Monomeric structure of an active form of bovine cytochrome c oxidase
Monomeric structure of an active form of bovine cytochrome c oxidase
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DOI：
10.1073/pnas.1907183116
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发表时间：
2019-09
影响因子：
11.1
通讯作者：
K. Shinzawa-Itoh;T. Sugimura;T. Misaki;Y. Tadehara;Shogo Yamamoto;M. Hanada;N. Yano;T. Nakagawa;Shigefumi Uene;T. Yamada;H. Aoyama;E. Yamashita;T. Tsukihara;S. Yoshikawa;K. Muramoto
中科院分区：
文献类型：
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作者：
K. Shinzawa-Itoh;T. Sugimura;T. Misaki;Y. Tadehara;Shogo Yamamoto;M. Hanada;N. Yano;T. Nakagawa;Shigefumi Uene;T. Yamada;H. Aoyama;E. Yamashita;T. Tsukihara;S. Yoshikawa;K. Muramoto
线粒体细胞色素c氧化酶（CcO）的x射线晶体学分析基于其二聚体形式。最近的低温电镜结构显示，CcO以其单体形式存在于呼吸超复合体中。本研究采用双极性稳定的CcO，结果表明单体活性高于二聚体。这里测定的单体晶体结构表明，其中一个质子转移途径的局部结构不同于二聚体。晶体结构也表明心磷脂位于超配合物的界面区。综上所述，这些结果表明，依赖于心磷脂的单体状态、二聚体状态和超复合体状态的CcO参与了呼吸电子传递的调节。细胞色素c氧化酶（Cytochrome c oxidase, CcO）是呼吸链中的一种膜酶，通过质子泵将电子和质子通过该酶耦合在膜上，从而催化氧还原。在迄今报道的所有晶体中，牛CcO以二聚体形式存在，具有相同的单体间接触，而原核生物的CcO和相关酶以单体形式存在。最近对线粒体呼吸超复合体的结构分析表明，CcO单体与复合体I和复合体III结合，表明单体状态在功能上很重要。在本研究中，我们制备了单聚和二聚的牛CcO，并使用双酚类进行稳定，结果表明该单体具有较高的活性。此外，使用新合成的洗涤剂，我们测定了单体的氧化和还原结构，分辨率分别为1.85和1.95 Å。单体和二聚体的结构比较表明，在单体CcO中，在质子转移途径（称为k途径）的入口表面形成了水分子的氢键网络，而在二聚体CcO中，该网络发生了变化。基于这些结果，我们提出单体是活化形式，而二聚体可以被视为线粒体膜的生理备用形式。我们还根据电子密度和磷原子的反常散射效应确定了磷脂的结构。在超络合物的界面区发现了两种心磷脂。我们讨论了单体CcO、二聚体CcO和超络合物的形成，以及它们在调节CcO活性中的作用。
Significance X-ray crystallographic analyses of mitochondrial cytochrome c oxidase (CcO) have been based on its dimeric form. Recent cryo-electron microscopy structures revealed that CcO exists in its monomeric form in the respiratory supercomplex. This study, using amphipol-stabilized CcO, shows that the activity of monomer is higher than that of the dimer. The crystal structure of monomer determined here shows that the local structure of one of the proton transfer pathways differs from that in the dimer. The crystal structure also shows that cardiolipins are located at the interface region in the supercomplex. Taken together, these results suggest that CcO in the monomeric state, dimeric state, and supercomplex state depending on cardiolipins are involved in regulation of respiratory electron transport. Cytochrome c oxidase (CcO), a membrane enzyme in the respiratory chain, catalyzes oxygen reduction by coupling electron and proton transfer through the enzyme with a proton pump across the membrane. In all crystals reported to date, bovine CcO exists as a dimer with the same intermonomer contacts, whereas CcOs and related enzymes from prokaryotes exist as monomers. Recent structural analyses of the mitochondrial respiratory supercomplex revealed that CcO monomer associates with complex I and complex III, indicating that the monomeric state is functionally important. In this study, we prepared monomeric and dimeric bovine CcO, stabilized using amphipol, and showed that the monomer had high activity. In addition, using a newly synthesized detergent, we determined the oxidized and reduced structures of monomer with resolutions of 1.85 and 1.95 Å, respectively. Structural comparison of the monomer and dimer revealed that a hydrogen bond network of water molecules is formed at the entry surface of the proton transfer pathway, termed the K-pathway, in monomeric CcO, whereas this network is altered in dimeric CcO. Based on these results, we propose that the monomer is the activated form, whereas the dimer can be regarded as a physiological standby form in the mitochondrial membrane. We also determined phospholipid structures based on electron density together with the anomalous scattering effect of phosphorus atoms. Two cardiolipins are found at the interface region of the supercomplex. We discuss formation of the monomeric CcO, dimeric CcO, and supercomplex, as well as their role in regulation of CcO activity.