Cytochrome c (cytc) is a heme protein of 12 kDa that transfers electrons in the mitochondrial respiratory chain. Increased cytc peroxidase activity leads to cardiolipin (CL) oxidation, a hallmark of early apoptosis stage. Here, we aimed to investigate the interaction between cytc with cardiolipin hydroperoxide (CLOOH) in a mimetic mitochondrial membrane. Cytc-CL peroxidase reaction occurred at faster rates with CLOOH than with H₂O₂. Moreover, liposomes containing CLOOH promoted increased protein aggregation with minor or no release of cytc from the membrane. Dimeric and trimeric cytc species were observed in the first 15 min, followed by increased formation of high-molecular-weight aggregates afterwards. nLC-MS/MS analysis identified several Lys and His residues covalently modified by lipid aldehydes that showed mass increments corresponding to 4-hydroxynonenal (HNE), 4-oxononenal (ONE), hexanoyl, heptenal and octenal addition. Noteworthy, most modifications were observed at Lys and His residues located at A-site (K73, K87, K88), L-site (H26, H33, and K27) membrane binding sites. Further, dityrosine cross-linked peptides were also characterized at residues Y48-Y74, Y48-Y97 and Y74-Y97. Collectively, our findings show that CLOOH causes irreversible protein damage and crosslinking of cytc in the membrane.

细胞色素c（cytc）是一种12 kDa的血红素蛋白，可在线粒体呼吸链中转移电子。cytc过氧化物酶活性的增加导致心磷脂（CL）氧化，这是早期凋亡阶段的标志。在这里，我们旨在研究模拟线粒体膜中cytc与心磷脂氢过氧化物（CLOOH）之间的相互作用。Cytc-CL过氧化物酶反应与CLOOH的反应速率比与H ₂ O _2的反应速率更快。而且，含有CLOOH的脂质体促进了蛋白质聚集的增加，而cytc从膜上很少或没有释放。在最初的15分钟内观察到二聚体和三聚体cytc物种，其后高分子量聚集体的形成增加。nLC-MS / MS分析鉴定了被脂质醛共价修饰的几个Lys和His残基，其质量增量分别对应于4-羟基壬烯醛（HNE），4-氧酮醛酮（ONE），己酰基，庚烯和辛烯醛的添加。值得注意的是，在位于A位（K73，K87，K88），L位（H26，H33和K27）膜结合位点的Lys和His残基上观察到大多数修饰。此外，还对残基Y48-Y74，Y48-Y97和Y74-Y97表征了二酪氨酸交联肽。总的来说，