Electrochemical and ultraviolet/visible/infrared spectroscopic analysis of heme a and a3 redox reactions in the cytochrome c oxidase from Paracoccus denitrificans:: Separation of heme a and a3 contributions and assignment of vibrational modes
Electrochemical and ultraviolet/visible/infrared spectroscopic analysis of heme a and a3 redox reactions in the cytochrome c oxidase from Paracoccus denitrificans:: Separation of heme a and a3 contributions and assignment of vibrational modes
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DOI：
10.1021/bi982282
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发表时间：
1999-02-09
期刊：
影响因子：
2.9
通讯作者：
Mäntele, W
中科院分区：
文献类型：
--
作者：
Hellwig, P;Grzybek, S;Mäntele, W
采用电化学和紫外/可见/红外（UV/vis/IR）联合光谱法对反硝化副球菌细胞色素c氧化酶进行了研究。氧化电化学氧化还原滴定的全局拟合分析分别分离了与血红素a和氧化还原跃迁耦合的光谱贡献。同时调整紫外/可见光（900-400 nm）和红外（1800-1250 cm(-1)）中所有波长（1800-1250 cm）的中点电位和自定义数量的氧化还原成分（这里是血红素a和a(3)）的振幅，得到所选氧化还原电位数量的不同光谱。假设有两个氧化还原组分，得到了在-0.03 +/- 0.01 V和0.22 +/- 0.04 V（引用vs Ag/AgCl）下的氧化还原电位谱。本文采用的方法可以在不添加任何抑制剂的情况下，将血红素信号从天然细胞色素c氧化酶的电化学诱导可见差异光谱中分离出来。分离的血红素a和as紫外/可见差异光谱基本上与Babcock提出的高/低自旋和5/6配位血红素a/a(3)模型化合物的光谱相对应[（1988）在共振拉曼光谱的生物应用（Spiro， T., Ed.） Wiley and Sons, New York]。在此基础上计算了两种血红素的单分量傅里叶变换红外（FTIR）差谱，从而揭示了多肽主链的重组、血红素和单个氨基酸在辅助因子（血红素a/a(3)、Cu-A和Cu-B）的电子转移以及质子转移等耦合过程的贡献。提出了血红素振动模式的初步分配，并讨论了多肽主链重组和单个氨基酸（特别是Asp， Glu， Arg或Tyr）扰动的信号分配。
Cytochrome c oxidase from Paracoccus denitrificans was studied with a combined electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectroscopic approach. Global fit analysis of oxidative electrochemical redox titrations was used to separate the spectral contributions coupled to heme a and as redox transitions, respectively. Simultaneous adjustment of the midpoint potentials and of the amplitudes for a user-defined number of redox components (here heme a and a(3)) at all wavelengths in the UV/vis (900-400 nm) and at all wavenumbers in the infrared (1800-1250 cm(-1)) yielded difference spectra for the number of redox potentials selected. With an assumption of two redox components, two spectra for the redox potential at -0.03 +/- 0.01 V and 0.22 +/- 0.04 V (quoted vs Ag/AgCl) were obtained. The method used here allows the separation of the heme signals from the electrochemically induced visible difference spectra of native cytochrome c oxidase without the addition of any inhibitors. The separated heme a and as UV/vis difference spectra essentially correspond to spectra obtained for high/low-spin and 5/6-coordinated heme a/a(3) model compounds presented by Babcock [(1988) in Biological Applications of Resonance Raman Spectroscopy (Spiro, T., Ed.) Wiley and Sons, New York]. Single-component Fourier transform infrared (FTIR) difference spectra were calculated for both hemes on the basis of these fits, thus revealing contributions from the reorganization of the polypeptide backbone, from the hemes, and from single amino acids upon electron transfer of the cofactors (heme a/a(3), Cu-A, and Cu-B), as well from coupled processes such as proton transfer. A tentative assignment of heme vibrational modes is presented and the assignment of the signals to the reorganization of the polypeptide backbone and to perturbations of single amino acids, in particular Asp, Glu, Arg, or Tyr, is discussed.