Calcium binding to the chloroplast andE. coli (CF0) F0 subunit III (c) of the ATP-synthase

Abstract

Subunit III and c, the 8 kDa components of the chloroplast CF0, andE. coli H+ channel complexes respectively, were isolated and purified for the purpose of studying their Ca++-binding properties. Purified subunit III or c as well as the unfractionated organic-solvent soluble preparation from chloroplasts were used in a45Ca++-ligand blot assay known to detect high affinity Ca++-binding sites in proteins. Both subunit III and c showed strong45Ca++-binding. None of the other CF0 subunits bound Ca++ and of the CF1 only a weak binding was detected in the region of the α,β subunits. The Ca++-binding was inhibited after treating the proteins in solution by derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La3+. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu 61 (Asp 61 inE. 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 the carboxyl group of the subunit III (or c) C-terminal provide some of seven essential oxygen ligands normally required for defining a Ca++-binding site in proteins. Based on the accepted models for the hairpin conformation of the subunit III (c), it seems clear that the Ca++-binding site can form on the lumenal side of the membrane in the functional CF0 structures or on the periplasmic side of theE. coli membrane. A working hypothesis we are testing is that Ca++-binding to the CF0 (or F0) can form an easily reversible gating site such as to enhance the probability for membrane-localized H+ gradients being coupled to ATP formation under moderate energization loads, but under excess energization the local H+ ion concentration may build up high enough to displace the bound Ca++, resulting in delocalization of the H+ gradient. The latter situation seems, in chloroplasts at least, to function as a signal for over-energization; i.e., excess light absorption, a potential stress situation for plants. Lumenal acidification appears to be a trigger for initiating stress alleviation responses.