The Properties And Transfer [2Fe-2S] Cluster Analysis Of MiNT

Iron-sulfur clusters are involved in a variety of crucial physiological processes as protein cofactors,such as aerobic respiration,DNA replication and repair,and cofactor biosynthesis.Cells assemble iron and sulfur into iron and sulfur clusters and transfer them to receptor proteins for biological function.NEET protein is an iron-sulfur cluster protein with the CDGSH motif as its amino acid sequence.There are three NEET proteins in human cells:mNT（mitoNEET）and NAF-1（Nutrient-deprivation autophagy factor-1）,which contain a single CDGSH motif,and MiNT,which contains two CDGSH motifs.In this paper,the basic properties of MiNT protein and the redox properties of MiNT protein[2Fe-2S]cluster were studied.Finally,the transfer of MiNT protein[2Fe-2S]cluster to receptor protein was preliminatively explored.Firstly,the effect of MiNT protein on pioglitazone hydrochloride（PIO）was studied by fluorescence spectrometry.The binding ratio was 1:1 and the binding constant was K_a=1.31×10⁵ M^-1.The Trp and PIO distance r of the MiNT-H75C protein was r=2.03 nm.Pioglitazone hydrochloride（PIO）occupied the hydrophobic cavity of MiNT protein,and the degree of protein hydrophobic structure exposure decreased significantly.Pioglitazone hydrochloride（PIO）has a stabilizing effect on MiNT protein[2Fe-2S]cluster.Secondly,different reducing agents were used to reduce the[2Fe-2S]in MiNT protein with sodium disulfite（Dith）and dithiothreitol（DTT）.It was found that Dith can completely reduce the iron in iron-sulfur clusters,while DTT can partially reduce it.The process is reversible.Pioglitazone hydrochloride may regulate the function of MiNT protein by blocking the reduction of[2Fe-2S]cluster in mercapto-mediated proteins.Finally,MiNT-C60M-C62M was prepared by site-specific mutation.Expression and purification by iron-sulfur clusters FDX1 protein receptor proteins,iron-sulfur clusters are obtained by trichloroacetic acid acidification of the protein:apo-FDX1protein.The transfer of iron-sulfur clusters from MiNT and MiNT-C60M-C62M to apo-FDX1 has been studied using UV-visible absorption spectroscopy.The transfer rates of MiNT and MiNT-C60M-C62M to the receptor proteins are different.Under anaerobic conditions,in the presence of DTT,apo-MiNT can assemble iron-sulfur clusters with Fe²⁺and S^2-in vitro and bind them to the cluster binding domain of the protein.The characteristic absorption peak of the reconstructed MiNT protein iron-sulfur cluster is 420 nm,indicating that the iron in the cluster is reduced.