Exploring the proton pump mechanism of cytochrome c oxidase in real time
Exploring the proton pump mechanism of cytochrome c oxidase in real time
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DOI：
10.1073/pnas.0608794104
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发表时间：
2007-02-20
影响因子：
11.1
通讯作者：
Verkhovsky, Michael I.
中科院分区：
文献类型：
--
作者：
Belevich, Ilya;Bloch, Dmitry A.;Verkhovsky, Michael I.
细胞色素c氧化酶催化地球上大部分的生物氧消耗，这是一个负责有氧生物体能量供应的过程。这种显著的膜结合酶还通过作为氧化还原连接的质子泵发挥作用，将来自O-2还原的自由能转化为电化学质子梯度。虽然几种氧化酶的结构是已知的，但氧化还原连接的质子易位的分子机制仍然难以捉摸。在这里，相关的内部电子和质子转移反应进行了跟踪真实的时间后，激光激活电子注入到氧化酶的光谱和电测量技术。所观察到的动力学建立了长期寻求的质子泵机制的反应序列，并描述了它的一些热力学性质。10 μ s的电子转移到血红素a提高了“泵位点”的pK（a），该位点在150 μ s内被来自膜内部的质子加载。这种负载增加了血红素a和a（3）的氧化还原电位，这使得它们之间的电子以相同的速率平衡。然后，在0.8 ms内，另一个质子从内部转移到血红素a（3）/Cu-B中心，电子转移到Cu-B。最后，在2.6 ms内，预加载的质子从泵位点释放到膜的相对侧。
Cytochrome c oxidase catalyzes most of the biological oxygen consumption on Earth, a process responsible for energy supply in aerobic organisms. This remarkable membrane-bound enzyme also converts free energy from O-2 reduction to an electrochemical proton gradient by functioning as a redox-linked proton pump. Although the structures of several oxidases are known, the molecular mechanism of redox-linked proton translocation has remained elusive. Here, correlated internal electron and proton transfer reactions were tracked in real time by spectroscopic and electrometric techniques after laser-activated electron injection into the oxidized enzyme. The observed kinetics establish the long-sought reaction sequence of the proton pump mechanism and describe some of its thermodynamic properties. The 10-mu s electron transfer to heme a raises the pK(a) of a "pump site," which is loaded by a proton from the inside of the membrane in 150 mu s. This loading increases the redox potentials of both hemes a and a(3), which allows electron equilibration between them at the same rate. Then, in 0.8 ms, another proton is transferred from the inside to the heme a(3)/Cu-B center, and the electron is transferred to Cu-B. Finally, in 2.6 ms, the preloaded proton is released from the pump site to the opposite side of the membrane.