Selectivity of modification when latent and activated forms of the chloroplast F 1-ATPase are inactivated by 7-chloro-4-nitrobenzofurazan

Abstract

The characteristics and specificity of inactivation of the chloroplast F 1-ATPase (CF 1) with 7-chloro-4-nitrobenzofurazan (Nbf-Cl) have been investigated. Inactivation of the octylglucoside-dependent Mg 2+-ATPase activity of latent CF 1 by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. Following inactivation of CF 1 with [ 14C]Nbf-Cl, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the majority of the radioactive reagent incorporated is present in the β subunit. Treatment of the enzyme with [ 14C]Nbf-Cl following dithiothreitol heat activation, led to similar labeling of the β subunit and substantial incorporation of 14C into the γ subunit. On complete inactivation, about 4 mol of Nbf-S-Cys is formed per mole of dithiothreitol-heat-activated CF 1. Incorporation of 14C into the γ subunit is prevented by prior treatment of the latent CF 1 or of the dithiothreitol-heat-activated CF 1 with iodoacetamide. Following incubation of the dithiothreitol-heat-activated CF 1 with iodoacetamide, complete inactivation of the octylglucoside-dependent Mg 2+-ATPase activity by Nbf-Cl can be correlated with the formation of about 1.2 mol of Nbf-O-Tyr per mole of enzyme. After stabilization of the [ 14C]Nbf-O-Tyr derivative by treatment with sodium dithionite, a labeled peptide was purified. Automatic Edman degradation of this peptide revealed the sequence V-X-V-P-A-D-(D). The majority of the radioactivity was cleaved in the second cycle, the position occupied in CF 1 by Tyr-β-328, which is homologous to Tyr-β-311, the residue reactive with Nbf-Cl in the beef heart mitochondrial F 1-ATPase. When CF 1, modified at Tyr-β-328 with Nbf-Cl, is incubated at pH 9.0, the Nbf-O-Tyr adduct is hydrolyzed, leading to concomitant recovery of the ATPase activity. In double labeling experiments, two-dimensional isoelectric focusing in the presence of urea followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that 2-azido-ADP, covalently bound at the tight ADP binding site, and the tyrosine modified by [ 14C]Nbf-Cl are located in different β subunits.