A radical on the Met-Tyr-Trp modification required for catalase activity in catalase-peroxidase is established by isotopic labeling and site-directed mutagenesis

摘要

A transient tyrosyl-like radical with a narrow doublet X-band EPR signal is present during catalase turnover by M. tuberculosis catalase-peroxidase (KatG). Labeling of KatG with β-methylene deuterated tyrosine causes a collapse of the doublet to a singlet, while for 3,5- ring-deuterated tyrosine labeled enzyme, no changes occur in the EPR signal. Except for the replacement Tyr229Phe, all other single tyrosine mutants of KatG exhibit the same narrow doublet EPR signal and catalase activity similar to wild-type enzyme. These findings confirm that this catalytically competent radical is associated with Tyr229, whose 3′, 5′ protons are replaced due to crosslinks with neighboring Met255 and Trp107 side chains in the post-translationally modified enzyme containing a distal side Met255Tyr229Trp107 adduct.