Effect of proline content and histidine ligation on the dynamics of Ω-loop D and the peroxidase activity of iso-1-cytochrome c
Effect of proline content and histidine ligation on the dynamics of Ω-loop D and the peroxidase activity of iso-1-cytochrome c
复制标题
脯氨酸含量和组氨酸连接对 β-loop D 动力学和 iso-1-细胞色素 c 过氧化物酶活性的影响
DOI：
10.1016/j.jinorgbio.2023.112474
复制
发表时间：
2024
影响因子：
3.9
通讯作者：
Bowler, Bruce E.
中科院分区：
文献类型：
--
作者：
Martin, William J.;McClelland, Levi J.;Nold, Shiloh M.;Boshae, Kassandra L.;Bowler, Bruce E.
为了研究脯氨酸残基如何影响细胞色素c的Ω环D（残基70至85）的动态，我们在存在和不存在K73H突变的情况下制备了酵母iso-1-细胞色素c（iso-1-Cytc）的G83P和G83A变体。 Ω-loop D 在细胞凋亡中控制 Cytcan 的电子传递功能和 Cytcused 的过氧化物酶活性方面非常重要，因为它提供 Met80 血红素配体。 G83P 和 G83A 突变对 iso-1-Cytcin 存在或不存在 K73H 突变的整体稳定性没有影响。然而，这两种突变都会破坏 His73 介导的碱性构象异构体相对于天然状态的稳定性。 pH跳跃停流实验表明，G83P突变显着增强了His73介导的碱性转变的动力学。门控电子转移研究表明，动力学的增强是由于返回天然状态的速率增加，而 G83P 突变导致 Met80 连接的损失率没有变化。因此，G83P 取代不会使天然状态的构象变硬。由于当 Cytc 与含心磷脂的膜结合时会发生双组氨酸血红素连接，因此我们研究了 His73 连接对 Cytc 过氧化物酶活性的影响，Cytc 通过引起心磷脂氧化而充当细胞凋亡的早期信号。我们发现 His73 碱性构象异构体抑制 Cytc 的过氧化物酶活性。因此，在与心磷脂结合后，Cytcformed 的双组氨酸连接状态是细胞凋亡中 Cytcearly 过氧化物酶活性的负效应子。
To study how proline residues affect the dynamics of Ω-loop D (residues 70 to 85) of cytochromec, we prepared G83P and G83A variants of yeast iso-1-cytochromec(iso-1-Cytc) in the presence and absence of a K73H mutation. Ω-loop D is important in controlling both the electron transfer function of Cytcand the peroxidase activity of Cytcused in apoptosis because it provides the Met80 heme ligand. The G83P and G83A mutations have no effect on the global stability of iso-1-Cytcin presence or absence of the K73H mutation. However, both mutations destabilize the His73-mediated alkaline conformer relative to the native state. pH jump stopped-flow experiments show that the dynamics of the His73-mediated alkaline transition are significantly enhanced by the G83P mutation. Gated electron transfer studies show that the enhanced dynamics result from an increased rate of return to the native state, whereas the rate of loss of Met80 ligation is unchanged by the G83P mutation. Thus, the G83P substitution does not stiffen the conformation of the native state. Because bis-His heme ligation occurs when Cytcbinds to cardiolipin-containing membranes, we studied the effect of His73 ligation on the peroxidase activity of Cytc, which acts as an early signal in apoptosis by causing oxygenation of cardiolipin. We find that the His73 alkaline conformer suppresses the peroxidase activity of Cytc. Thus, the bis-His ligated state of Cytcformed upon binding to cardiolipin is a negative effector for the peroxidase activity of Cytcearly in apoptosis.