Redox titration of all electron carriers of cytochrome c oxidase by Fourier transform infrared spectroscopy
Redox titration of all electron carriers of cytochrome c oxidase by Fourier transform infrared spectroscopy
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DOI：
10.1021/bi060257v
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发表时间：
2006-05-02
期刊：
影响因子：
2.9
通讯作者：
Verkhovsky, MI
中科院分区：
文献类型：
--
作者：
Gorbikova, EA;Vuorilehto, K;Verkhovsky, MI
采用衰减全反射傅里叶变换红外光谱法（ATR-FTIR）对拟球藻细胞色素c氧化酶进行电化学氧化还原滴定。大多数差异红外吸收特征可分为四组，它们与氧化还原相关。酶的四个氧化还原中心的转换。红外光谱法的优点是允许人们测量氧化还原中心的独立变化，这些变化不能很好地分离，甚至不能通过其他光谱技术观察到。我们发现了12个红外波段，滴定与最高观察到的中点氧化还原电位（E-m = 412 mV，pH 6.5），并在碱性区域的pH依赖性为52 mV每pH单位。这些条带被指定与CUB中心相关联。我们将条带：分配给CUA中心，其显示250 mV的pH无关性E-m。另外两组红外微分带反映了两个血红素基团的氧化还原跃迁，表现出更复杂的行为。它们每个都包括两个部分，分别对应于高电位和低电位的氧化还原转变。对于代表血红素a的条带，高电位组分与低电位组分的比例约为0.05。3：2;对于血红素a（3），该比例为约。2比3。考虑到血红素之间的氧化还原相互作用，这些比率在血红素之间产生9 mV的E-m差（在pH 8.0下，血红素a为359 mV;血红素a（3）为350 mV）。发现血红素之间的氧化还原相互作用的程度（在pH 8.0下为-115 mV）是pH依赖性的。两种血红素的E-m值的pH依赖性是相同的，并且比理论值小约两倍，这表明酸/碱基团在还原任一血红素时结合质子。所应用的方法允许在四个组中的每一个中的红外波段的血红素的振动，氧化还原中心的配体，氨基酸残基，和/或蛋白质骨架的分配。例如，在1737/1746 cm（-1）处的已知谱带位移对应于质子化谷氨酸E278与血红素a的氧化还原相关。
Electrochemical redox titrations of cytochrome c oxidase from Paraccocus denitrificans were performed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The majority of the differential infrared absorption features may be divided into four groups, which correlate with the redox. transitions of the four redox centers of the enzyme. Infrared spectroscopy has the advantage of allowing one to measure independent alterations in redox centers, which are not well separated, or even observed, by other spectroscopic techniques. We found 12 infrared bands that titrated with the highest observed midpoint redox potential (E-m = 412 mV at pH 6.5) and which had a pH dependence of 52 mV per pH unit in the alkaline region. These bands were assigned to be linked to the CUB center. We assigned bands: to the CUA center that showed a pH-independent E-m of 250 mV. Two other groups of infrared differential bands reflected redox transitions of the two heme groups and showed a more complex behavior. Each of them included two parts, corresponding to high- and low-potential redox transitions. For the bands; representing heme a, the ratio of high- to low-potential components was ca. 3:2; for heme a(3) this ratio was ca. 2:3. Taking into account the redox interactions between the hemes, these ratios yielded a difference in E-m of 9 mV between the hemes (359 mV for heme a; 350 mV for heme a(3) at pH 8.0). The extent of the redox interaction between the hemes (-115 mV at pH 8.0) was found to be pH-dependent. The pH dependence of the E-m values for the two hemes was the same and about two times smaller than the theoretical one, suggesting that an acid/base group binds a proton upon reduction of either heme. The applied approach allowed assignment of infrared bands in each of the four groups to vibrations of the hemes, ligands of the redox centers, amino acid residues, and/or protein backbone. For example, the well-known band shift at 1737/1746 cm(-1) corresponding to the protonated glutamic acid E278 correlated with oxidoreduction of heme a.