Two-Electron Reduction versus One-Electron Oxidation of the Type 3 Pair in the Multicopper Oxidases.
Two-Electron Reduction versus One-Electron Oxidation of the Type 3 Pair in the Multicopper Oxidases.
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DOI：
10.1021/jacs.5b04136
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发表时间：
2015-07-15
影响因子：
15
通讯作者：
Solomon EI
中科院分区：
文献类型：
--
作者：
Kjaergaard CH;Jones SM;Gounel S;Mano N;Solomon EI
多铜氧化酶（MCO）利用电子穿梭1型Cu（T1）位点结合单核2型（T2）和双核3型（T3）位点，排列在三核铜簇（TNC）中，将O2还原为H2O。O2的还原在有限的过电位下发生，这表明活性位点中的所有铜都可以通过高电位电子供体还原。在MCO中观察到两种形式的静息酶：交替静息形式（AR），其中三种TNC Cu中只有一种被氧化，以及静息氧化形式（RO），其中所有三种TNC Cu都被氧化。与AR形式相反，我们表明，在RO形式的高电位MCO，双核T3 Cu（II）的网站可以通过700 mV T1铜减少。系统的光谱评估表明，这是通过一个双电子过程，其中第一个电子的传递，形成一个高能量，亚稳定的半还原T3状态，其次是第二个能量有利的电子的快速传递，以充分减少T3网站。或者，当这种完全还原的双核T3位点通过T1 Cu氧化时，产生不同的生物学上有利的半氧化T3形式，即AR位点。DFT计算表明，蛋白质骨架在控制活性中心铜的环境中起着重要作用。这允许形成亚稳定的半还原状态，从而使酶完全还原活化用于催化。
Multicopper Oxidases (MCOs) utilize an electron shuttling Type 1 Cu (T1) site in conjunction with a mononuclear Type 2 (T2) and a binuclear Type 3 (T3) site, arranged in a trinuclear copper cluster (TNC), to reduce O2 to H2O. Reduction of O2 occurs with limited overpotential indicating that all the coppers in the active site can be reduced via high-potential electron donors. Two forms of the resting enzyme have been observed in MCOs: the Alternative Resting form (AR), where only one of the three TNC Cu’s is oxidized, and the Resting Oxidized form (RO), where all three TNC Cu’s are oxidized. In contrast to the AR form, we show that in the RO form of a high-potential MCO, the binuclear T3 Cu(II) site can be reduced via the 700 mV T1 Cu. Systematic spectroscopic evaluation reveals that this proceeds by a two-electron process, where delivery of the first electron, forming a high energy, meta-stable half reduced T3 state, is followed by the rapid delivery of a second energetically favorable electron to fully reduce the T3 site. Alternatively, when this fully reduced binuclear T3 site is oxidized via the T1 Cu, a different thermodynamically favored half oxidized T3 form, i.e. the AR site, is generated. This behavior is evaluated by DFT calculations, which reveal that the protein backbone plays a significant role in controlling the environment of the active site coppers. This allows for the formation of the meta-stable, half reduced state and thus the complete reductive activation of the enzyme for catalysis.