Exploring mitoNEET as a pyridoxal phosphate-dependent enzyme
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 CISD●PLP complex was observed and characterized by UV-visible spectroscopy for two CISD family members, mitoNEET and NAF-1. The kobs of CISD●PLP 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- /D-cysteine (L-Cys/D-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 = 2.8 ± 0.8 mM and a vmax= 4.3 ± 0.3 E-4 Abs/sec. MitoNEET●PLP was also found to react with the sulfur-containing oxidized glutathione (GSSG) as well as reduced glutathione (GSH). The conversion of these amino acids by mitoNEET●PLP represents the first evidence that mitoNEET is a PLP-dependent enzyme.