CALCIUM-BINDING TO THE SUBUNIT-C OF ESCHERICHIA-COLI ATP-SYNTHASE ANDPOSSIBLE FUNCTIONAL IMPLICATIONS IN ENERGY COUPLING

The 8-kDa subunit c of the E. coli F-0 ATP-synthase proton channel was tested for Ca++ binding activity using a Ca-45(++) ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl diflu oride membranes. The purified subunit c binds Ca-45(++) 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 carbony l group seems necessary for Ca++ binding capacity, shown by loss of Ca ++ binding following removal of the formyl group by mild acid treatmen t. The dicyclohexylcarbodiimide-reactive Asp-61 is not involved in the Ca++ binding, shown by Ca++ binding being retained in two E. coli mut ants, 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 increas e the Ca++ binding affinity to its F-0 binding site (chlorpromazine ph otoaffinity attachment) caused an inhibition of ATP formation driven b y a base-to-acid pH jump in whole cells. Inhibition was not observed w hen the Ca++ chelator EGTA was present with the cells during the chlor promazine photoaffinity treatment, An apparent Ca++ binding constant o n the site responsible for the UV plus chlorpromazine effect of near 8 0-100 nM was obtained using an EGTA-Ca++ buffer system to control free Ca++ concentration during the UV plus chlorpromazine treatment. The d ata are consistent with the notion that Ca++ bound to the periplasimic side of the E. coli F-0 proton channel can block H+ entry into the ch annel. A similar effect occurs in thylakoid membranes, but the Ca++ bi nding site is on the lumen side of the thylakoid, where Ca++ binding c an modulate acid-base jump ATP formation, The Ca++ binding to the F-0 and CF0 complexes is consistent with a pH-dependent gating mechanism f or control of H+ ion flux across the opening of the H+ channel.