X-ray structural analyses of azide-bound cytochrome c oxidases reveal that the H-pathway is critically important for the proton-pumping activity.
X-ray structural analyses of azide-bound cytochrome c oxidases reveal that the H-pathway is critically important for the proton-pumping activity.
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DOI：
10.1074/jbc.ra118.003123
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发表时间：
2018-09-21
期刊：
影响因子：
--
通讯作者：
Yoshikawa S
中科院分区：
文献类型：
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作者：
Shimada A;Hatano K;Tadehara H;Yano N;Shinzawa-Itoh K;Yamashita E;Muramoto K;Tsukihara T;Yoshikawa S
细胞色素 c 氧化酶 (CcO) 是细胞呼吸的末端氧化酶，可将 O2 还原为水并泵送质子。 X射线结构特征表明，CcO通过一种机制来泵送质子，该机制涉及质子传导路径（H-路径）的氢键网络中的泵送质子与铁位点血红素a（Fea2+）氧化时产生的净正电荷之间的静电排斥，从而在另一个铁位点血红素a3（Fea32+）处还原O2。用于泵送的质子从N侧通过H路径的水通道转移到氢键网络。质子向 N 侧的回漏被认为可以通过水通道的关闭来阻止。为了通过实验测试这一点，我们检查了叠氮化物结合的氧化牛 CcO 的 X 射线结构，发现叠氮化物衍生物（N3−–Fea33+、CuB2+–N3−）会诱导血红素 a3 平面的平移运动。这伴随着水通道的开放，表明 Fea3 和 H 通路紧密耦合。氧化状态下的通道打开可能会引起泵浦质子的回漏，从而降低酶周转过程中氢键网络中的质子水平。如果 Fea 与氢键网络中的质子发生静电相互作用，质子水平的降低会削弱 Fea 的电子亲和力。先前报道的叠氮化物诱导的 Fea 氧化还原电位降低支持静电相互作用的存在。总之，我们的结果表明 H 通路对于 CcO 的质子泵功能至关重要。
Cytochrome c oxidase (CcO) is the terminal oxidase of cellular respiration, reducing O2 to water and pumping protons. X-ray structural features have suggested that CcO pumps protons via a mechanism involving electrostatic repulsions between pumping protons in the hydrogen-bond network of a proton-conducting pathway (the H-pathway) and net positive charges created upon oxidation of an iron site, heme a (Fea2+), for reduction of O2 at another iron site, heme a3 (Fea32+). The protons for pumping are transferred to the hydrogen-bond network from the N-side via the water channel of the H-pathway. Back-leakage of protons to the N-side is thought to be blocked by closure of the water channel. To experimentally test this, we examined X-ray structures of the azide-bound, oxidized bovine CcO and found that an azide derivative (N3−–Fea33+, CuB2+–N3−) induces a translational movement of the heme a3 plane. This was accompanied by opening of the water channel, revealing that Fea3 and the H-pathway are tightly coupled. The channel opening in the oxidized state is likely to induce back-leakage of pumping protons, which lowers the proton level in the hydrogen-bond network during enzymatic turnover. The proton level decrease weakens the electron affinity of Fea, if Fea electrostatically interacts with protons in the hydrogen-bond network. The previously reported azide-induced redox-potential decrease in Fea supports existence of the electrostatic interaction. In summary, our results indicate that the H-pathway is critical for CcO's proton-pumping function.