Two Oxidation Sites for Low Redox Potential Substrates: A DIRECTED MUTAGENESIS,KINETIC, AND CRYSTALLOGRAPHIC STUDY ON PLEUROTUS ERYNGII VERSATILE PEROXIDASE*

Versatile peroxidase shares with manganese peroxidase and lignin peroxidase theability to oxidize Mn²⁺ and high redox potential aromaticcompounds, respectively. Moreover, it is also able to oxidize phenols (and lowredox potential dyes) at two catalytic sites, as shown by biphasic kinetics. Ahigh efficiency site (with 2,6-dimethoxyphenol andp-hydroquinone catalytic efficiencies of ∼70 and∼700 s⁻¹ mm⁻¹, respectively)was localized at the same exposed Trp-164 responsible for high redox potentialsubstrate oxidation (as shown by activity loss in the W164S variant). The secondsite, characterized by low catalytic efficiency (∼3 and ∼50s⁻¹ mm⁻¹ for2,6-dimethoxyphenol and p-hydroquinone, respectively) waslocalized at the main heme access channel. Steady-state and transient-statekinetics for oxidation of phenols and dyes at the latter site were improved whenside chains of residues forming the heme channel edge were removed in single andmultiple variants. Among them, the E140G/K176G, E140G/P141G/K176G, andE140G/W164S/K176G variants attained catalytic efficiencies for oxidation of2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) at the heme channelsimilar to those of the exposed tryptophan site. The heme channel enlargementshown by x-ray diffraction of the E140G, P141G, K176G, and E140G/K176G variantswould allow a better substrate accommodation near the heme, as revealed by theup to 26-fold lower K_m values (compared with nativeVP). The resulting interactions were shown by the x-ray structure of theE140G-guaiacol complex, which includes two H-bonds of the substrate with Arg-43and Pro-139 in the distal heme pocket (at the end of the heme channel) andseveral hydrophobic interactions with other residues and the heme cofactor.