Calcium gating of H+ fluxes in chloroplasts affects acid-base-driven ATP formation |
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Authors: | Dennis C. Wooten Richard A. Dilley |
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Affiliation: | (1) Department of Biological Sciences, Purdue University, 47907 West Lafayette, Indiana |
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Abstract: | In previous work, calcium ions, bound at the lumenal side of the CF0H+ channel, were suggested to keep a H+ flux gating site closed, favoring sequestered domain H+ ions flowing directly into the CF0-CF1 and driving ATP formation by a localized gradient. Treatments expected to displace Ca++ from binding sites had the effect of allowing H+ ions in the sequestered domains to equilibrate with the lumen, and energy coupling showed delocalized characteristics. The existence of such a gating function implies that a closed-gate configuration would block lumenal H+ ions from entering the CF0-CF1 complex. In this work that prediction was tested using as an assay the dark, acid-base jump ATP formation phenomenon driven by H+ ions derived from succinic acid loaded into the lumen.Chlorpromazine, a photoaffinity probe for many proteins having high-affinity Ca++-binding sites, covalently binds to the 8-kDa CF0 subunit in the largest amounts when there is sufficient Ca++ to favor the localized energy coupling mode, i.e., the gate closed configuration. Photoaffinity-bound chlorpromazine blocked 50% or more of the succinate-dependent acid-base jump ATP formation, provided that the ionic conditions during the UV photoaffinity treatment were those which favor a localized energy coupling pattern and a higher level of chlorpromazine labeling of the 8-kDa CF0 subunit. Thylakoids held under conditions favoring a delocalized energy coupling mode and less chlorpromazine labeling of the CF0 subunit did not show any inhibition of acid-base jump ATP formation.Chlorpromazine and calmidazolium, another Ca++-binding site probe, were also shown to block redox-derived H+ initially released into sequestered domains from entering the lumen, at low levels of domain H+ accumulation, but not at higher H+ uptake levels; ie., the closed gate state can be overcome by sufficiently acidic conditions. That is consistent with the observation that the inhibition of lumenal succinate-dependent ATP formation by photoaffinity-attached chlorpromazine can be reversed by lowering the pH of the acid stage from 5.5 to 4.5.The evidence is consistent with the concept that Ca++ bound at the lumenal side of the CF0 H+ channel can block H+ flux from either direction, consistent with the existence of a molecular structure in the CF0 complex having the properties of a gate for H+ flux across the inner boundary of the CF0. Such a gate could control the expression of localized or delocalized energy coupling gradients. |
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Keywords: | Energy coupling ATP formation H+ gradients Ca++ binding in thylakoids |
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