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
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
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位于多铜氧化酶三核铜中心外球的氨基酸，CueO 作为双氧四电子还原中假定的电子供体
DOI：
10.1016/j.bbapap.2017.04.005
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发表时间：
2017
期刊：
影响因子：
--
通讯作者：
Kataoka Kunishige
中科院分区：
文献类型：
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作者：
Sakurai Takeshi;Yamamoto Moe;Ikeno Shinsuke;Kataoka Kunishige
尽管对多铜氧化酶（MCO）的中间体I（过氧化物中间体）和中间体II（在三核铜中心（TNC）处具有O中心结构的天然中间体）进行了广泛的研究，但尚未完全理解多铜氧化酶（MCO）将分子氧还原为水的反应机理。我们对CueO中TNC外层的四个氨基酸Tyr 69、Cys 138、Trp 139和Tyr 496进行了Phe突变，目的是研究它们是否作为分子氧的第四电子供体发挥作用。光谱特性和酶活性的CueO的很少受到影响或不受影响的突变在这些假定的电子供体。在靶向的四个氨基酸中，Trp 139与其中一个T3 Cus处于d-π相互作用距离，并且通过使两个T3 Cus不等价来驱动水分子的逐步形成和释放。然而，在反应中间体的形成和衰变过程中，没有观察到来自Trp 139的自由基物种的贡献。本研究强烈地表明，位于TNC外层的氨基酸在三结构域MCO（CueO）将分子氧还原成水的过程中不被用作电子供体，这与细胞色素氧化酶和SLAC（一种两结构域MCO，其中已提出非配位Tyr残基参与反应）不同。Trp 139和Tyr 496位于三结构域多铜氧化酶CueO的三核铜中心的外配位层，以确定它们在分子氧的四电子还原中是否作为电子供体。的突变体和反应的表征并没有建议参与的目标氨基酸，表明CueO遵循不同的反应机制，从一个两个域的多铜氧化酶，SLAC，其中的反应参与的一个不协调的酪氨酸已被建议。
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-π 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.SummaryWe 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.