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 mitochondria] dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of center dot 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 center dot 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. C43 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 Zn2+-binding, which was reported functionally important for complex I assembly. C-79 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 center dot O-2(-) production, and mediating redox dysfunction of complex I.