Enzymatic Oxidation of Biological Thiols by MitoNEET

MitoNEET is a mitochondrial [2Fe‐2S] protein known for its involvement in mitochondrial bioenergetics, iron metabolism, and oxidative stress. The protein has been extensively characterized at the biochemical level since its discovery in 2004, but the mechanisms of physiological function(s) remain unresolved. Two observations seem especially relevant for understanding the physiological function of mitoNEET: (1) under anaerobic conditions in vitro thiol compounds such as glutathione (GSH) and cysteine (Cys) reduce the 2Fe‐2S cluster from the Fe +3 Fe +3 to the Fe 3+ Fe 2+ state, and (2) mitoNEET expression levels increase in cells when the glutathione redox balance is shifted towards the oxidized state. We hypothesize that mitoNEET’s in vivo function can be elucidated by bridging these two observations and postulate that mitoNEET is an enzyme responsible for the oxidation of thiol groups on either low molecular‐weight compounds or cysteine residues on target proteins involved in redox signaling. To test this hypothesis, we monitored thiol oxidation by following oxygen consumption rates and demonstrate that mitoNEET accelerates thiol oxidation of cysteine and glutathione (GSH). Four distinct experiments comparing the reactivity of Fe 3+ in the form of FeCl 3 to mitoNEET demonstrate that the observed thiol oxidation by mitoNEET was not due to free Fe 3+ contamination. These observations were: (1) rates of cysteine oxidation (8 mM) catalyzed by mitoNEET (1.5 mM) or Fe 3+ (1.5 mM) show different optima in the pH range 6.4 to 9.4 and the mitoNEET‐catalyzed reaction is 137% faster at pH 8.4 than the rate observed for Fe 3+ alone. (2) Oxidation rates of the nonphysiological compound, n‐acetylcysteine (NAC) by mitoNEET are 181% higher compared to Fe 3+ at a pH of 9.0. (3) MitoNEET‐mediated glutathione oxidation remained unaffected by addition of cysteine while oxidation catalyzed by free Fe 3+ decreased after addition of cysteine. (4) Dimedone, a probe used to inhibit thiol oxidation from sulfenic acid to sulfinic and sulfonic acid, was 25% more effective at inhibiting cysteine oxidation when free Fe 3+ was the catalyst compared to mitoNEET. These results support our hypothesis that mitoNEET possesses an active site which functions to oxidize biological thiols. While the in vivo substrate is currently unknown, we have narrowed down potential candidates to small thiol containing compounds and/or cysteine residues on specific target proteins. We are currently investigating how the enzymatic activity may be regulated by endogenous compounds and through pharmacological compounds targeting mitoNEET. Support or Funding Information This work was supported by NSF CHE‐1806266 to M.A.M and M.E.K and UofL Mentored Undergraduate Research and Creative Activities Grant to R.A.S.