Does Tyrosine Protect S. Coelicolor Laccase from Oxidative Degradation?

We have investigated the roles of tyrosine (Tyr) and tryptophan (Trp) residues in the four-electron reduction of oxygen catalyzed by Streptomyces coelicolor laccase (SLAC). During normal enzymatic turnover in laccases, reducing equivalents are delivered to a type 1 Cu center (Cu_T1) and then are transferred over 13 Å to a trinuclear Cu site (TNC: (Cu_T3)₂Cu_T2) where O₂ reduction occurs. The TNC in SLAC is surrounded by a large cluster of Tyr and Trp residues that can provide reducing equivalents when the normal flow of electrons is disrupted. Canters and coworkers have shown that when O₂ reacts with a reduced SLAC variant lacking the Cu_T1 center, a Tyr108^· radical near the TNC forms rapidly. We have found that ascorbate reduces the Tyr108^· radical in wild-type SLAC about 10 times faster than it reacts with the Cu_T1²⁺ center, possibly owing to radical transfer along a Tyr/Trp chain. Aerobic oxidation of two reduced SLAC mutants (Y108F and W132F) leads to the formation of a long-lived (~15 min) Tyr^· radical with distinct absorption at 408 nm. The diffusion of redox equivalents away from the primary enzymatic pathway in SLAC may indicate a poorly optimized enzyme or a mechanism to protect against protein damage.