Increased cysteine sulfonation of Complex III with destruction of heme c mediates cardiac reperfusion injury via impairing catalytic function and enhancing FADH 2 ‐linked superoxide production

One consequence of myocardial ischemia‐reperfusion injury (I/R) is oxidative damage, which can cause mitochondrial dysfunction. Such I/R‐induced mitochondrial dysfunction results in impaired state 3 respiration and overproduction of superoxide, which can potentially induce protein cysteine sulfonation (PrSO 3 H) in mitochondria. Herein we employed UV/VIS spectroscopy and LC‐MS/MS analysis to study the mechanism of I/R‐mediated oxidative injury of complex III in mitochondria from rat hearts subjected to 30‐min of ischemia and 24‐h of reperfusion in vivo. In the I/R region, the activity of complex III was decreased by 31.0±4.1 % (n=7, p <0.001). Visible spectroscopic and immunoblotting analysis indicated that I/R mediated destruction of heme c ( c‐ type heme) without affecting protein level of cytochrome c ( cyt c ) in the mitochondria. I/R thus mediates dysfunction of cyt c via heme destruction. However, no significant impairment of complex III activity and heme c destruction was observed in mitochondria isolated from the risk region of rat heart subjected to 30 min ischemia (no reperfusion) in vivo despite a decreased state 3 respiration. LC‐MS/MS analysis showed enhanced formation of PrSO 3 H, which was consistent with mitochondrial redox state following I/R (EPR measurement of cyclic hydroxylamine oxidation). Mitochondria from I/R regions had intensely increased oxidative modification with sulfonation of cysteine (at C 236 ) of 2Fe‐2S cluster of Rieske iron‐sulfur protein (uqcrfs1), which would impair the main electron transfer pathway controlled by the Q‐cycle mechanism of complex III. LC‐MS/MS analysis also indicated that I/R intensely increased complex III PrSO 3 H at the subunits of hinge protein, cytochrome c 1 , core 1 and core 2. Carbamidomethylated C 122 /C 125 of the cytochrome c 1 via alkylating complex III was exclusively detected in the post‐ischemic mitochondria by LC‐MS/MS, indicating I/R mediates cleavage of thioether bonds between heme c 1 and apocytochrome c 1 . FADH 2 ‐linked · O 2 − generation by mitochondrial SCR (supercomplex hosting complex II and complex III) was induced by succinate and evaluated by EPR spin trapping with DEPMPO. FADH 2 ‐linked · O 2 − generation by normal mitochondrial SCR was minimized in the presence of a functional cyt c . Whereas, FADH 2 ‐linked · O 2 − generation by the post‐ischemic mitochondrial SCR was intensely increased by 123% in the presence of heme‐depleted cyt c , and further enhanced by 227% under the conditions of increasing pO 2 in vitro (from 0.23 mM O 2 to 0.8 mM O 2 ), suggesting that physiologic conditions of hyperoxygenation during reperfusion with heme‐depleted cyt c mediated overproduction of FADH 2 ‐linked · O 2 − by the post‐ischemic mitochondria. In conclusion, reperfusion‐induced PrSO 3 H with heme depletion of cyt c controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction via overproducing · O 2 − in the post‐ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may necessitate lessening of PrSO 3 H and preservation of functional integrity of heme groups in mitochondria. Support or Funding Information NIH RO1HL083237Enhancement of FADH2‐linked superoxide generation by post‐ischemic mitochondrial SCR ( C vs B) in the presence of post‐ischemic cyt c ( D vs B ) and hyperoxygenation ( E vs D ).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .