Mitochondrial complex I in the post-ischemic heart: reperfusion-mediated oxidative injury and protein cysteine sulfonation

A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of O₂^-. The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to injury. Herein we employed LC-MS/MS to identify protein sulfonation of complex I in mitochondria from the infarct region of rat hearts subjected to 30-min of coronary ligation and 24-h of reperfusion in vivo as well as the mitochondria of sham controls. Mitochondrial preparations from the I/R regions had enhanced sulfonation levels on the cysteine ligands of iron‑sulfur clusters, including N3 (C₄₂₅), N1b (C₉₂), N4 (C₂₂₆), N2 (C₁₅₈/C₁₈₈), and N1a (C₁₃₄/C₁₃₉). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus sulfonation of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and an antioxidative function. Increased C₁₃₄/C₁₃₉ sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O₂^- generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C₁₂₅, C₁₈₇, C₂₀₆, C₂₃₈, C₂₅₅, C₂₈₆), 75 kDa (C₃₆₇, C₅₅₄, C₅₆₄, C₇₂₇), 49 kDa (C₁₄₆, C₃₂₆, C₃₄₇), and PSST (C₁₈₈). These results were consistent with the consensus indicating that 51 kDa and 75 kDa are two of major subunits hosting regulatory thiols, and their enhanced sulfonation by I/R predisposed the myocardium to further oxidant stress with impaired ubiquinone reduction. MS analysis further showed I/R-mediated enhanced sulfonation at the supernumerary subunits of 42 kDa (C₆₇, C₁₁₂, C₁₈₃, C₂₅₃), 15 kDa (C₄₃), and 13 kDa (C₇₉). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C₄₃ of the 15 kDa subunit forms an intramolecular disulfide bond with C₅₆, which was reported to stabilize complex I structure. C₇₉ of the 13 kDa subunit is involved in Zn²⁺-binding, which was reported functionally important for complex I assembly. C₇₉ sulfonation by I/R was found to impair Zn²⁺-binding. No significant enhancement of protein sulfonation was observed in mitochondrial complex I from the rat heart subjected to 30-min ischemia alone in vivo despite a decreased state 3 respiration, suggesting that the physiologic conditions of hyperoxygenation during reperfusion mediated an increase in complex I sulfonation and oxidative injury. In conclusion, sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increasing ^•O₂^- production, and mediating redox dysfunction of complex I.