Amino acids located in the outer-sphere of the trinuclear copper center in a multicopper oxidase, CueO as the putative electron donor in the four-electron reduction of dioxygen

作者：Sakurai Takeshi^*; Yamamoto Moe; Ikeno Shinsuke; Kataoka Kunishige

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2017, 1865(8): 997-1003.

DOI：10.1016/j.bbapap.2017.04.005

• Muticopper oxidase
• CueO
• Trinuclear copper center
• Outer-coordination sphere
• Dioxygen reduction
• Electron donor

The reaction mechanism of multicopper oxidase (MCO) to reduce dioxygen to water has not been fully understood yet in spite of extensive studies including on the intermediate I (peroxide intermediate) and intermediate II (native intermediate with an O-centered structure at the trinuclear copper center (TNC)). We performed the Phe mutations at the four amino acids, Tyr69, Cys138, Trp139, and Tyr496 located in the outer sphere of TNC in CueO at the aim of studying whether they play a role as the fourth electron donor to dioxygen or not. Spectral properties and enzymatic activities of CueO were sparingly affected or not affected by the mutations at these putative electron donors. Of the targeted four amino acids Trp139 is in a d-pi interaction distance with one of T3Cus and drives stepwise formation and release of water molecules by making two T3Cus non-equivalent. However, contribution of a radical species derived from Trp139 has not been observed in the formation and decay processes of the reaction intermediates. The present study strongly suggests that the amino acids located in the outer-sphere of TNC are not utilized as electron donor in the reduction of dioxygen to water by the three-domain MCO, CueO, differing from cytochrome oxidase and SLAC, a two-domain MCO, in which reaction participation of an uncoordinated Tyr residue has been proposed.
Summary: We performed the Phe mutations at the four amino acids, Tyr69, Cys138, Trp139 and Tyr496 located in the outer-coordination sphere of the trinuclear copper center in a three-domain multicopper oxidase, CueO to ascertain whether they function as an electron donor or not in the four-electron reduction of dioxygen. Characterizations of the mutants and reactions did not suggest participation of the targeted amino acids, indicating that CueO follows a different reaction mechanism from that of a two-domain multicopper oxidase, SLAC, in which reaction participation of an uncoordinated Tyr has been suggested.

• 出版日期2017-8