EPR evidence of cyanide binding to the Mn(Mg) center of cytochrome c oxidase: support for Cu(A)-Mg involvement in proton pumping.
EPR evidence of cyanide binding to the Mn(Mg) center of cytochrome c oxidase: support for Cu(A)-Mg involvement in proton pumping.
复制标题
氰化物与细胞色素 c 氧化酶的 Mn(Mg) 中心结合的 EPR 证据：支持 Cu(A)-Mg 参与质子泵送。
DOI：
10.1021/bi801391r
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发表时间：
2009
期刊：
影响因子：
2.9
通讯作者：
Ferguson-Miller,Shelagh
中科院分区：
文献类型：
--
作者：
Sharpe,MartynA;Krzyaniak,MatthewD;Xu,Shujuan;McCracken,John;Ferguson-Miller,Shelagh
我们检查了细胞色素氧化酶中Mg（Mn）位点的阴离子结合行为，以测试该中心在质子泵中可能发挥的作用。在富含Mn（II）的培养基中生长的球形红细菌用EPR可检测的Mn（II）离子取代了固有的Mg（II）离子，而活性没有变化。由于它的紧密接近和共享的配体，氧化的CuAis自旋耦合到Mn（II）离子，影响EPR光谱。牛andR.S.氧化酶晶体结构的检查揭示了在附近的Mg（II）的网站是一致的三个水配体的Mg（Mn）中心时，CuAis氧化的氢键模式。在还原结构中，CuAligand E198附近的一个水分子移动得更近，似乎被转化为离子键合的水合氢离子，而与Mg（Mn）键合的第二个水分子显示出转化为氢氧化物的证据。隐含的质子运动的一部分，提出了一个氧化还原连接出口泵质子从双核中心到出口途径。为了测试该模型，氰化物和叠氮化物被添加到氧化和还原形式的酶，和Mn（II）CW-EPR和ESEEM光谱记录。叠氮化物的加入使氧化型和还原型酶的CW-EPR谱都变宽。氰化物的加入通过增加Mn（II）的零场分裂和加宽谱线形状而影响还原型细胞色素氧化酶的Mn（II）CW-EPR谱，但对氧化酶没有影响。ESEEM测量支持的差异能力的锰（II）结合氰化物的还原状态的细胞色素氧化酶。在Mg/Mn位点的阴离子结合的这一新的观察结果是感兴趣的掩埋位点的可访问性和其在氧化还原依赖的质子泵的潜在作用。
We examined the anion binding behavior of the Mg(Mn) site in cytochromecoxidase to test a possible role of this center in proton pumping.Rhodobacter sphaeroidesgrown in a Mn(II)-rich medium replaces the intrinsic Mg(II) ion with an EPR-detectable Mn(II) ion without change in activity. Due to its close proximity and a shared ligand, oxidized CuAis spin-coupled to the Mn(II) ion, affecting the EPR spectrum. An examination of both bovine andR.s.oxidase crystal structures reveals a hydrogen-bonding pattern in the vicinity of the Mg(II) site that is consistent with three water ligands of the Mg(Mn) center when CuAis oxidized. In the reduced structure, one water molecule in the vicinity of the CuAligand, E198, moves closer, appearing to be converted into an ionically bonded hydronium ion, while a second water molecule bonded to Mg(Mn) shows evidence of conversion to a hydroxide. The implied proton movement is proposed to be part of a redox-linked export of a pumped proton from the binuclear center into the exit pathway. To test the model, cyanide and azide were added to the oxidized and reduced forms of the enzyme, and Mn(II) CW-EPR and ESEEM spectra were recorded. Addition of azide broadened the CW-EPR spectra for both oxidized and reduced enzyme. Cyanide addition affected the Mn(II) CW-EPR spectrum of reduced cytochromecoxidase by increasing Mn(II) zero field splitting and broadening the spectral line shapes but had no effect on oxidized enzyme. ESEEM measurements support a differential ability of Mn(II) to bind cyanide in the reduced state of cytochromecoxidase. This new observation of anion binding at the Mg/Mn site is of interest in terms of accessibility of the buried site and its potential role in redox-dependent proton pumping.