Characterization of copper atoms in bilirubin oxidase by spectroscopic analyses.
Bilirubin oxidase [EC 1.3.3.5], purified from the culture medium of Myrothecium verrucaria, was found to contain two blue copper atoms per protein molecule with a molecular weight of ca. 52 kDa. The two copper atoms were estimated to be in the all cupric state by the cuproine colorimetric method and also atomic absorption analysis. We could remove the reduce cuprous ions from the holo enzyme by adding ascorbate, followed by a KCN solution, yielding an apo-enzyme with no activity. The apo-enzyme can be reconstituted with Cu or other divalent cations such as Co, Fe, and Cd, with accompanying recovery of the enzyme activity. The activity recovery depended upon the species of cation employed; Cu being most effective, an almost 100% recovery, and Cd the least, only a 25% recovery. We could obtain information on the copper ions and their coordination structure by spectroscopic analyses of the apo- and reconstituted enzymes, obtaining such as absorption, CD, MCD, and XPS spectra. The bilirubin oxidase catalyzed-reaction was a second order reaction with respect to copper bound with protein. The donor set was of the CuSS*N2 (S = Cys, S* = Met, N = His) type, i.e., the same as in the case of blue copper proteins. On studying the Co-substituted enzyme, it was revealed that the copper site of the enzyme had a 4-coordinated structure.
Gotoh
Y
Y
Department of Chemistry, College of Science and Engineering, Aoyama Gakuin University, Tokyo.
Kondo
Y
Y
Kaji
H
H
Takeda
A
A
Samejima
T
T
D001059:Apoproteins / Q000032:analysis; D002413:Cations, Divalent / Q000032:analysis; D002942:Circular Dichroism; D003300:Copper / Q000032:analysis*; D003904:Mitosporic Fungi / Q000201:enzymology*; D010088:Oxidoreductases / Q000032:analysis*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011485:Protein Binding; D013052:Spectrometry, X-Ray Emission; D013054:Spectrophotometry, Atomic
D001059:Apoproteins; D002413:Cations, Divalent; D003300:Copper; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C042401:bilirubin oxidase