Identification of the lysine residue to which the 4-nitrobenzofurazan group migrates after the bovine mitochondrial F1-ATPase is inactivated with 7-chloro-4-nitro14Cbenzofurazan.

When bovine heart mitochondrial F1-ATPase, taken as alpha 3 beta 3 gamma delta epsilon with a molecular weight of 375,000, was inactivated by greater than 90% with a 4-fold molar excess of 7-chloro-4-nitro14Cbenzofurazan at pH 7.4, 1.15 mol of 4-nitrobenzofurazan 14CNbf were incorporated per mol of enzyme. Reactivation of a sample of the modified enzyme with dithiothreitol removed 0.82 mol of 14CNbf/mol of the F1-ATPase indicating that, of the 1.15 mol of 14CNbf incorporated, 0.82 mol were present on tyrosine residues and 0.33 mol on lysine residues. Incubation of the modified enzyme at pH 9.0 for 18 h at 23 degrees C led to an increase of 0.64 mol of 14CNbf-N'-Lys/mol of the F1-ATPase which occurred as a consequence of an O----N migration. About 15% enzyme reactivation occurred simultaneously with the migration indicating that the fraction of the 14CNbf group originally present on tyrosine which did not migrate was lost by hydrolysis. Examination of a tryptic digest of the labeled enzyme after the O----N migration by reversed-phase high-pressure liquid chromatography revealed a single major radioactive peptide. The labeled tryptic fragment was purified and subjected to automatic Edman degradation. This analysis revealed that Lys-beta-162 was specifically labeled during the O----N migration of the 14CNbf group.