Introduction of tryptophan near heme greatly increased the dye-decolorizing activity of cytochrome P460 from Nitrosomonas europaea.

Dye-decolorizing peroxidases (DyPs) are promising for the bioremediation of diverse synthetic dyes in wastewater; however, their practical application is severely limited by their acidic pH optima and inefficient functional expression in Escherichia coli, which often results in heme-free forms. To overcome these limitations, we focused on P460 from Nitrosomonas europaea, a c-type hemoprotein containing a covalently attached heme that exhibits guaiacol peroxidase activity under near-neutral conditions. Recombinant P460 was evaluated by monitoring the degradation of Reactive Blue 19 (RB19). P460 efficiently degraded RB19 at pH 8.0, retaining high activity under conditions where DyPs are inactive. To efficiently degrade bulky dyes, we engineered a surface radical site by substituting a solvent-exposed Ala47 with tryptophan, based on the long-range electron transfer mechanism observed in DyPs. The resulting A47W mutant exhibited a markedly enhanced degradation rate of 2.3 s-1 at 5 μM RB19, approximately 20-fold higher than that of the wild type (0.11 s-1). Kinetic analyses of the reaction with H2O2 revealed that, unlike the wild-type, the A47W mutant did not form the high-valent two-electron oxidized species (Compound I); instead, only a one-electron oxidized species was detected. These results indicate that electron transfer from the engineered tryptophan forms a tryptophan radical (a Compound ES-like intermediate), leading to the improvement in dye-decolorizing activity. Our findings demonstrate that P460 circumvents the pH limitations of DyPs, while rational engineering successfully imparts potent activity toward bulky dyes. This suggests that the P460 platform could lead to the realization of an effective E. coli-based dye-decolorizing system.