Increased cysteine sulfonation of Complex III with destruction of heme c mediates cardiac reperfusion injury via impairing catalytic function and enhancing FADH2‐linked superoxide production

Abstract

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 (PrSO3H) 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 PrSO3H, 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 C236) 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 PrSO3H at the subunits of hinge protein, cytochrome c1, core 1 and core 2. Carbamidomethylated C122/C125 of the cytochrome c1 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 c1 and apocytochrome c1. FADH2-linked ·O2− generation by mitochondrial SCR (supercomplex hosting complex II and complex III) was induced by succinate and evaluated by EPR spin trapping with DEPMPO. FADH2-linked ·O2− generation by normal mitochondrial SCR was minimized in the presence of a functional cyt c. Whereas, FADH2-linked ·O2− 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 pO2 in vitro (from 0.23 mM O2 to 0.8 mM O2), suggesting that physiologic conditions of hyperoxygenation during reperfusion with heme-depleted cyt c mediated overproduction of FADH2-linked ·O2− by the post-ischemic mitochondria. In conclusion, reperfusion-induced PrSO3H with heme depletion of cyt c controls oxidative injury of the complex III, and aggravates mitochondrial dysfunction via overproducing ·O2− in the post-ischemic heart. Preservation of mitochondrial function and ultimately the salvage of ischemic tissue may necessitate lessening of PrSO3H and preservation of functional integrity of heme groups in mitochondria. Support or Funding Information NIH RO1HL083237 Enhancement 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.