Luntz T L, Schejter A, Garber E A, Margoliash E
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
Proc Natl Acad Sci U S A. 1989 May;86(10):3524-8. doi: 10.1073/pnas.86.10.3524.
The tyrosine-67 to phenylalanine mutated rat cytochrome c is similar to the unmutated protein in its spectral, reduction potential, and enzymic electron-transfer properties. However, the loss of the 695-nm band, characteristic of the ferric form of the normal low-spin physiologically active configuration, occurs 1.2 pH units higher on the alkaline side and 0.7 pH unit lower on the acid side. Similarly, the heme iron-methionine-80 sulfur bond is more stable to temperature, with the midpoint of the transition being 30 degrees C higher, corresponding to an increase in delta H of 5 kcal/mol (1 cal = 4.184 J), partially mitigated by an increase of 11 entropy units in delta S. Urea has only slightly different effects on the two proteins. These phenomena are best explained by considering that the loss of one of the three hydrogen-bonding side chains, tyrosine-67, asparagine-52, and threonine-78, which hold an internal water molecule on the "left, lower front" side of the protein [Takano, T. & Dickerson, R. E. (1981) J. Mol. Biol. 153, 95-115], is sufficient to prevent its inclusion in the mutant protein, leading to a more stable structure, and, as indicated by preliminary proton NMR two-dimensional phase-sensitive nuclear Overhauser effect spectroscopy analyses, a reorganization of this area. This hypothesis predicts that elimination of the hydrogen-bonding ability of residue 52 or 78 would also result in cytochromes c having similar properties. It is not obvious why the space-filling structure involving the internalized water molecule that leads to a destabilization energy of about 3 kcal/mol should be subject to extreme evolutionary conservation, when a more stable and apparently fully functional structure is readily available.
酪氨酸-67突变为苯丙氨酸的大鼠细胞色素c在光谱、还原电位和酶促电子转移特性方面与未突变的蛋白质相似。然而，正常低自旋生理活性构型的铁离子形式特有的695 nm波段的消失，在碱性一侧发生的pH值高1.2个单位，在酸性一侧低0.7个单位。同样，血红素铁-甲硫氨酸-80硫键对温度更稳定，转变中点高30℃，对应于ΔH增加5千卡/摩尔（1卡 = 4.184焦耳），部分被ΔS增加11熵单位所缓解。尿素对这两种蛋白质的影响只有轻微差异。这些现象最好通过考虑蛋白质“左下方前部”一侧保持一个内部水分子的三个氢键侧链（酪氨酸-67、天冬酰胺-52和苏氨酸-78）之一的缺失来解释，这种缺失足以阻止其包含在突变蛋白中，导致结构更稳定，并且如初步的质子核磁共振二维相敏核Overhauser效应光谱分析所示，该区域发生了重组。该假设预测，消除残基52或78的氢键能力也将导致细胞色素c具有类似特性。目前尚不清楚为什么涉及内化水分子的空间填充结构会导致约3千卡/摩尔的去稳定化能量，而当更容易获得更稳定且显然功能完全的结构时，这种结构却受到极端的进化保守。
