CALCIUM GATING OF H-BASE-DRIVEN ATP FORMATION( FLUXES IN CHLOROPLASTSAFFECTS ACID)

In previous work, calcium ions, bound at the lumenal side of the CFoH channel, were suggested to keep a H+ flux gating site closed, favorin g sequestered domain H+ ions flowing directly into the CFo-CF1 and dri ving ATP formation by a localized Delta(mu)(similar to)H(+) gradient. Treatments expected to displace Ca++ from binding sites had the effect of allowing H+ ions in the sequestered domains to equilibrate with th e lumen, and energy coupling showed delocalized characteristics. The e xistence of such a gating function implies that a closed-gate configur ation would block lumenal H+ ions from entering the CFo-CF1 complex. I n this work that prediction was tested using as an assay the dark, aci d-base jump ATP formation phenomenon driven by H+ ions derived from su ccinic acid loaded into the lumen. Chlorpromazine, a photoaffnity prob e for many proteins having high-affinity Ca++ binding sites, covalentl y binds to the 8-kDa CFo 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 blo cked 50% or more of the succinate-dependent acid-base jump ATP formati on, provided that the ionic conditions during the UV photoaffinity tre atment were those which favor a localized energy coupling pattern and a higher level of chlorpromazine labeling of the 8-kDa CFo subunit. Th ylakoids held under conditions favoring a delocalized energy coupling mode and less chlorpromazine labeling of the CFo subunit did not show any inhibition of acid-base jump ATP formation. Chlorpromazine and cal midazolium, another Ca++ -binding site probe, were also shown to block redox-derived H+ initially released into sequestered domains from ent ering the lumen, at low levels of domain H+ accumulation, but not at h igher H+ uptake levels; ie., the closed gate state can be overcome by sufficiently acidic conditions. That is consistent with the observatio n 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 CFo H+ channel can block H+ flux from either direction, consistent with the existence of a molecular structure in the CFo complex having the properties of a gate for H+ flux across the inner boundary of the CFo. Such a gate c ould control the expression localized or delocalized Delta (similar to ) H-mu(+) energy coupling gradients.