Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, NY 10461, United States.
J Inorg Biochem. 2010 Mar;104(3):318-23. doi: 10.1016/j.jinorgbio.2009.11.011. Epub 2009 Dec 3.
The structural and functional properties of active site mutants of cytochrome c oxidase from Paracoccus denitrificans (PdCcO) were investigated with resonance Raman spectroscopy. Based on the Fe-CO stretching modes and low frequency heme modes, two conformers (alpha- and beta-forms) were identified that are in equilibrium in the enzyme. The alpha-conformer, which is the dominant species in the wild-type enzyme, has a shorter heme a(3) iron-Cu(B) distance and a more distorted heme, as compared to the beta-conformer, which has a more relaxed and open distal pocket. In general, the mutations caused a decrease in the population of the alpha-conformer, which is concomitant with a decreased in the catalytic activity, indicating that the alpha-conformer is the active form of the enzyme. The data suggest that the native structure of the enzyme is in a delicate balance of intramolecular interactions. We present a model in which the mutations destabilize the alpha-conformer, with respect to the beta-conformer, and raise the activation barrier for the inter-conversion between the two conformers. The accessibility of the two conformers in the conformational space of CcO plausibly plays a critical role in coupling the redox reaction to proton translocation during the catalytic cycle of the enzyme.
用共振拉曼光谱法研究了脱氮副球菌（PdCcO）细胞色素 c 氧化酶活性部位突变体的结构和功能特性。基于 Fe-CO 伸缩模式和低频血红素模式，鉴定出两种处于平衡状态的构象（α-和β-构象）。与β-构象相比，α-构象的血红素 a(3)铁-Cu(B)距离更短，血红素变形更严重，是野生型酶中的主要构象。与β-构象相比，β-构象具有更松弛和开放的远端口袋。一般来说，这些突变导致α-构象的比例降低，同时催化活性也降低，这表明α-构象是酶的活性形式。数据表明，酶的天然结构处于分子内相互作用的微妙平衡之中。我们提出了一个模型，即突变使α-构象相对于β-构象不稳定，并增加了两种构象之间相互转化的活化能垒。在 CcO 的构象空间中，两种构象的可及性可能在酶催化循环中氧化还原反应与质子转移的偶联中起着关键作用。
