The interaction of cytochrome c with cardiolipin (CL) is a critical step in the initial stages of apoptosis and is mediated by a positively charged region on the protein surface comprising several lysine residues (site A). Here, the interaction of wt S. cerevisiae cytochrome c (ycc) and its K72A/K73A, K72A/K79A, K73A/K79A and K72A/K73A/K79A variants with CL was studied through UV–Vis and MCD spectroscopies at pH 7 and molecular dynamics (MD) simulations, to clarify the role of the mutated lysines. Moreover, the influence of the lipid to protein ratio on the interaction mechanism was investigated using low (0.5–10) and high (5–60) CL/ycc molar ratios, obtained with small and gradual or large and abrupt CL additions, respectively. Although all proteins bind to CL, switching from the native low-spin His/Met-ligated form to a low-spin bis-His conformer and to a high-spin species at larger CL concentrations, the two schemes of CL addition show relevant differences in the CL/ycc molar ratios at which the various conformers appear, due to differences in the interaction mechanism. Extended lipid anchorage and peripheral binding appear to prevail at low and high CL/ycc molar ratios, respectively. Simultaneous deletion of two or three surface positive charges from Site A does not abolish CL binding, but instead increases protein affinity for CL. MD calculations suggest this unexpected behavior results from the mutation-induced severe weakening of the H-bond connecting the N_ε of His26 with the backbone oxygen of Glu44, which lowers the conformational stability compared to the wt species, overcoming the decreased surface electrostatic interaction.

中文翻译：

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酿酒酵母iso-1细胞色素c及其表面赖氨酸-丙氨酸变体与心磷脂的结合：电荷效应和脂蛋白比率的作用。

细胞色素c与心磷脂（CL）的相互作用是细胞凋亡初期的关键步骤，并由包含多个赖氨酸残基的蛋白质表面上带正电荷的区域（位点A）介导。在这里，wt 。酿酒酵母细胞色素c的相互作用（ycc）及其带有CL的K72A / K73A，K72A / K79A，K73A / K79A和K72A / K73A / K79A变体通过pH值为7的UV-Vis和MCD光谱学和分子动力学（MD）模拟研究，以阐明突变的赖氨酸。此外，使用低（0.5–10）和高（5–60）的CL / ycc摩尔比研究了脂质与蛋白质的比例对相互作用机理的影响，分别通过添加少量和逐渐添加或大量添加和突然添加CL来获得。尽管所有蛋白质均与CL结合，但从天然的低旋转His / Met连接形式转变为低旋转的bis-他的构象异构体和在较高的CL浓度下属于高自旋物种，两种CL的添加方案由于相互作用机理的差异，显示了各种构象异构体出现的CL / ycc摩尔比的相关差异。在低和高CL / ycc摩尔比下，延长的脂质锚定和周围结合似乎占优势。从位点A同时删除两个或三个表面正电荷不会消除CL结合，但会增加蛋白质对CL的亲和力。MD计算表明从H键连接的N突变诱导的严重削弱这个意外的行为结果_ε His26与Glu44的主链氧，这降低相比重量物种的构象稳定性，克服降低的表面的静电相互作用。