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

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Abstract

The 8-kDa subunit c of theE. coli F0 ATP-synthase proton channel was tested for Ca++ binding activity using a45Ca++ ligand blot assay after transferring the protein from SDS-PAGE gels onto polyvinyl difluoride membranes. The purified subunit c binds45Ca++ 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 twoE. coli mutants, Asp61→Asn and Asp61→Gly. The Ca++ binding is pH dependent in both theE. 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 theE. 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++ binding can modulate acid-base jump ATP formation. The Ca++ 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.

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Authors and Affiliations

  1. Department of Biological Sciences, Purdue University, 47907, West Lafayette, Indiana

    Stanislav D. Zakharov, Xia Li, Taya P. Red'ko & Richard A. Dilley

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  1. Stanislav D. Zakharov
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  2. Xia Li
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  3. Taya P. Red'ko
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  4. Richard A. Dilley
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Additional information

This work was supported in part by grants from the Department of Energy and the U.S. Department of Agriculture.

On leave from the Institute of Soil Science and Photosynthesis, Russian Academy of Science, Pushchino, Russia.

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Zakharov, S.D., Li, X., Red'ko, T.P. et al. Calcium binding to the subunit c ofE. coli ATP-synthase and possible functional implications in energy coupling. J Bioenerg Biomembr 28, 483–494 (1996). https://doi.org/10.1007/BF02110438

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  • DOI: https://doi.org/10.1007/BF02110438

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