Dynamics of the His79-heme alkaline transition of yeast iso-1-cytochrome c probed by conformationally gated electron transfer with Co(II)bis(terpyridine).
Dynamics of the His79-heme alkaline transition of yeast iso-1-cytochrome c probed by conformationally gated electron transfer with Co(II)bis(terpyridine).
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通过 Co(II)bis(三联吡啶) 构象门控电子转移探测酵母 iso-1-细胞色素 c 的 His79-血红素碱性转变动力学。
DOI：
10.1021/ja405725f
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发表时间：
2013
影响因子：
15
通讯作者：
Bowler,BruceE
中科院分区：
文献类型：
--
作者：
Cherney,MelisaM;Junior,CarolynC;Bergquist,BryanB;Bowler,BruceE
细胞色素c的碱性构象可能参与其电子传递和凋亡功能。我们使用钴（II）双（三联吡啶），钴（三联吡啶）22+，作为构象门控电子转移（门控ET）实验的试剂，研究碱性构象转变的K79 H变体的酵母异-1-cytochromec表达在大肠杆菌，WT* K79 H，丙氨酸在位置72和酿酒酵母，yK 79 H，三甲基赖氨酸（Tml）在位置72。Co（terpy）22+非常适合于这些变体的His 79介导的碱性构象转变的100 ms至1 s的时间尺度。通过Co（terpy）22+的His 79-血红素碱性构象的还原主要通过门控ET发生，其涉及转化为天然状态，然后还原，其中一小部分His 79-血红素碱性构象直接通过Co（terpy）22+还原。门控ET实验表明，形成的His 79-血红素碱性构象的机制只涉及两个可电离的基团。在以前的工作中，我们表明，His 73介导的碱性构象转变的机制需要三个电离基团。因此，血红素裂隙开放的机制取决于介导该过程的配体的位置。门控ET研究提供的微观速率常数表明，突变Tml 72（γ K79 H变体）在血红素缝隙环的Ala 72（WT* K79 H变体）影响血红素缝隙开放的动力学通过一个小的不稳定的天然构象和过渡态相对于His 79-血红素碱性构象。先前的pH跳跃数据表明，Tml 72 →Ala突变主要稳定了His 79介导的碱性构象转变的过渡态。
Alkaline conformers of cytochromecmay be involved in both its electron transport and apoptotic functions. We use cobalt(II)bis(terpyridine), Co(terpy)22+, as a reagent for conformationally gated electron-transfer (gated ET) experiments to study the alkaline conformational transition of K79H variants of yeast iso-1-cytochromecexpressed in Escherichia coli, WT*K79H, with alanine at position 72 and Saccharomyces cerevisiae, yK79H, with trimethyllysine (Tml) at position 72. Co(terpy)22+is well-suited to the 100 ms to 1 s time scale of the His79-mediated alkaline conformational transition of these variants. Reduction of the His79-heme alkaline conformer by Co(terpy)22+occurs primarily by gated ET, which involves conversion to the native state followed by reduction, with a small fraction of the His79-heme alkaline conformer directly reduced by Co(terpy)22+. The gated ET experiments show that the mechanism of formation of the His79-heme alkaline conformer involves only two ionizable groups. In previous work, we showed that the mechanism of the His73-mediated alkaline conformational transition requires three ionizable groups. Thus, the mechanism of heme crevice opening depends upon the position of the ligand mediating the process. The microscopic rate constants provided by gated ET studies show that mutation of Tml72 (yK79H variant) in the heme crevice loop to Ala72 (WT*K79H variant) affects the dynamics of heme crevice opening through a small destabilization of both the native conformer and the transition state relative to the His79-heme alkaline conformer. Previous pH jump data had indicated that the Tml72→Ala mutation primarily stabilized the transition state for the His79-mediated alkaline conformational transition.