Role of lysines in cytochrome c-cardiolipin interaction.
Role of lysines in cytochrome c-cardiolipin interaction.
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DOI：
10.1021/bi400324c
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发表时间：
2013-06
期刊：
影响因子：
2.9
通讯作者：
F. Sinibaldi;B. Howes;E. Droghetti;F. Polticelli;M. Piro;D. Di pierro;L. Fiorucci;M. Coletta;G. Smulevich;R. Santucci
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文献类型：
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作者：
F. Sinibaldi;B. Howes;E. Droghetti;F. Polticelli;M. Piro;D. Di pierro;L. Fiorucci;M. Coletta;G. Smulevich;R. Santucci
细胞色素c在凋亡过程中发生结构变化;这种变化与蛋白质与心磷脂（构成线粒体膜的磷脂之一）形成复合物时发生的修饰有关。虽然已经进行了几项研究，以确定参与细胞色素c-心磷脂相互作用的蛋白质的网站（S），到目前为止，该托管区域的位置（S）仍然是未知的，是一个有争议的问题。为了更深入地了解反应机制，我们研究了Lys 72，Lys 73和Lys 79残基在细胞色素c-心磷脂相互作用中的作用，因为这些侧链似乎对细胞色素c-心磷脂识别至关重要。制备了马心细胞色素c的Lys 72 Asn、Lys 73 Asn、Lys 79 Asn、Lys 72/73 Asn和Lys 72/73/79 Asn突变体，并通过圆二色谱、紫外-可见和共振拉曼光谱对其进行了表征，研究了突变体对心磷脂相互作用的影响。突变体的特征在于具有非天然轴向协调的亚群，并且比野生型蛋白质更不稳定。此外，缺少Lys 72和/或Lys 79的突变体不结合心磷脂，而缺少Lys 73的突变体虽然与磷脂形成复合物，但不显示任何过氧化物酶活性。这些观察结果表明，Lys 72，Lys 73和Lys 79残基稳定的天然轴向Met 80-Fe（III）的协调以及细胞色素c的三级结构。此外，虽然Lys 72和Lys 79对于细胞色素c-心磷脂识别是关键的，但是Lys 72、Lys 73和Lys 79的同时存在对于心磷脂结合的细胞色素c的过氧化物酶活性是必需的。
Cytochrome c undergoes structural variations during the apoptotic process; such changes have been related to modifications occurring in the protein when it forms a complex with cardiolipin, one of the phospholipids constituting the mitochondrial membrane. Although several studies have been performed to identify the site(s) of the protein involved in the cytochrome c-cardiolipin interaction, to date the location of this hosting region(s) remains unidentified and is a matter of debate. To gain deeper insight into the reaction mechanism, we investigate the role that the Lys72, Lys73, and Lys79 residues play in the cytochrome c-cardiolipin interaction, as these side chains appear to be critical for cytochrome c-cardiolipin recognition. The Lys72Asn, Lys73Asn, Lys79Asn, Lys72/73Asn, and Lys72/73/79Asn mutants of horse heart cytochrome c were produced and characterized by circular dichroism, ultraviolet-visible, and resonance Raman spectroscopies, and the effects of the mutations on the interaction of the variants with cardiolipin have been investigated. The mutants are characterized by a subpopulation with non-native axial coordination and are less stable than the wild-type protein. Furthermore, the mutants lacking Lys72 and/or Lys79 do not bind cardiolipin, and those lacking Lys73, although they form a complex with the phospholipid, do not show any peroxidase activity. These observations indicate that the Lys72, Lys73, and Lys79 residues stabilize the native axial Met80-Fe(III) coordination as well as the tertiary structure of cytochrome c. Moreover, while Lys72 and Lys79 are critical for cytochrome c-cardiolipin recognition, the simultaneous presence of Lys72, Lys73, and Lys79 is necessary for the peroxidase activity of cardiolipin-bound cytochrome c.