Glynn Laboratory of Bioenergetics, Institute of Structural and Molecular Biology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
Proc Natl Acad Sci U S A. 2011 May 24;108(21):8634-8. doi: 10.1073/pnas.1019419108. Epub 2011 May 4.
Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm(-1) range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(-), and 3,607(+) cm(-1). A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D(2)O and H(2)(18)O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.
尽管细胞色素 c 氧化酶中的内部电子转移和氧还原化学已得到相当深入的了解，但将这些过程与跨膜门控质子转移偶联的相关基团和途径仍不清楚。已经从晶体结构模型中确定了几种可能的途径；这些途径涉及亲水性残基与结构水的组合，这些结构水可能在催化循环中重新排列以形成瞬时质子转移途径。然而，迄今为止，不同氧化酶在不同氧化还原或配位状态下的原子结构比较并未就哪种途径起作用或其在催化过程中的动态变化细节提供一致的答案。为了提供解决此问题的实验手段，已使用 3,560-3,800 cm(-1) 范围内的 FTIR 光谱来检测牛细胞色素 c 氧化酶中可能在催化过程中发生变化的弱氢键合水分子。完整的氧化还原光谱在 3,674(+)、3,638(+)、3,620(-)和 3,607(+) cm(-1) 处至少显示了四个信号。在亚铁-CO 化合物的光解光谱中观察到更复杂的信号集，该反应模拟了催化氧结合步骤，并且它们的 D(2)O 和 H(2)(18)O 灵敏度证实它们是由水分子重排引起的。用高斯组件拟合表明，多达八个水分子参与了光解跃迁。在来自 Paracoccus denitrificans 的细菌 CcO 的亚铁-CO 化合物的光解光谱中也观察到类似的信号。这些水的变化与亲水性通道和质子/电子偶联机制的作用有关。
