Increased Protein Cysteine Sulfonation with Heme Destruction of Mitochondrial Complex III Mediates Cardiac Reperfusion Injury

A serious consequence of myocardial ischemia‐reperfusion injury (IR) is oxidative damage, which causes mitochondrial dysfunction. Such IR induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide. The cascading reactive oxygen species can propagate and potentially induce protein cysteine sulfonation (PrCSO 3 H) of mitochondrial proteins. Herein we employed UV/VIS spectroscopy and LC‐MS/MS analysis to study the mechanism of IR‐mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30 min of ischemia (coronary ligation) and 24 h of reperfusion in vivo. In the IR region, the activity of complex III was decreased by 31.0±4.1 % ( n =7, p <0.001). Visible spectroscopic analysis indicated that IR mediated destruction of heme b and hemes ( c+c 1 ) in the mitochondria, thus supporting IR‐induced impairment of the enzymatic activity of complex III. However, no significant impairment of complex III activity and heme destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min coronary ligation in vivo despite a decreased state 3 respiration detected in the ischemic mitochondria. LC‐MS/MS analysis showed formation of PrCSO 3 H, which was consistent with a redox analysis of mitochondria following IR (EPR measurement of oxidation of cyclic hydroxylamine). Mitochondria from IR regions had intensely increased oxidative modification with S‐sulfonation level on the cysteine ligand (at C 236 ) of 2Fe‐2S cluster of Rieske iron‐sulfur protein ( uqcrfs1 ), thus impairing the main electron transfer pathway controlled by the Q‐cycle mechanism of complex III. LC‐MS/MS analysis also indicated that IR‐mediated increased PrCSO 3 H of hinge protein at C 65 (subunit 8, uqcrh ) and cytochrome c 1 subunit at C 140 and C 220 , which weakened the interaction of hinge protein with cytochrome c 1 and proper complex formation between cytochrome c 1 and cytochrome c . LC‐MS/MS analysis further identified 4‐HNE (4‐hydroxylnonenal) adduct of cytochrome c 1 at C 140 , suggesting involvement of IR‐induced lipid peroxidation in heme destruction and PrCSO 3 H of complex III. LC‐MS/MS analysis also showed that IR‐enhancing PrCSO 3 H of core 1 subunit ( uqcrc1 at C 69 , C 268 , C 380 , C 410 , C 445 , C 453 ) and core 2 subunit ( uqcrc2 at C 191 ), which may affect the binding of subunit 9 inhibitory peptide to core 1 and core 2 subunits and reactivate the MPP (matrix processing peptidase) activity of the core 1/core 2 subunits of mammalian complex III. LC‐MS/MS analysis of ischemic mitochondria has indicated a modest reduction from the basal level of complex III PrCSO 3 H detected in the mitochondria of sham control heart, suggesting that physiologic conditions of hypoxia during ischemia suppressed PrCSO 3 H, and physiologic conditions of hyperoxygenation during reperfusion mediated enhancement of complex III PrCSO 3 H. In conclusion, reperfusion‐mediated heme destruction with increased PrCSO 3 H controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction in the post‐ischemic heart . Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may related to the preservation of functional integrity of heme groups and protein structure within mitochondrial complex III. Support or Funding Information NHLBI/NIH, HL083237 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .