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 connecting two polypeptide chains; either a covalent dimer or a heterodimer with glutamate dehydrogenase. We have also assessed the reactivity of non-ligating residues, both in proximity to the cluster and over the global structure. According to crystal structures, Lys55 is in a hydrogen bond network with the ligating histidine residue of the opposite protomer. We have found that Lys55 has the propensity to form imines with the reactive electrophile 4-oxononenal and the cofactor pyridoxal phosphate (PLP) as characterized with UV-visible spectroscopy, gel electrophoresis, and proteomic analysis. Similar molecules, 4-hydroxynonenal and pyridoxal respectively, did not form an imine at Lys55. PLP is a cofactor for enzymes that metabolize amino acids, so the mitoNEET-PLP complex so the amino acids alanine, serine, phosphoserine and cysteine were screened against the mitoNEET-PLP complex. Only cysteine addition caused the formation of the canonical external aldimine with a Km = 2.4 <u>+</u> 0.3 mM. Taken together, these results indicate that the metal cluster may be acting to activate surrounding residues as part of the structure/function relationship of mitoNEET.

Revealing reactive residues in the metalloprotein mitoNEET

ACS Spring Meeting 2021

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