The nature of the reaction of an essential tyrosine residue of bovine heart mitochondrial ATPase with 4-chloro-7-nitrobenzofurazan and related compounds.

Bovine heart mitochondrial ATPase is inhibited after covalent modification with 4-chloro-7-nitrobenzofuroxan. The kinetics of the reaction are indistinguishable from those for the reaction of an essential tyrosine residue of the ATPase with 4-chloro-7-nitrobenzofurazan that have been described previously [Ferguson et al. (1975) Eur. J. Biochem. 54, 117-126]. 4-Fluoro-7-nitrobenzofurazan inhibits the ATPase with a pseudo-first-order rate constant that is tenfold greater than that for 4-chloro-7-nitrobenzofurazan. These data indicate that the rate-limiting step for reaction of the enzyme with these reagents is formation of a Meisenheimer complex at the C-4 position and that the modified tyrosine is probably on the surface of the protein. No evidence was found for more complex patterns of reactivity of 4-chloro-7-nitrobenzofurazan and its analogues. Both ammonium 4-chloro-7-sulphobenzofurazan and ammonium 4-fluoro-7-sulphobenzofurazan fail to react with the ATPase. The utility of these reagents as alternatives to the nitro derivatives may be limited owing to their slow reaction rates. After modification on tyrosine by 4-chloro-7-nitrobenzofurazan, the nitrobenzofurazan group can be transferred by an intramolecular process to lysine [Ferguson et al. (1975) Eur. J. Biochem. 54, 127-133]. ATPase with the lysine thus modified is shown to be reactive towards 4-chloro-7-nitrobenzofurazan in a manner indistinguishable from the native enzyme. This indicates that the intramolecular transfer occurs at sufficient distance to avoid steric hindrance to the second reaction, and that the lysine does not participate in a neighbouring group effect to enhance the reactivity of the tyrosine.