Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, University of Montana, Missoula, MT 59812, United States.
Biochemistry. 2013 Feb 5;52(5):837-46. doi: 10.1021/bi301599g. Epub 2013 Jan 23.
Trimethyllysine 72 (Tml72) of yeast iso-1-cytochrome c lies across the surface of the heme crevice loop (Ω-loop D, residues 70-85) like a brace. Lys72 is oriented similarly in horse cytochrome c (Cytc). To determine whether this residue affects the dynamics of opening the heme crevice loop, we have studied the effect of a Tml72 to Ala substitution on the formation of the His79-heme alkaline conformer near neutral pH using a variant of iso-1-Cytc including K72A and K79H mutations. Guanidine hydrochloride denaturation shows that the Tml72 to Ala substitution within error does not affect the global stability of the protein. The effect of the Tml72 to Ala substitution on the thermodynamics of the His79-heme alkaline transition is also small. However, pH-jump kinetic studies of the His79-heme alkaline transition show that both the forward and backward rates of conformational change are increased by the Tml72 to Ala substitution. The barrier for opening the heme crevice is reduced by 0.5 kcal/mol and for closing the heme crevice by 0.3 kcal/mol. The ability of Tml72 to modulate the heme crevice dynamics may indicate a crucial role in regulating function, such as in the peroxidase activity seen in the early stages of apoptosis.
酵母同工型 1-细胞色素 c 的三甲基赖氨酸 72（Tml72）位于血红素裂隙环（Ω-环 D，残基 70-85）的表面，就像一个支撑物。马细胞色素 c（Cytc）中的 Lys72 也有类似的取向。为了确定该残基是否会影响血红素裂隙环的打开动力学，我们研究了 Tml72 到 Ala 取代对在中性 pH 附近形成 His79-血红素碱性构象的影响，使用包括 K72A 和 K79H 突变的同工型 1-Cytc 的变体。盐酸胍变性表明，Tml72 到 Ala 取代在误差范围内不会影响蛋白质的整体稳定性。Tml72 到 Ala 取代对 His79-血红素碱性转变热力学的影响也很小。然而，His79-血红素碱性转变的 pH 跳跃动力学研究表明，Tml72 到 Ala 取代均增加了构象变化的正向和反向速率。打开血红素裂隙的势垒降低了 0.5 kcal/mol，关闭血红素裂隙的势垒降低了 0.3 kcal/mol。Tml72 调节血红素裂隙动力学的能力可能表明它在调节功能方面起着关键作用，例如在细胞凋亡早期观察到的过氧化物酶活性中。
