MitoNEET is a protein that contains a [2Fe-2S] cluster that is ligated by a 3Cys-1His residue motif. Prior results indicated heightened reactivity of residues near the cluster, even across the dimer interface. The ligating cysteine residues (Cys72, 74, and 83) can form disulfide bonds with other polypeptide chains in a sequence specific manner. This work investigated the reactivity of mitoNEET towards, acyl-CoAs (palmitoyl-CoA, myristoyl-CoA, and acetyl-CoA), a group of biologically-relevant molecules containing sulfur atoms. For all the substrates investigated, Ellman’s reagent (DTNB) was used in a spectrophotometric assay to quantify the free thiols produced from the candidate acyl-CoA in the absence or presence of mitoNEET. MitoNEET did catalyze the liberation of free thiols from palmitoyl-CoA, myristoyl-CoA and acetyl-CoA. We hypothesize that the products of these reactions would be CoASH (reactive towards DTNB) and the free fatty acids. Additionally, kinetic analyses were quantified over 12 hrs. While these reactions are slow, they are the first example of mitoNEET having enzymatic activity as an acyl-CoA thioesterase enzyme. The reactivity of mitoNEET was also investigated with palmitoyl-CoA and GAPDH. This assay interrogated the hypothesis that mitoNEET could act as a protein acyl transferase. Proteomics at the Indiana University School of Medicine found that GAPDH did cause an increase in autopalmitoylation on specific cysteine and lysine residues of and by mitoNEET.

Characterization of the Interactions between MitoNEET and Acyl-CoAs

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