Modulating O2 Reactivity in a Fungal Flavoenzyme INVOLVEMENT OF ARYL-ALCOHOL OXIDASE PHE-501 CONTIGUOUS TO CATALYTIC HISTIDINE

Aryl-alcohol oxidase (AAO) is a tlavoenzyme responsible for activation of O-2 to H2O2 in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations show O-2 access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O-2 reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substitution of Phe-501 with tryptophan. The high oxygen catalytic efficiency of F501W, similar to 2-fold that of native AAO and similar to 120-fold that of F501A, seems related to a higher O-2 availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O-2 inside the active-site cavity of AAO (and several in sitico Phe-501 variants) yielded >60% O-2 population at 3-4 angstrom from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O-2 reactivity of AAO decreased when the access channel was enlarged and increased when it was constricted by introducing a tryptophan residue. This is because the side chain of Phe-501, contiguous to the catalytic histidine (His-502 in AAO), helps to position O-2 at an adequate distance from flavin C4a (and His-502 N epsilon). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O-2 reactivity of this flavoenzyme.