Electron Tranfer between Multi-copper Enzyme and Fe (CN) _6^<4->

胆红素氧化酶是催化胆红素氧化为胆绿素的酶，是一种多铜酶，分别含有1：1：2比例的1型、2型和3型铜。亚铁氰化物（Fe（CN）_6 ~（2-）<4->）是胆红素氧化酶的良好底物。米氏常数（K_m）和摩尔活性（K_<cat>）为3.0 × 10 - 4 <-3>M和300 sec-4<-1>。Cr（CN）_6 ~<3->（2-）与Fe（CN）_6 ~（2-）结构相似<4->，竞争性抑制Fe（CN）_6 ~（2-）的氧化<4->。Cr（CN）_6^2的缓蚀常数<3->为3.0 × 10 ~（10）<-3>M，与Fe（CN）_6^2的K_m值基本相同<4->。这些结果表明Cr（CN）_6 ~（2-<3->）与胆红素氧化酶中Fe（CN）_6 ~（2-）的结合位点<4->结合。克隆了胆红素氧化酶基因，并测定了其核苷酸序列。根据胆红素氧化酶的核苷酸序列，测定了胆红素氧化酶的氨基酸序列。确定了与铜离子配位的氨基酸残基，并推测了底物结合位点周围的氨基酸残基。

Bilirubin oxidase is the enzyme that catalyses the oxidation of bilirubin to biliverudin and a multi-copper enzyme containing type 1, type 2, and type 3 copper in the ratio of 1 : 1 : 2, respectively. Ferrocyanide ion (Fe (CN) _6^<4->) is a good substrate for bilirubin oxidase. Michaelis-Menten constant (K_m) and molar activity (K_<cat>) are 3.0*10^<-3>M and 300 sec^<-1>. Cr (CN) _6^<3-> that is structually similar to Fe (CN) _6^<4-> inhibits competitively the oxidation of Fe (CN) _6^<4->. The inhibition constant of Cr (CN) _6^<3-> is 3.0*10^<-3> M that is almost same value of K_m of Fe (CN) _6^<4->2. These results indicate that Cr (CN) _6^<3-> binds the binding site of Fe (CN) _6^<4-> in bilirubin oxidase. The gene of bilirubin oxidase was cloned and the nucleotide sequence of bilirubin oxidase has been determined. On the basis of the nucleotide sequence of bilirubin oxidase, the complete amino acid sequence of bilirubin oxidase has been determined. The amino acid residues that coordinate to cupric ions were decided and those that are around substrate binding site also are also guessed.