Bandi Swati, Bowler Bruce E
Department of Chemistry & Biochemistry, Center for Biomolecular Structure and Dynamics, University of Montana , Missoula, Montana 59812, United States.
Biochemistry. 2015 Mar 10;54(9):1729-42. doi: 10.1021/bi501252z. Epub 2015 Feb 24.
An A81H variant of yeast iso-1-cytochrome c is prepared to test the hypothesis that the steric size of the amino acid at sequence position 81 of cytochrome c, which has evolved from Ala in yeast to Ile in mammals, slows the dynamics of the opening of the heme crevice. The A81H mutation is used both to increase steric size and to provide a probe of the dynamics of the heme crevice through measurement of the thermodynamics and kinetics of the His81-mediated alkaline conformational transition of A81H iso-1-cytochrome c. Thermodynamic measurements show that the native conformer is more stable than the His81-heme alkaline conformer for A81H iso-1-cytochrome c. ΔGu°(H2O) is approximately 1.9 kcal/mol for formation of the His81-heme alkaline conformer. By contrast, for K79H iso-1-cytochrome c, the native conformer is less stable than the His79-heme alkaline conformer. ΔGu°(H2O) is approximately -0.34 kcal/mol for formation of the His79-heme alkaline conformer. pH jump and gated electron transfer kinetics demonstrate that this stabilization of the native conformer in A81H iso-1-cytochrome c arises primarily from a decrease in the rate constant for formation of the His81-heme alkaline conformer, kf,His81, relative to kf,His79 for formation of the His79-heme alkaline conformer, which forms by a mechanism similar to that observed for the His81-heme alkaline conformer. The result is discussed in terms of the effect of global protein stability on protein dynamics and in terms of optimization of the sequence of cytochrome c for its role as a peroxidase in the early stages of apoptosis in higher eukaryotes.
制备酵母同工酶-1-细胞色素c的A81H变体，以检验以下假设：细胞色素c序列第81位氨基酸的空间大小，从酵母中的丙氨酸演变为哺乳动物中的异亮氨酸，减缓了血红素裂隙打开的动力学。A81H突变既用于增加空间大小，又通过测量A81H同工酶-1-细胞色素c的His81介导的碱性构象转变的热力学和动力学，来探测血红素裂隙的动力学。热力学测量表明，对于A81H同工酶-1-细胞色素c，天然构象体比His81-血红素碱性构象体更稳定。形成His81-血红素碱性构象体时，ΔGu°(H2O)约为1.9千卡/摩尔。相比之下，对于K79H同工酶-1-细胞色素c，天然构象体比His79-血红素碱性构象体不稳定。形成His79-血红素碱性构象体时，ΔGu°(H2O)约为-0.34千卡/摩尔。pH跃变和门控电子转移动力学表明，A81H同工酶-1-细胞色素c中天然构象体的这种稳定性主要源于形成His81-血红素碱性构象体的速率常数kf,His81相对于形成His79-血红素碱性构象体的kf,His79的降低，His79-血红素碱性构象体的形成机制与His81-血红素碱性构象体类似。从全局蛋白质稳定性对蛋白质动力学的影响以及细胞色素c序列在高等真核生物凋亡早期作为过氧化物酶的作用优化方面讨论了该结果。
