Changing the invariant proline-30 of rat and Drosophila melanogaster cytochromes c to alanine or valine destabilizes the heme crevice more than the overall conformation.
Changing the invariant proline-30 of rat and Drosophila melanogaster cytochromes c to alanine or valine destabilizes the heme crevice more than the overall conformation.
复制标题
将大鼠和果蝇细胞色素 c 的不变脯氨酸 30 更改为丙氨酸或缬氨酸会比整体构象更不稳定地破坏血红素缝隙。
DOI：
10.1073/pnas.87.22.8697
复制
发表时间：
1990
影响因子：
11.1
通讯作者：
Margoliash,E
中科院分区：
文献类型：
--
作者：
Koshy,TI;Luntz,TL;Schejter,A;Margoliash,E
将脯氨酸-30转化为丙氨酸或缬氨酸的黑腹果蝇和大鼠细胞色素c在一种面包酵母——酿酒酵母中表达出来，并在那里维持有氧生长。突变对光谱和氧化还原电位没有显著影响，但极大地改变了甲硫氨酸-80硫和血红素铁之间的键的稳定性，从四个标准来判断：(i)突变蛋白的铁形式的695 nm波段的碱性pKa值比野生型降低了近1个pH单位；（ii）酸性pKa值增加0.5 ~ 1.2 pH单位；（iii）与野生型相比，突变蛋白的695-nm波段在温度降低10-20℃时一半消失；（iv）突变蛋白的695 nm波段对尿素浓度敏感，但对其整体结构影响不大。缬氨酸取代的大鼠细胞色素c具有介于野生型和丙氨酸突变体之间的特性。不稳定的配位键位于距离突变位点很远的空间中。这表明，突变削弱了残基30的羰基与组氨酸-18的咪唑的亚氨基之间的氢键，改变了咪唑与血红素铁的键，而咪唑又通过反式效应削弱了血红素铁与另一个轴向配体——蛋氨酸-80的硫之间的键。或者，突变的影响可以沿着肽链变构传播。
Drosophila melanogaster and rat cytochromes c in which proline-30 was converted to alanine or valine were expressed in a strain of baker's yeast, Saccharomyces cerevisiae, where they sustained aerobic growth. The mutations had no significant effect on the spectra or redox potentials but altered drastically the stability of the bond between the methionine-80 sulfur and the heme iron, as judged by four criteria: (i) the alkaline pKa values of the 695-nm band of the ferric form of the mutant proteins decreased by almost 1 pH unit as compared to the wild types; (ii) the acid pKa values increased by 0.5 to 1.2 pH units; (iii) the 695-nm band half-disappeared at temperatures 10-20 degrees C lower in the mutant proteins than in the wild types; and (iv) the 695-nm band of the mutant proteins was susceptible to concentrations of urea that had little influence on their overall structure. The valine-substituted rat cytochrome c had properties intermediate between those of the wild type and the alanine mutant. The destabilized coordinative bond is located in space a long distance from the mutation site. It is suggested that the mutations weaken the hydrogen bond between the carbonyl of residue 30 and the imino group of the imidazole of histidine-18, modifying the bonding of the heme iron by that imidazole, which, in turn, through a trans effect, weakens the bond between the heme iron and the other axial ligand, the sulfur of methionine-80. Alternatively, the effect of the mutations may be propagated allosterically along the peptide chain.