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submitted on 18.07.2020 and posted on 20.07.2020by Patrycja Kielb, Harry B. Gray, Jay R. Winkler
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 (CuT1) and then are transferred over
13 Å to a trinuclear Cu site (TNC: (CuT3)2CuT2)
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 CuT1
center, a Tyr108·
radical near the TNC forms rapidly. We have found that ascorbate reduces the
in wild-type SLAC about 10 times faster than it reacts with the CuT12+
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.