ABSTRACT
Tryptophan and tyrosine can form radical intermediates that enable long-range, multistep electron transfer (ET) reactions in proteins. This report describes the mechanisms of formation and quenching of a neutral tryptophan radical in azurin, a blue-copper protein that contains native tyrosine (Y108 and Y72) and tryptophan (W48) residues. A long-lived neutral tryptophan radical W48⢠is formed upon UV-photoexcitation of a zinc(II)-substituted azurin mutant in the presence of an external electron acceptor. The quantum yield of W48⢠formation (Φ) depends upon the tyrosine residues in the protein. A tyrosine-deficient mutant, Zn(II)Az48W, exhibited a value of Φ = 0.080 with a Co(III) electron acceptor. A nearly identical quantum yield was observed when the electron acceptor was the analogous tyrosine-free, copper(II) mutant; this result for the Zn(II)Az48W:Cu(II)Az48W mixture suggests there is an interprotein ET path. A single tyrosine residue at one of the native positions reduced the quantum yield to 0.062 (Y108) or 0.067 (Y72). Wild-type azurin with two tyrosine residues exhibited a quantum yield of Φ = 0.045. These data indicate that tyrosine is able to quench the tryptophan radical in azurin.
