Calcium binding to the subunit c of E. coli ATP-synthase and possible functional implications in energy coupling

The 8-kDa subunit c of the E. coli F0 ATP-synthase proton channel was tested for Ca++ binding activity using a 45Ca++ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds 45Ca++ strongly with Ca++ binding properties very similar to those of the 8-kDa CF0 subunit III of choloroplast thylakoid membranes. The N-terminal f-Met carbonyl group seems necessary for Ca++ binding capacity, shown by loss of Ca++ binding following removal of the formyl group by mild acid treatment. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca++ binding, shown by Ca++ binding being retained in two E. coli mutants, Asp61-->Asn and Asp61-->Gly. The Ca++ binding is pH dependent in both the E. coli and thylakoid 8-kDa proteins, being absent at pH 5.0 and rising to a maximum near pH 9.0. A treatment predicted to increase the Ca++ binding affinity to its F0 binding site (chlorpromazine photoaffinity attachment) caused an inhibition of ATP formation driven by a base-to-acid pH jump in whole cells. Inhibition was not observed when the Ca++ chelator EGTA was present with the cells during the chlorpromazine photoaffinity treatment. An apparent Ca++ binding constant on the site responsible for the UV plus chlorpromazine effect of near 80-100 nM was obtained using an EGTA-Ca++ buffer system to control free Ca++ concentration during the UV plus chlorpromazine treatment. The data are consistent with the notion that Ca++ bound to the periplasimic side of the E. coli F0 proton channel can block H+ entry into the channel. A similar effect occurs in thylakoid membranes, but the Ca++ binding site is on the lumen side of the thylakoid, where Ca+2 binding can modulate acid-base jump ATP formation. The Ca+2 binding to the F0 and CF0 complexes is consistent with a pH-dependent gating mechanism for control of H+ ion flux across the opening of the H+ channel.