MitoNEET is a homodimeric protein located on the outer mitochondrial membrane where each monomer contains a [2Fe-2S] cluster within a CDGSH Iron-Sulfur Domain (CISD). CISD family members mitoNEET and NAF-1 bind pyridoxal 5’-phosphate (PLP), a biologically relevant lysine modifier. PLP was found to selectively modify Lys55 of mitoNEET over the other 10 available lysine residues. Lys55 is in a hydrogen-bonding network with His87, one of the residues that ligates the metal cluster. The formation of PLP-CISD complex was observed and characterized by UV-visible spectroscopy for two CISD family members, mitoNEET and NAF-1. The kobs of PLP-CISD formation for mitoNEET and NAF-1 were measured to be 0.019 + 0.002/s and 0.08 + 0.02/s respectively. This complex was successfully reacted with L-cysteine (L-Cys). The reaction rates were measured for a kinetics curve of the mitoNEET-PLP complex with L-Cys and found to fit a Michaelis-Menten model with a KM = 2.78 mM. The conversion of L-Cys by mitoNEET represents the first evidence that mitoNEET is a PLP-dependent enzyme.

Characterization of the PLP-dependent functions of CISD proteins

2019 Spring National Meeting

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