Time-resolved step-scan Fourier transform infrared spectroscopy of the CO adducts of bovine cytochrome c oxidase and of cytochrome bo3 from Escherichia coli
Time-resolved step-scan Fourier transform infrared spectroscopy of the CO adducts of bovine cytochrome c oxidase and of cytochrome bo3 from Escherichia coli
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DOI：
10.1021/bi010823g
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发表时间：
2002-02-26
期刊：
影响因子：
2.9
通讯作者：
Dyer, RB
中科院分区：
文献类型：
--
作者：
Bailey, JA;Tomson, FL;Dyer, RB
我们利用低温差示傅里叶变换红外光谱和时间分辨步进扫描傅里叶变换红外光谱（TR-FTIR）来探讨家庭铜呼吸氧化酶的氧化还原相关质子泵机制。这些技术被用来探测血红素a（3）-Cu-B双核中心的结构和动力学，以及响应于CO从血红素Fe的光解离及其随后与Cu-B的结合和解离的偶联蛋白质结构。之前的低温（80 K）FTIR CO光解离差异结果是针对大肠杆菌的泛醇氧化酶细胞色素bo（3）获得的[Puustinen，A.，等人（1997）Biochemistry 36，13195-13200]。这些数据揭示了Cu-B和谷氨酸E286之间的连接性，谷氨酸E286是一种与质子泵有关的残基。在本工作中，在低温条件下使用牛细胞色素aa（3）的CO加合物观察到了相同的现象，显示了等效残留物（E242）对bo（3）的扰动。此外，使用时间分辨（5 μ s分辨率）步进扫描FTIR光谱在室温下，我们观察到相同的光谱扰动在细胞色素aa 3和bo（3）。此外，我们观察到的第二羧酸侧链的扰动的证据，在较高的频率在两种酶在室温下。在低温差谱中没有出现高频特征，表明微扰是一个激活过程。我们假设高频红外特征是由于E62（bo（3）中的E89）的扰动，E62是质子K通道开口附近的残基，是酶功能所需的。这些结果相对于质子泵机制的影响进行了讨论。最后，观察到bo（3）中超过60%的Cu-B-CO信号的快速损失，并归因于酶的一种或多种额外构象。这种快速构象被提出来占与O-2在流动闪蒸实验中的不受抑制的反应。
We have used cryogenic difference FTIR and time-resolved step-scan Fourier transform infrared (TR-FTIR) spectroscopies to explore the redox-linked proton-pumping mechanism of home-copper respiratory oxidases. These techniques are used to probe the structure and dynamics of the heme a(3)-Cu-B binuclear center and the coupled protein structures in response to the photodissociation of CO from heme Fe and its subsequent binding to and dissociation from Cu-B. Previous cryogenic (80 K) FTIR CO photodissociation difference results were obtained for cytochrome bo(3), the ubiquinol oxidase of Escherichia coli [Puustinen, A., et al. (1997) Biochemistry 36, 13195-13200]. These data revealed a connectivity between Cu-B and glutamic acid E286, a residue which has been implicated in proton pumping. In the current work, the same phenomenon is observed using the CO adduct of bovine cytochrome aa(3) under cryogenic conditions, showing a perturbation of the equivalent residue (E242) to that in bo(3). Furthermore, using time-resolved (5 mus resolution) step-scan FTIR spectroscopy at room temperature, we observe the same spectroscopic perturbation in both cytochromes aa3 and bo(3). In addition, we observe evidence for perturbation of a second carboxylic acid side chain, at higher frequency in both enzymes at room temperature. The high-frequency feature does not appear in the cryogenic difference spectra, indicating that the perturbation is an activated process. We postulate that the high-frequency IR feature is due to the perturbation of E62 (E89 in bo(3)), a residue near the opening of the proton K-channel and required for enzyme function. The implications of these results with respect to the proton-pumping mechanism are discussed. Finally, a fast loss of over 60% of the Cu-B-CO signal in bo(3) is observed and ascribed to one or more additional conformations of the enzyme. This fast conformer is proposed to account for the uninhibited reaction with O-2 in flow-flash experiments.