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

Manganese and copper were released from spinach chloroplasts by NaCN‐treatment, though iron was not affected. The Hill reaction activity was also inhibited by this treatment, but was partially recovered by the addition of either Mn 2+ or Cu 2+ but not of Fe 3+ The interaction of Mn 2+ with manganese‐depleted chloroplasts by NaCN‐treatment was studied using 54 Mn 2+ . A Scatchard plot shows the high and low affinity binding sites of Mn 2+ on NaCN‐treated chloroplast membrane; high affinity binding being specific for NaCN‐treated chloroplast with a binding constant, K H , of 1.9 × 105 M −1 , and a maximum binding number, N H , of 0.0016g‐atom per mole of chlorophyll. The low binding site was also found on untreated chloroplasts; its binding constant, K L , being 1.2 × 10 4 M −1 , and its maximum binding number, N L , of 0.0112 g‐atom per mole of chlorophyll at pH 8.2. N H was proportional to the degree of the removal of Mn by NaCN‐treatment and was constant at pH 4–9. N L 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 Mn 2+ . Within I min in the dark the binding reaction reached equilibrium in the absence of pyrophosphate, however, 20 min were required to transform into pyrophosphate‐resistant form. The pH dependence of the binding of Mn 2+ with pK 2 7.2 and the ineffectiveness of p ‐chloromercuribenzoate suggest the possible ligand of Mn 2+ is the imidazole nitrogen of the histidine residue.