Transferring asymmetrical [2Fe2S] clusters from the mitochondrial protein MiNT to acceptor

NEET protein is an evolutionarily conserved protein in almost all kingdoms of life. As an important member of the NEET (Asn-Glu-Glu-Thr) superfamily, MiNT (Miner2) involved in regulating iron and reactive oxygen species (ROS) homeostasis. It contains two CDGSH (consensus sequence: C-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H) domains used for binding [2Fe2S] clusters. To elucidate the heterogeneities of the two clusters, residues of His75 and His113 were mutated to Cys, respectively. The inequality of the two [2Fe-2S] in maintaining protein conformation and in cluster transferring was studied, in which the different contributions from two [2Fe-2S] were especially followed. Results revealed that [2Fe2S]₂ located in the second CDGSH domain made less exposure of hydrophobic on MiNT than that of [2Fe-2S]₁ did. Based on fluorescence resonance energy transfer (FRET), we constructed a novel system used to explore transferring [2Fe2S] from MiNT to acceptor protein. The [2Fe2S]₁ and [2Fe-2S]₂ were successively transferred to mitochondrial matrix ferredoxins (Fdx). Its transferring velocities were calculated to be Ks = 0.027 S^-1 and 0.017 S^-1. Such knowledge will shed light on how sequence conservation influences specific biological functions and how MiNT mediates the utilization of Fe in cell cycle.