Gorbikova Elena A, Vuorilehto Kai, Wikström Mårten, Verkhovsky Michael I
Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), FIN-00014 Helsinki, Finland.
Biochemistry. 2006 May 2;45(17):5641-9. doi: 10.1021/bi060257v.
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 Cu(B) center. We assigned bands to the Cu(A) 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.
采用衰减全反射傅里叶变换红外光谱（ATR-FTIR）对反硝化副球菌细胞色素c氧化酶进行了电化学氧化还原滴定。大多数红外吸收差异特征可分为四组，这与该酶四个氧化还原中心的氧化还原转变相关。红外光谱的优势在于能够测量氧化还原中心的独立变化，而这些变化用其他光谱技术难以很好地分辨，甚至无法观测到。我们发现有12条红外谱带，其滴定的氧化还原中点电位最高（在pH 6.5时E(m)=412 mV），且在碱性区域每pH单位的pH依赖性为52 mV。这些谱带被确定与Cu(B)中心相关。我们将显示pH独立的E(m)为250 mV的谱带归属于Cu(A)中心。另外两组红外差异谱带反映了两个血红素基团的氧化还原转变，呈现出更为复杂的行为。每组都包含两部分，分别对应高电位和低电位的氧化还原转变。对于代表血红素a的谱带，高电位与低电位成分的比例约为3:2；对于血红素a(3)，该比例约为2:3。考虑到血红素之间的氧化还原相互作用，这些比例得出血红素之间E(m)的差值为9 mV（在pH 8.0时，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程度（在pH 8.0时为-115 mV）与pH相关。两个血红素E(m)值的pH依赖性相同，且约为理论值的一半，这表明在任何一个血红素还原时，一个酸/碱基团会结合一个质子。所采用的方法能够将四组中的每条红外谱带归属于血红素、氧化还原中心配体、氨基酸残基和/或蛋白质主链的振动。例如，对应于质子化谷氨酸E278的1737/1746 cm(-)(1)处著名的谱带位移与血红素a的氧化还原相关。
