Biochemical Control of the Mitochondrial Protein MitoNEET by Biological Thiols and Lipid-derived Electrophiles

MitoNEET is a mitochondrial [2Fe-2S] protein known for its involvement in cellular metabolism, iron regulation, and oxidative stress. The protein has been associated with diseases ranging from diabetes to Parkinson's disease which has prompted development of compounds designed to selectively target mitoNEET. Unfortunately, drug development is limited due to a lack of understanding on the mechanistic level how mitoNEET integrates into pathophysiological processes. In particular, biological compounds that govern mitoNEET function are still ill defined. We demonstrate an oxygen-dependent reaction with biological thiols catalyzed by mitoNEET. Furthermore, we observed that formation of a covalently linked mitoNEET homodimer is controlled by both thiols and lipid-derived electrophiles. Finally, we demonstrate that reduced glutathione (L-GSH) regulates the reactivity of two lipid-derived biomarkers of oxidative stress, 4-HNE and 4-ONE, towards mitoNEET. We find that exposure to L-GSH prior to treatment with either of the electrophilic aldehydes prevents the formation of the covalently linked mitoNEET dimer. Meanwhile, addition of L-GSH after electrophile treatment recovers mitoNEET from the 4-HNE induced modification but not from the modification induced by 4-ONE. Our results collectively suggest that the thiol-electrophile redox balance governing ferroptotic cell death also controls mitoNEET's state at multiple biochemical levels. These results indicate a possible role for mitoNEET in thiol-mediated oxidative stress and may inform about development of probes designed to modulate mitoNEET activity to improve pathophysiological states.