7-Chloro-4-nitrobenzofurazan inactivates chloroplast H(2+)-ATPase by modification of different tyrosines, depending on the presence of ATP.

The addition of 7-chloro-4-nitrobenzofurazan (NBD) to isolated CF1 at pH 7.5 leads to one tyrosine-bound NBD molecule per CF1 in one of the three beta-subunits, concomitantly with the inhibition of the ATPase activity. Addition of ADP prior to NBD-incubation protected the ATPase activity and reduced binding of NBD to beta-subunits. The addition of MgATP prior to modification did not result in protection against modification of the beta-subunit as well as preservation of activity. Cleavage of the NBD-labelled subunits with cyanogen bromide, followed by analysis of the labelled peptides, led to detection of a 14C-labelled peptide of 7 kDa in both cases (+/- ATP-preincubation). Sequence analysis of this peptide showed that in ATP-incubated CF1, tyr beta 328 was modified with NBD-Cl, whereas the ATP free sample contained no NBD bound to this tyrosine. Further digestion of the labelled peptide with protease V8 (Staphylococcus aureus) followed by sequence analysis of the radioactive labelled peptide, led to the detection of beta-tyr362 as the modified amino acid in case of ATP-free CF1. Both tyrosines are closely related to a proposed nucleotide binding region of beta.