Davies A M, Guillemette J G, Smith M, Greenwood C, Thurgood A G, Mauk A G, Moore G R
School of Chemical Sciences, University of East Anglia, Norwich, U.K.
Biochemistry. 1993 May 25;32(20):5431-5. doi: 10.1021/bi00071a019.
Heme propionate-7 in cytochrome c is an ionizable group located in a region of the protein that is inaccessible to bulk solvent. Electrostatic stabilization of this functional group appears to be achieved through interaction of heme propionate-7 with several amino acid residues that occur within hydrogen-bonding distance of it. To investigate the functional and spectroscopic roles of the amino acid residues that contribute to the immediate environment of heme propionate-7, the following variant forms of yeast (Saccharomyces cerevisiae) cytochrome c have been prepared and characterized by electrochemical and spectrochemical analyses: Arg38Ala, Tyr48Phe, Ala38Phe, Tyr48Phe/Trp59Phe, and Arg38Ala/Tyr48Phe/Trp59Phe. For each protein, the dependence of midpoint reduction potential and NMR spectrum on pH was determined, and the UV (250-450 nm) circular dichroic (CD) spectrum was measured. All of the variant proteins exhibited decreased reduction potentials with the greatest difference (-65 to -70 mV) exhibited by the multiply mutated proteins. The electrostatic properties of the variant proteins as reflected by the oxidation-state dependence of the His-39 pKa value were similar to those of the wild-type protein. Previous indirect assignments of minima in the CD spectrum of cytochrome c at 282 and 289 nm to Trp-59 are confirmed by spectra of the variant cytochromes in which this residue is replaced by Phe. The present results establish that the electrochemical effects of eliminating hydrogen-bonding interactions with heme propionate-7 are not additive and that the functional modulation of cytochrome c through regulation of the heme propionate-7 dielectric environment involves a complex combination of solvation effects and electrostatic or hydrogen-bonding interactions.
细胞色素c中的血红素丙酸酯-7是一个可电离基团，位于蛋白质中一个与大量溶剂无法接触的区域。该官能团的静电稳定似乎是通过血红素丙酸酯-7与几个在其氢键距离内出现的氨基酸残基相互作用来实现的。为了研究对血红素丙酸酯-7直接环境有贡献的氨基酸残基的功能和光谱作用，制备了以下酵母（酿酒酵母）细胞色素c的变体形式，并通过电化学和光谱化学分析进行了表征：Arg38Ala、Tyr48Phe、Ala38Phe、Tyr48Phe/Trp59Phe和Arg38Ala/Tyr48Phe/Trp59Phe。对于每种蛋白质，测定了中点还原电位和NMR谱对pH的依赖性，并测量了紫外（250 - 450 nm）圆二色（CD）谱。所有变体蛋白的还原电位均降低，多重突变蛋白表现出最大差异（-65至-70 mV）。由His-39 pKa值的氧化态依赖性反映的变体蛋白的静电性质与野生型蛋白相似。细胞色素c在282和289 nm处CD谱最小值先前的间接归属为Trp-59，这一归属通过该残基被Phe取代的变体细胞色素的光谱得到证实。目前的结果表明，消除与血红素丙酸酯-7的氢键相互作用的电化学效应不是累加的，并且通过调节血红素丙酸酯-7介电环境对细胞色素c进行功能调节涉及溶剂化效应与静电或氢键相互作用的复杂组合。
