Anions binding to bilirubin oxidase from Trachyderma tsunodae K-2593

Abstract

Bilirubin oxidase obtained from Trachyderma tsunodae K-2593 is a multi-copper enzyme which has type 1:type 2:type 3 copper atoms in the ratio 1:1:2. Anion binding properties of bilirubin oxidase were investigated to determine the structural and functional properties of bilirubin oxidase. The anions N 3 −, SCN −, F −, Cl −, and Br − were non-competitive inhibitors against the substrate, bilirubin ditaurate, and the inhibition constants of the anions were in the following order: N 3 − <SCN − ≪F − <Cl − <Br −. With the addition of N 3 − or SCN − to resting bilirubin oxidase, the absorption around 610 nm which is characteristic of type 1 cupric ion increased, whereas the absorption shoulder around 330 nm which is characteristic of type 3 cupric ion slightly decreased. The addition of F −, on the contrary, resulted respectively in the decreasing and increasing of the absorption bands around 610 and 330 nm. The kinetics of anions binding to bilirubin oxidase was also followed by the absorbance change at 610 nm by a micro-stopped flow apparatus at 25°C. The increases in the absorbance were observed in the reactions of bilirubin oxidase with N 3 − and SCN −, and a decrease was observed in the reaction with F −. These results were consistent with those observed by the spectrophotometric method. The EPR spectra clearly showed that the anions bound to the type 2/type 3 cluster site of resting bilirubin oxidase and not to type 1 copper. The redox potential of type 1 copper in bilirubin oxidase was measured under anaerobic conditions using Li 2[Co(II)(2,6-pyridinedicarboxylate) 2] as a mediator and was high (615 mV at pH 6.8), so that the type 1 copper of the enzyme could be easily reduced. The results of the full oxidation of the copper ions in the enzyme by H 2O 2 showed that part (13% of type 1 copper) of the copper ions in the enzyme had been present in the reduced state. The absorbance changes in type 1 and type 3 copper with the addition of anions were interpreted by the disproportionation of electrons among the copper ions in bilirubin oxidase. The opposite absorbance changes due to N 3 − and F − binding to bilirubin oxidase, around 610 and 330 nm, were attributed to the opposite changes in the redox potentials of the type 2/type 3 cluster site by the binding of these anions to the cluster site.