Mn-Depleted Chloroplasts

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Abstract
Mn and Cu were released from spinach chloroplasts by NaCN-treatment, though Fe was not affected. The Hill reaction activity was also inhibited by this treatment, but was partially recovered by the addition of either Mn2+ or Cu2+, but not of Fe3+. The interaction of Mn2+ with Mn-depleted chloroplasts by NaCN-treatment was studied using 54Mn2+. A Scatchard plot showed the high and low affinity binding sites of Mn2+ on NaCN-treated chloroplast membrane; high affinity binding being specific for NaCN-treated chloroplast with a binding constant, KH, of 1.9 .times. 105 M-1 and a maximum binding number, NH, of 0.0016 g-atom/mole of chlorophyll. The low binding site was also found on untreated chloroplasts; its binding constant, KL, being 1.2 .times. 104 M-1, and its maximum binding number, NL, of 0.0112 g-atom/mole of chlorophyll at pH 8.2. NH was proportional to the degree of the removal of Mn by NaCN-treatment and was constant at pH 4-9. NL markedly increased at a high pH with a midpoint of pH 7.9, indicating the exposure of a new, similar binding site. Light illumination partially inhibited the binding of Mn2+. Within 1 min in the dark, the binding reaction reached equilibrium in the absence of pyrophosphate; 20 min were required to transform into pyrophosphate-resistant form. The pH dependence of the binding of Mn2+ with pKa 7.2 and the ineffectiveness of p-chloro-mercuribenzoate suggested the possible ligand of Mn2+ was the imidazole nitrogen of the histidine residue.