Blair D F, Ellis W R, Wang H, Gray H B, Chan S I
J Biol Chem. 1986 Sep 5;261(25):11524-37.
The cytochrome a and a3 sites in uninhibited, detergent-solubilized cytochrome c oxidase have been studied under a wide range of conditions using thin-layer spectroelectrochemistry. The observed absorbance changes at the alpha and Soret absorbance maxima have been used together to estimate the extents of reduction of cytochromes a and a3, using the absorbance properties of these cytochromes deduced from previous measurements employing ligand inhibition of cytochrome a3. The resulting Nernst plots, combined with the results of parallel studies on the carbon monoxide-inhibited enzyme (Ellis, W. R., Jr., Wang, H., Blair, D. F., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 161-167; Wang, H., Blair, D. F., Ellis, W. R., Jr., Gray, H. B., and Chan, S. I. (1986) Biochemistry 25, 167-171), indicate that the cytochrome a site participates in anticooperative thermodynamic interactions which involve all three of the other metal sites in the protein. Using an analysis which resolves the intrinsic thermodynamic properties of the cytochromes from the effects of the intersite interactions, the pH, temperature, and ionic strength dependences of the cytochrome reduction potentials have been measured. The standard entropy of reduction of cytochrome a in the native enzyme is large and negative, in agreement with measurements on the carbon monoxide-inhibited enzyme. The reduction potential of cytochrome a is only moderately (less than -30 mV/pH unit) dependent upon pH, which implies that its reduction is linked to the uptake, on the average, of only about 0.5 protons at pH 7.0, and significantly less at the higher pH values relevant to the mitochondrial matrix. The thermodynamic properties of cytochrome a3 were found to be different in the two enzyme batches studied: in one batch, the cytochrome a3 reduction potential decreased steeply (about -56 mV/pH unit) with increasing pH, indicating stoichiometric (1 H+/e-) coupling of protonation to reduction. In the other batch, the cytochrome a3 potential was insensitive to pH below pH 7.5 and decreased at higher pH values in a manner suggesting coupling to an ionizable group with pKa near 7.8. The temperature dependence of the cytochrome a3 reduction potential indicates that its standard entropy of reduction is more positive than that of myoglobin, another high-spin metalloprotein heme, and significantly more positive than that of cytochrome a.(ABSTRACT TRUNCATED AT 400 WORDS)
利用薄层光谱电化学技术，在多种条件下对未受抑制、经去污剂增溶的细胞色素c氧化酶中的细胞色素a和a3位点进行了研究。利用先前通过细胞色素a3的配体抑制测量得出的这些细胞色素的吸光度特性，将在α和Soret吸光度最大值处观察到的吸光度变化结合起来，以估计细胞色素a和a3的还原程度。所得的能斯特图，结合对一氧化碳抑制的酶的平行研究结果（埃利斯，W.R.，Jr.，王，H.，布莱尔，D.F.，格雷，H.B.，和陈，S.I.（1986年）《生物化学》25，161 - 167；王，H.，布莱尔，D.F.，埃利斯，W.R.，Jr.，格雷，H.B.，和陈，S.I.（1986年）《生物化学》25，167 - 171），表明细胞色素a位点参与了反协同热力学相互作用，该相互作用涉及蛋白质中其他三个金属位点。通过一种从位点间相互作用的影响中解析细胞色素内在热力学性质的分析方法，测量了细胞色素还原电位对pH、温度和离子强度的依赖性。天然酶中细胞色素a还原的标准熵很大且为负值，这与对一氧化碳抑制的酶的测量结果一致。细胞色素a的还原电位仅适度地（小于 - 30 mV/pH单位）依赖于pH，这意味着在pH 7.0时其还原平均仅与约0.5个质子的摄取相关联，而在与线粒体基质相关的较高pH值下摄取的质子明显更少。在所研究的两批酶中，发现细胞色素a3的热力学性质不同：在一批中，细胞色素a3的还原电位随着pH升高而急剧下降（约 - 56 mV/pH单位），表明质子化与还原存在化学计量关系（1 H⁺/e⁻）。在另一批中，细胞色素a3的电位在pH低于7.5时对pH不敏感，而在较高pH值下以一种表明与pKa接近7.8的可电离基团偶联的方式下降。细胞色素a3还原电位的温度依赖性表明，其还原的标准熵比另一种高自旋金属蛋白血红素肌红蛋白的更正值，且比细胞色素a的明显更正值。（摘要截短于400字）
