135 - Covalent Modification and Aggregation of Cytochrome c Induced by Cardiolipin Hydroperoxide

Abstract

Cytochrome c (cyt c) is a heme protein of 12 kDa that transfers electrons in the mitochondrial respiratory chain. Several in vitro studies have shown that increased cyt c peroxidase activity leads to cardiolipin (CL) oxidation, a hallmark of early stages of apoptosis. Apart from CL hydroperoxide (CL-OOH) and hydroxide (CL-OH) formation, it is also known that cyt c aggregates when interacting with H2O2. Protein aggregation can play an important role in cell signaling, but a definitive characterization remains to be performed. Here we aimed to investigate the role of CL-OOH in cyt c post-translational modifications and aggregation. Analysis of free cyt c after ultracentrifugation showed that cyt c remains anchored to liposomes (composed of phosphatidylcholine and CL (80:20 mol/mol) containing 20% and 100% of the CL as CL-OOH. This was confirmed using a high-ionic strength KCl-based medium, leading to the hypothesis that CL-OOH may induce covalent binding of cyt c to the membrane. This binding was further demonstrated to be time-dependent, with dimeric and trimeric species observed in the first 15 min and increased aggregates formation afterwards. We estimated this reaction rate at 9.58 ± 0.16 x 102k x M-1 x s-1, which is 100 times faster than that with H2O2. Data on nano-liquid chromatography coupled to mass spectrometry (Q-TOF) have revealed both ONE and HNE modification sites on lysine (K27, K73 and K88) and arginine residues as well as dityrosine cross-linking between Y74 and Y94 residues. These cross-linking pathways may play a role in cyt c oligomerization. Our findings thus suggest that CL-OOH can induce cyt c covalent binding to membranes and protein cross-linking. Furthermore, formation of K27, K73 and K88 adducts by CL-OOH derived electrophiles represents potential sites for lipid-protein interaction. Collectively, our results may shed light into the role of CL-OOH in cell death signaling in biological systems. Acknowledgements FAPESP, CNPq, CAPES, INCT/NAP/CEPID Redoxoma.