Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pK_a and reduction potential in O₂ reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O₂ reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pK_a of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O₂ reduction activity and the fraction of H₂O formation with the pK_a of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H₂O₂, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.