文摘
Tyrosine oxidation鈥搑eduction involves proton-coupled electron transfer (PCET) and a reactive radical state. These properties are effectively controlled in enzymes that use tyrosine as a high-potential, one-electron redox cofactor. The 伪3Y model protein contains Y32, which can be reversibly oxidized and reduced in voltammetry measurements. Structural and kinetic properties of 伪3Y are presented. A solution NMR structural analysis reveals that Y32 is the most deeply buried residue in 伪3Y. Time-resolved spectroscopy using a soluble flash-quench generated [Ru(2,2鈥?bipyridine)3]3+ oxidant provides high-quality Y32鈥揙鈥?absorption spectra. The rate constant of Y32 oxidation (kPCET) is pH dependent: 1.4 脳 104 M鈥? s鈥? (pH 5.5), 1.8 脳 105 M鈥? s鈥? (pH 8.5), 5.4 脳 103 M鈥? s鈥? (pD 5.5), and 4.0 脳 104 M鈥? s鈥? (pD 8.5). kH/kD of Y32 oxidation is 2.5 卤 0.5 and 4.5 卤 0.9 at pH(D) 5.5 and 8.5, respectively. These pH and isotope characteristics suggest a concerted or stepwise, proton-first Y32 oxidation mechanism. The photochemical yield of Y32鈥揙鈥?is 28鈥?8% versus the concentration of [Ru(2,2鈥?bipyridine)3]3+. Y32鈥揙鈥?decays slowly, t1/2 in the range of 2鈥?0 s, at both pH 5.5 and 8.5, via radical鈥搑adical dimerization as shown by second-order kinetics and fluorescence data. The high stability of Y32鈥揙鈥?is discussed relative to the structural properties of the Y32 site. Finally, the static 伪3Y NMR structure cannot explain (i) how the phenolic proton released upon oxidation is removed or (ii) how two Y32鈥揙鈥?come together to form dityrosine. These observations suggest that the dynamic properties of the protein ensemble may play an essential role in controlling the PCET and radical decay characteristics of 伪3Y.