Nold Shiloh M, Lei Haotian, Mou Tung-Chung, Bowler Bruce E
Department of Chemistry and Biochemistry, University of Montana , Missoula, Montana 59812, United States.
Center for Bimolecular Structure and Dynamics, University of Montana , Missoula, Montana 59812, United States.
Biochemistry. 2017 Jul 5;56(26):3358-3368. doi: 10.1021/acs.biochem.7b00342. Epub 2017 Jun 21.
We test the hypothesis that Lys72 suppresses the intrinsic peroxidase activity of human cytochrome c, as observed previously for yeast iso-1-cytochrome c [McClelland, L. J., et al. (2014) Proc. Natl. Acad. Sci. U. S. A. 111, 6648-6653]. A 1.25 Å X-ray structure of K72A human cytochrome c shows that the mutation minimally affects structure. Guanidine hydrochloride denaturation demonstrates that the K72A mutation increases global stability by 0.5 kcal/mol. The K72A mutation also increases the apparent pK of the alkaline transition, a measure of the stability of the heme crevice, by 0.5 unit. Consistent with the increase in the apparent pK, the rate of formation of the dominant alkaline conformer decreases, and this conformer is no longer stabilized by proline isomerization. Peroxidase activity measurements show that the K72A mutation increases k by 1.6-4-fold at pH 7-10, an effect larger than that seen for the yeast protein. X-ray structures of wild type and K72A human cytochrome c indicate that direct interactions of Lys72 with the far side of Ω-loop D, which are seen in X-ray structures of horse and yeast cytochrome c and could suppress peroxidase activity, are lacking. Instead, we propose that the stronger effect of the K72A mutation on the peroxidase activity of human versus yeast cytochrome c results from relief of steric interactions between the side chains at positions 72 and 81 (Ile in human vs Ala in yeast), which suppress the dynamics of Ω-loop D necessary for the intrinsic peroxidase activity of cytochrome c.
赖氨酸72（Lys72）会抑制人细胞色素c的内在过氧化物酶活性，正如之前在酵母异-1-细胞色素c中观察到的那样[麦克莱兰，L. J.等人（2014年）《美国国家科学院院刊》111卷，6648 - 6653页]。赖氨酸72突变为丙氨酸（K72A）的人细胞色素c的1.25埃X射线结构表明，该突变对结构的影响极小。盐酸胍变性实验表明，K72A突变使整体稳定性提高了0.5千卡/摩尔。K72A突变还使碱性转变的表观pK值增加了0.5个单位，这是血红素裂隙稳定性的一种度量。与表观pK值的增加相一致，主要碱性构象体的形成速率降低，并且该构象体不再因脯氨酸异构化而稳定。过氧化物酶活性测量结果表明，在pH值为7至10时，K72A突变使反应速率常数k增加了1.6至4倍，这一效应比在酵母蛋白中观察到的更大。野生型和K72A人细胞色素c的X射线结构表明，不存在赖氨酸72与Ω环D远侧的直接相互作用，而在马和酵母细胞色素c的X射线结构中可以看到这种相互作用，它可能会抑制过氧化物酶活性。相反，我们认为，K72A突变对人细胞色素c过氧化物酶活性的影响比对酵母细胞色素c的影响更强，其原因是72位和81位侧链之间（人细胞色素c中为异亮氨酸，酵母细胞色素c中为丙氨酸）的空间相互作用得到缓解，这种相互作用抑制了细胞色素c内在过氧化物酶活性所需的Ω环D的动力学。
