The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process
The low-spin heme of cytochrome c oxidase as the driving element of the proton-pumping process
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DOI：
10.1073/pnas.2635097100
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发表时间：
2003-12-23
影响因子：
11.1
通讯作者：
Yoshikawa, S
中科院分区：
文献类型：
--
作者：
Tsukihara, T;Shimokata, K;Yoshikawa, S
线粒体细胞色素C氧化酶在细胞的有氧呼吸中起着至关重要的作用，在这个过程中将氧气还原为水，并将质子泵过线粒体内膜。位于酶表面附近的天冬氨酸残基Asp-51经历了氧化还原耦合的X射线结构变化，这表明该残基在氧化还原驱动的质子泵中发挥了作用。然而，尚未获得这种残基参与质子泵浦的功能或机械证据。我们报道，牛酶的Asp-51--&Gt；Asn突变取消了其质子泵功能，而不损害氧气还原活性。改进的X射线结构(在完全氧化/还原状态下的分辨率为1.8/1.9-A)表明，该酶的低自旋血红素氧化产生的净正电荷推动活性质子从线粒体内部通过水通道和氢键网络通过串联的网络传输到Asp-51，并且酶的还原诱导质子从天冬氨酸向线粒体外部喷射。氢键网络中的一个肽键严重地抑制了通过该网络的反向质子转移。由低自旋血红素的羟法尼乙基引起的水通道容量的氧化还原耦合变化表明，该通道起到了有效的质子收集区域的作用。红外结果表明，Asp-51的构象只受低自旋血红素的氧化态控制。这些结果表明，低自旋的血红素驱动质子泵浦过程。
Mitochondrial cytochrome c oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 --> Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-A resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.