Gene Technical Approach for Substrate Specificity of Multi-copper Enzymes.

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We isolated the clone of bilirubin oxidase (BOD) obtained from Trachyderma Tsunodae K-2593 and deduced the amino acid sequence. The sequence has submitted to the DDDBJ/EMBL/GenBank database as AB 006824. On the basis of EPR, visible spectra, and the copper content of BOD, it was shown that of BOD is multi-copper enzyme, which has one type 2 copper, one type 1 copper, and two type 3 copper. The coordination residues of type 1, type2 and type 3 copper was predicted of homology of the amino acid sequence between BOD and ascorbate oxidase obtained from zucchini. Every ligand of type 2 and type 3 copper ions in BOD was shown to be His and was completely consistent with those of zucchini ascorbate oxidase. The ligand of type 1 copper in BOD was Cys, His, His, and Phe. Met which coordinates with type 1 copper in zucchini ascorbate oxidase was replaced by Phe in BOD. The redox potential of type 1 copper of BOD was measured under anaerobic conditions and was 642 mV that was much higher than that zucchini ascorbate oxidase. The higher redox potential of type 1 copper is because the hydrophobicity of Phe around type 1 copper in BOD raises the redox potential. The various anions influenced the visible spectra of type 1 copper in BOD. NィイD23ィエD2ィイD1-ィエD1 and SCNィイD1-ィエD1 gave the increase of the visible absorption spectra, but FィイD1-ィエD1 gave the decrease of it. EPR spectra of the adducts of these anions with BOD clearly indicated that these anion bind to type 2 copper in BOD. These behaviors were interpreted by the disproportination of the electron between type 1 copper and type 2/type 3 cluster. We tried the expression of the gene of BOD in Ecoli, but it was not succeeded.