The protein mitoNEET was initially identified as a drug target for type-2 diabetes. However, the lack of a clear cellular function has stymied efforts towards this end. MitoNEET is a homodimer and ligates one [2Fe-2S] cluster per monomer. The metal cluster of mitoNEET is ligated by three cysteine residues and one histidine residue, a ligation pattern that is unique to the CISD protein family. We have shown that the ligating cysteine residues are reactive towards the formation of a disulfide bond between two cysteine residues and connecting two polypeptide chains; either a covalent dimer or a heterodimer with the enzyme glutamate dehydrogenase. Other reports showed that cysteine can reduce the [2Fe-2S] cluster protein under anoxic condition. In contrast, when cysteine was added to mitoNEET under normoxic conditions and observed by an oxygen electrode, the solution consumed oxygen at an enzymatic rate. The products of this reaction were unknown. UV-visible spectroscopy and Ellman’s reagent were used in order to quantify the amount of free thiols present with cysteine alone, mitoNEET alone, and cysteine with mitoNEET. We demonstrate that approximately 25% of the free thiols is consumed in the presence of Ellman’s reagent, mitoNEET, and cysteine when cysteine is in a large molar excess. The biological thiols homocysteine, glutathione, and cysteamine were also considered. This information, along with supporting research, supports that the unique structure of mitoNEET can lead to concurrent consumption of both molecular oxygen and the thiol group of cysteine. Taken together, these results indicate that the metal cluster may be serving to balance the free thiol/disulfide ratio in cellular homeostasis.

Reactivity of the Metalloprotein MitoNEET towards Biological Thiols

ACS Fall 2021

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