The type I Cu site in the Cys457Ser mutant of Myrothecium verrucaria bilirubin oxidase was vacant, but the trinuclear center composed of a type II Cu and a pair of type III Cu's was fully occupied by three Cu ions. Cys457Ser could react with dioxygen, affording reaction intermediate I with absorption maxima at 340, 470, and 675 nm. This intermediate corresponds to that obtained from laccase, whose type I Cu is cupric and type II and III Cu's are cuprous [Zoppellaro, G., Sakurai, T., and Huang, H. (2001) J. Biochem. 129, 949-953] or whose type I Cu is substituted with Hg [Palmer, A. E., Lee, S. K., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 6591-6599]. Another type I Cu mutant, Met467Gln, with modified spectroscopic properties and redox potential, afforded reaction intermediate II with absorption maxima at 355 and 450 nm. This intermediate corresponds to that obtained during the reaction of laccase [Sundaram, U. M., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 12525-12540; Huang, H., Zoppellaro, G., and Sakurai, T. (1999) J. Biol. Chem. 274, 32718-32724]. According to a three-dimensional model of bilirubin oxidase, Asp105 is positioned near the trinuclear center. Asp105Glu and Asp105Ala exhibited 46 and 7.5% bilirubin oxidase activity compared to the wild-type enzyme, respectively, indicating that Asp105 conserved in all multi-copper oxidases donates a proton to reaction intermediates I and II. In addition, this amino acid might be involved in the formation of the trinuclear center and in the binding of dioxygen based on the difficulties in incorporating four Cu ions in Asp105Ala and Asp105Asn and their reactions with dioxygen.
