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 <i>Streptomyces coelicolor</i> laccase (SLAC). During normal enzymatic turnover in laccases, reducing equivalents are delivered to a type 1 Cu center (Cu<sub>T1</sub>) and then are transferred over 13 Å to a trinuclear Cu site (TNC: (Cu<sub>T3</sub>)<sub>2</sub>Cu<sub>T2</sub>) where O<sub>2</sub> 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 O<sub>2</sub> reacts with a reduced SLAC variant lacking the Cu<sub>T1</sub> center, a Tyr108<sup>·</sup> radical near the TNC forms rapidly. We have found that ascorbate reduces the Tyr108<sup>·</sup><sup> </sup>radical in wild-type SLAC about 10 times faster than it reacts with the Cu<sub>T1</sub><sup>2+</sup> 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<sup>·</sup><sup> </sup>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.