PLANT PHYSIOLOGY , Vol 115, Issue 4 1549-1555, Copyright © 1997 by American Society of Plant Biologists
Modification of Sulfhydryl Groups in the [gamma]-Subunit of Chloroplast-Coupling Factor 1 Affects the Proton Slip through the ATP Synthase
Y. Evron and U. Pick
Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
A large proton leak not coupled to ATP synthesis (slip) occurs at alkaline
pH through the chloroplast ATP synthase (Y. Evron, M. Avron [1990] Biochim
Biophys Acta 1019: 115-120). The involvement of the ATP synthase
[gamma]-subunit in the regulation of proton conductance was analyzed by
measuring the effect of thiolalkylating agents on proton slip. Alkylation
by N-ethylmaleimide of [gamma]-cysteine (Cys)-89, which is exposed upon
energization of thylakoids, increases the slip only at alkaline pH. The
slip is partially suppressed by low concentrations of adenine nucleotides
and is completely eliminated by venturicidin, a blocker of the hydrophobic
polypeptide complex of the chloroplast ATP synthase (CF0). Conversely,
cross-linking of [gamma]-Cys-89 with [gamma]-Cys-322 renders the ATP
synthase leaky to protons and insensitive to ATP also at neutral pH. The
accessibility of [gamma]-Cys-89 to alkylation by fluorescein maleimide is
completely suppressed by N,N-dicyclohexylcarbodiimide and by venturicidin,
which block proton conductance through CF0 and increase the pH gradient.
These results suggest that the [gamma]-subunit has a dominant role in
proton gating through the ATP synthase and responds to changes in pH and
ligands taking place on either side of the thylakoid membrane. It is
proposed that the conformational changes that induce the proton slip and
the exposure of [gamma]-Cys-89 reflect the conversion of the enzyme from a
catalytically latent to an active state, and depend on the deprotonation of
a stromal site at alkaline pH and on protonation of an intrathylakoid inner
site upon energization. Therefore, conditions that induce the
conformational activation also provide the driving force for ATP synthesis.
