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

Abstract 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 O 2 − . 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 (C 425 ), N1b (C 92 ), N4 (C 226 ), N2 (C 158 /C 188 ), and N1a (C 134 /C 139 ). 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 C 134 /C 139 sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O 2 − generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C 125 , C 187 , C 206 , C 238 , C 255 , C 286 ), 75 kDa (C 367 , C 554 , C 564 , C 727 ), 49 kDa (C 146 , C 326 , C 347 ), and PSST (C 188 ). 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 (C 67 , C 112 , C 183 , C 253 ), 15 kDa (C 43 ), and 13 kDa (C 79 ). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C 43 of the 15 kDa subunit forms an intramolecular disulfide bond with C 56 , which was reported to stabilize complex I structure. C 79 of the 13 kDa subunit is involved in Zn 2+ -binding, which was reported functionally important for complex I assembly. C 79 sulfonation by I/R was found to impair Zn 2+ -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 • O 2 − production, and mediating redox dysfunction of complex I.