Identification of the essential tyrosine residue in the beta subunit of bovine heart mitochondrial F1-ATPase that is modified by 7-chloro-4-nitro[14C]benzofurazan

Abstract

Inactivation of the bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, with a 4-fold molar excess of 7-chloro-4-nitro[14C]benzofurazan at pH 7.5, led to the incorporation of 1.42 g atoms of 14C/mol. Treatment of the inactivated enzyme with dithiothreitol removed 0.99 g atom of 14C/mol of enzyme which was accompanied by reactivation of the ATPase. Therefore, of the 1.42 mol of 7-chloro-4-nitro-[14C]benzofurazan incorporated per mol of bovine heart mitochondrial F1-ATPase, 0.43 mol was present on lysine residues and 0.99 mol was present on tyrosine residues. When the inactivated enzyme was treated with 10 mM sodium dithionite at pH 6.0, 10% of the activity was recovered which was accompanied by a 10% loss in covalently bound 14C. Following dithionite treatment, that part of the 14C which remained covalently bound could not be removed by subsequent treatment of the labeled enzyme with dithiothreitol. It is presumed that dithionite reduces the 4-nitro group of the covalently bound reagent, converting it to 4-amino[14C]benzofurazan derivatives at lysine and tyrosine residues. The moles of 4-amino[14C]benzofurazan incorporated per mol of the isolated subunits were: alpha, 0.18; beta, 0.30; gamma, 0.03; and delta plus epsilon, less than 0.01. Gel filtration of a cyanogen bromide digest of the labeled beta subunit on Sephadex G-75 resolved a major 14C peak which contained 83% of the 14C recovered. The major, radioactive tryptic fragment derived from this peak was purified by gel filtration on Sephadex G-75 followed by reversed phase high performance liquid chromatography. Automatic Edman degradation of this peptide showed that the 14C was released at the position occupied by beta-Tyr-311.