Increased Cysteine Sulfonation of Complex I, Complex III, and Aconitase is Associated with Mitochondrial Dysfunction in the Post‐ischemic Heart
Authors: Kang Patrick T. , Chen Chwen‐Lih , Lin Paul , Zhang Liwen , Chen Yeong‐Renn 
Published: November 2023

A serious consequence of myocardial ischemia‐reperfusion injury (IR) is oxidative damage which causes mitochondrial dysfunction. Such IR induced mitochondrial dysfunction is observed as impaired state 3 respiration, decreased ATP generation, and overproduction of superoxide. The cascading reactive oxygen species can propagate cysteine oxidation on broader array of mitochondrial enzymes and add insult to injury. Herein we employed LC/MS/MS analysis to identify the oxidative post‐translational modifications (OPTM) focusing on three key mitochondrial enzymes: the 75 kDa iron‐sulfur protein (NDUS1) and 51 kDa FMN binding protein (Ndufv1) of Complex I, the core 1 subunit (QCR1) of Complex III, and aconitase (ACON) in the mitochondria of sham control rat heart and IR mitochondria isolated from the infarct region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the IR region, we observed an increasing trend of cysteine oxidation, which was consistent with a redox analysis of mitochondria following IR (EPR measurement of oxidation of cyclic hydroxylamine). Mitochondrial preparations from IR regions had increased OPTM with S‐sulfonation (C‐SO3) levels of NDUS1 at the cystenyl residues of C367, C554, C564, Nsufv1 at C125, C187, C206, C238, C425, QCR1 at the residues of C69, C410, C453, and ACON at the residues of C448, C451. To address the functional impact of each cysteine oxidation site, we compared enzymatic activity and results of in vitro protein cysteine sulfonation. A significant impairment in the enzymatic activity of Complex I (by 25.9±1.8%) was detected in the mitochondria of post‐ischemic heart. According to our constructed homology models of Complex I NDUS1 and Ndufv1, most cysteine sites were all surface exposed and were not involved in the [Fe‐S] cluster coordinating. Thiol disulfide exchange mediated S‐glutathionylation of C367, C187, C206 decreased superoxide production by Complex I, and preserved enzymatic function. Sulfonation of C367 (C367‐SO3), C187, C206 thus potentially enhanced Complex I‐mediated superoxide generation during IR injury. As indicated by in vitro protein cysteine sulfonation, C554‐SO3 and C206‐SO3 were mediated by protein thiyl radical mechanism in vitro. C425 is one of the ligands for the 4Fe‐4S cluster (N3 center) of Complex I Ndufv1. Sulfonation of C206, C425, C554 and C564 therefore could represent the consequence of IR‐induced oxidative damage of Complex I. Like IR‐induced C425‐SO3 of Complex I, aconitase exhibited increased [4Fe‐4S] coordinating C448‐ and C451‐ sulfonation, impaired its enzymatic activity (by 71.5±2.5%) and enhanced its pro‐oxidant activity by IR. Similarly, QCR1 is the core 1 subunit of Complex III, and its enzymatic activity was reduced by 36.5±4.1% after IR, strongly suggesting the increased sulfonation of C69, C410 and C453 impairing QCR1 function of Complex III. In conclusion, suflonation of specific cysteinyl residues of Complex I, Complex III, and aconitase mediates IR‐induced mitochondrial dysfunction via impaired enzymatic activities, diminished respiration, increased superoxide production, and enhanced pro‐oxidant activity.

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NIH Grant‐HL083237