Site-directed mutagenesis of a possible type 1 copper ligand of bilirubin oxidase; a Met467Gln mutant shows stellacyanin-like properties

In our previous paper, we reported a mutant of recombinant Myrothecium verr ucaria bilirubin oxidase, in which the Met467 residue was replaced by Gly [ Shimizu, A, et at. (1999) Biochemistry 38, 3034-3042]. This mutant displaye d a remarkable reduction in enzymatic activity and an evident decrease in t he intensity of the absorption band around 600 nm (type 1 charge transfer t ransition). In this study, we report the preparation of three Met467 mutant s (Met467Gln, Met467His, and Met467Arg) and characterize their enzymatic ac tivities, midpoint potentials, and absorption and ESR spectra. Met467His an d Met467Arg show no enzymatic activity and a great reduction in the intensi ty of the absorption band around 600 nm. Furthermore, their ESR spectra sho w no type 1 copper signal, but only a type 2 copper signal; however, oxidat ion by ferricyanide caused the type 1 copper signal to appear. On the other hand, Met467Gln as expressed shows both type 1 and type 2 copper signals i n its ESR spectrum, the type 1 copper atom parameters being very different from usual blue copper proteins but very similar to those of stellacyanin. The enzymatic activity of the Met467Gln mutant for bilirubin is quite low ( 0.3%), but the activity for potassium ferrocyanide is similar (130%) to tha t of the wild type enzyme. These results indicate that Met467 is important for characterizing the features of the type 1 copper of bilirubin oxidase.