Characterisation of phosphate binding to mitochondrial and bacterial membrane-bound ATP synthase by studies of inhibition with 4-chloro-7-nitrobenzofurazan.

The effect of phosphate on the inhibition by 4-chloro-7-nitrobenzofurazan of the ATPase activity of the proton-translocating ATP synthase in heart submitochondrial particles was investigated. Binding of phosphate protected strongly against the inhibition. A dissociation constant of 0.2 mM was determined for the enzyme X Pi complex and shown to be independent of pH in the range 7.0-8.0. The protective effect of phosphate was mimicked by arsenate but not by sulphate or malonate. Similar results were obtained for the enzyme from Paracoccus denitrificans. 2,4-Dinitrophenol enhanced phosphate binding to the mitochondrial enzyme since the protective effect of phosphate was increased. The data are compatible with protection arising from binding of phosphate to a catalytic site.