Gullo S M, Tayh J A, Li J, Baker G M
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Arch Biochem Biophys. 1993 Nov 15;307(1):78-84. doi: 10.1006/abbi.1993.1563.
Dilution of detergent-solubilized cytochrome c oxidase has been found to increase the pH sensitivity of the a3-CuB site. Comparison of a large number of preparations, each purified at pH 8.8, showed either one or two exponential reactivity with cyanide, indicating that pH was not the only factor regulating kinetic heterogeneity within the binuclear core. Differences in heme a concentration during the soluble stages of purification were shown to have a substantial effect on the distribution of both optical and kinetic states within the isolated enzyme. Similar dilution effects were also inducible after purification. In particular, the apparent pKa of the Soret band maximum was shifted to higher pH following dilution of the enzyme. The resulting increase in the 414-nm conformer was also associated with enhanced conversion to the slow cyanide-binding form, suggesting that a single protonation event may give rise to both optical and kinetic changes. Two lines of evidence were contrary to this: First, by appropriate choice of experimental conditions it was possible to observe 30-50% of the cytochrome a3 centers in the 414-nm state with no associated evidence of the slow conformer. Second, the addition of formate at pH 8.8 induced conversion to both slow and 414-nm conformers, but the rates and extents of conversion were not correlated. Separate structural events are therefore proposed to modulate the optical and kinetic properties of the binuclear core. Since acidic pH has the well-established ability to induce both rapid-->slow and 430-->414-nm conversions, we suggest that the effects of dilution and formate are to mimic an acid jump by facilitating proton uptake into the binuclear core.
已发现用去污剂增溶的细胞色素c氧化酶的稀释会增加a3-CuB位点的pH敏感性。对大量在pH 8.8下纯化的制剂进行比较，结果显示与氰化物反应呈一种或两种指数关系，这表明pH不是调节双核核心内动力学异质性的唯一因素。纯化的可溶性阶段血红素a浓度的差异对分离酶内光学和动力学状态的分布有重大影响。纯化后也可诱导出类似的稀释效应。特别是，酶稀释后，Soret带最大值的表观pKa向更高pH值移动。414nm构象体的相应增加也与向慢速氰化物结合形式的转化率提高有关，这表明单个质子化事件可能导致光学和动力学变化。有两条证据与此相反：第一，通过适当选择实验条件，可以观察到30%-50%的细胞色素a3中心处于414nm状态，而没有慢速构象体的相关证据。第二，在pH 8.8时添加甲酸盐会诱导向慢速和414nm构象体的转化，但转化的速率和程度并不相关。因此，提出了单独的结构事件来调节双核核心的光学和动力学性质。由于酸性pH具有诱导快速→慢速和430→414nm转化的既定能力，我们认为稀释和甲酸盐的作用是通过促进质子进入双核核心来模拟酸跃变。
