Roles of the indole ring of Trp396 covalently bound with the imidazole ring of His398 coordinated to type I copper in bilirubin oxidase.

Bilirubin oxidase has a post-translationally formed covalent-bond between the imidazole ring of His398 coordinated to type I copper and the indole ring of Trp396 located in the outer-coordination sphere. We performed point mutations at Trp396 with Ala, Thr, Phe, and Tyr with the aim of elucidating the role of the imidazole-indole moiety found only in bilirubin oxidase. The result showed shifts in the redox potential of type I copper towards negative direction by > 100 mV and decreases in cathodic current in electrochemistry, whereas optical and magnetic properties of type I copper were not affected or sparingly affected. Along with the conspicuous changes in redox properties enzymatic activities of the Trp396 mutants were prominently decreased. Further, chemical modification of the Trp residues with N-bromosuccinimide and photo-induced formylations of bilirubin oxidase exerted more pronounced effects on both redox properties and enzymatic activities compared to the Trp396 mutants. All these results unequivocally indicate that the covalent-bond formed between Trp396 and His398 plays a crucial role to enhance enzymatic activities of bilirubin oxidase by shifting the redox potential of type I Cu towards positive direction and also by functioning as the effective pathway of electron transport.