Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, 00133 Rome, Italy.
Biochemistry. 2013 Jul 2;52(26):4578-88. doi: 10.1021/bi400324c. Epub 2013 Jun 21.
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.
细胞色素 c 在凋亡过程中经历结构变化；这种变化与蛋白质与心磷脂形成复合物时发生的修饰有关，心磷脂是构成线粒体膜的磷脂之一。尽管已经进行了几项研究来确定与细胞色素 c-心磷脂相互作用有关的蛋白质的部位，但迄今为止，该宿主区域的位置仍未确定，这是一个争议的问题。为了更深入地了解反应机制，我们研究了 Lys72、Lys73 和 Lys79 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。通过圆二色性、紫外可见和共振拉曼光谱学对马心细胞色素 c 的 Lys72Asn、Lys73Asn、Lys79Asn、Lys72/73Asn 和 Lys72/73/79Asn 突变体进行了生产和表征，并研究了突变对变体与心磷脂相互作用的影响。这些突变体的特征是具有非天然轴向配位的亚群，并且比野生型蛋白质不稳定。此外，缺乏 Lys72 和/或 Lys79 的突变体不结合心磷脂，尽管缺乏 Lys73 的突变体与磷脂形成复合物，但没有显示任何过氧化物酶活性。这些观察结果表明，Lys72、Lys73 和 Lys79 残基稳定了天然轴向 Met80-Fe(III)配位以及细胞色素 c 的三级结构。此外，虽然 Lys72 和 Lys79 对细胞色素 c-心磷脂识别至关重要，但 Lys72、Lys73 和 Lys79 的同时存在对于结合心磷脂的细胞色素 c 的过氧化物酶活性是必需的。
