CALCIUM-BINDING TO THE CHLOROPLAST AND ESCHERICHIA-COLI (CF0) F-0 SUBUNIT-III (C) OF THE ATP-SYNTHASE

Subunit III and c, the 8 kDa components of the chloroplast CF0, and E. coli H+ channel complexes respectively, were isolated and purified fo r the purpose of studying their Ca++-binding properties. Purified subu nit III or c as well as the unfractionated organic-solvent soluble pre paration from chloroplasts were used in a Ca-45(++)-ligand blot assay known to detect high affinity Ca++-binding sites in proteins. Both sub unit III and c showed strong Ca-45(++)-binding. None of the other CF0 subunits bound Ca++ and of the CF1 only a weak binding was detected in the region of the alpha,beta subunits. The Ca++-binding was inhibited after treating the proteins in solution by derivatizing aqueously exp osed carboxyl groups with a water soluble carbodiimide plus a nucleoph ile, after de-formylation of the N-terminal methionine, or with a subs equent treatment with La3+. Dicyclohexylcarbodiimide treatment (no nuc leophile was added) of thylakoid membranes, which derivatizes the hydr ophobically located Glu 61 (Asp 61 in E. coli), did not inhibit the Ca ++-binding in either protein. The data indicate that for both proteins the carbonyl group of the formylated N-terminal Met-1 and probably th e carboxyl group of the subunit III (or c) C-terminal provide some of seven essential oxygen ligands normally required for defining a Ca++-b inding site in proteins. Based on the accepted models for the hairpin conformation of the subunit III (c), it seems clear that the Ca++-bind ing site can form on the lumenal side of the membrane in the functiona l CF0 structures or on the periplasmic side of the E. coli membrane. A working hypothesis we are testing is that Ca++-binding to the CF0 (or F-0) can form an easily reversible gating site such as to enhance the probability for membrane-localized H+ gradients being coupled to ATP for mation under moderate energization loads, but under excess energiz ation the local H+ ion concentration may build up high enough to displ ace the bound Ca++, resulting in delocalization of the H+ gradient. Th e latter situation seems, in chloroplasts at least, to function as a s ignal for over-energization; i.e., excess light absorption, a potentia l stress situation for plants. Lumenal acidification appears to be a t rigger for initiating stress alleviation responses.