Study on dioxygen reduction by mutational modifications of the hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase

• Morishita, Hirotoshi

• Kurita, Daisuke

• Kataoka, Kunishige

• Sakurai, Takeshi

Abstract

The hydrogen bond network leading from bulk water to the trinuclear copper center in bilirubin oxidase is constructed with Glu463 and water molecules to transport protons for the four-electron reduction of dioxygen. Substitutions of Glu463 with Gln or Ala were attributed to virtually complete loss or significant reduction in enzymatic activities due to an inhibition of the proton transfer steps to dioxygen. The single turnover reaction of the Glu463Gln mutant afforded the highly magnetically interacted intermediate II (native intermediate) with a broad g = 1.96 electron paramagnetic resonance signal detectable at cryogenic temperatures. Reactions of the double mutants, Cys457Ser/Glu463Gln and Cys457Ser/Glu463Ala afforded the intermediate I (peroxide intermediate) because the type I copper center to donate the fourth electron to dioxygen was vacant in addition to the interference of proton transport due to the mutation at Glu463. The intermediate I gave no electron paramagnetic resonance signal, but the type II copper signal became detectable with the decay of the intermediate I. Structural and functional similarities between multicopper oxidases are discussed based on the present mutation at Glu463 in bilirubin oxidase. © 2014 Elsevier Inc. All rights reserved.

Journal

• Biochemical and Biophysical Research Communications

  Biochemical and Biophysical Research Communications 450 (1), 767-772, 2014-07-18

  Academic Press / Elsevier

Details

• CRID: 1050845760872584832
• NII Article ID: 120005522959
• NII Book ID: AA00564395
• ISSN: 0006291X
• Web Site: http://hdl.handle.net/2297/40208
• Text Lang: en
• Article Type: journal article
• Data Source:
  • IRDB
  • CiNii Articles
  • KAKEN