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

Yang Yu †, Xiaoxuan Lv ‡¶, Jiasong Li ‡¶, Qing Zhou ‡¶, Chang Cui ∥, Parisa Hosseinzadeh §, Arnab Mukherjee ∥, Mark J. Nilges ⊥, Jiangyun Wang *‡¶, and Yi Lu *†∥§

‡ Laboratory of Non-Coding RNA, Institute of Biophysics, Chinese Academy of Sciences, Chaoyang District, Beijing, 100101, P. R. China

¶ University of Chinese Academy of Sciences, Beijing 100049, P. R. China

†Center of Biophysics and Computational Biology, ∥Department of Chemistry, §Department of Biochemistry,⊥Illinois EPR Research Center, University of Illinois at Urbana−Champaign, Urbana, Illinois 61801, United States

J. Am. Chem. Soc., 2015, 137 (14), pp 4594–4597

DOI: 10.1021/ja5109936

Publication Date (Web): February 11, 2015

Abstract

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa 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 pKa 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 pKa 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.