Characterization of pathways for the Fe-S protein biogenesis in the human cytoplasm

Human cytosolic monothiolglutaredoxin-3 (GLRX3) is a protein essential for the maturation of cytosolic [4Fe−4S] proteins. We show here that dimeric cluster-bridged GLRX3 transfers its [2Fe−2S]2+ clusters to the human P-loop NTPase NUBP1, an essential early component of the cytosolic iron−sulfur assembly (CIA) machinery. Specifically, we observed that [2Fe−2S]2+ clusters are transferred from GLRX3 to monomeric apo NUBP1 and reductively coupled to form [4Fe−4S]2+ clusters on both N-terminal CX13CX2CX5C and C-terminal CPXC motifs of NUBP1 in the presence of glutathione that acts as a reductant. In this process, cluster binding to the C-terminal motif of NUBP1 promotes protein dimerization, while cluster binding to the N-terminal motif does not affect the quaternary structure of NUBP1. The cluster transfer/assembly process is not complete on both N- and C-terminal motifs and indeed requires a reductant stronger than GSH to increase its efficiency. We also showed that the [4Fe−4S]2+ cluster formed at the N-terminal motif of NUBP1 is tightly bound, while the [4Fe−4S]2+ cluster bound at the C-terminal motif is labile. Our findings provide the first evidence for GLRX3 acting as a [2Fe−2S] cluster chaperone in the early stage of the CIA machinery. Iron-sulfur (Fe-S) clusters are among the most versatile cofactors in biology. Although Fe-S clusters formation can be achieved spontaneously in vitro with inorganic iron and sulfur sources, the in vivo behaviour is more complex and requires the so-called Fe-S biogenesis machineries. In the cytosol, the biogenesis of Fe-S proteins is assisted by the cytosolic Fe-S protein assembly machinery, which comprises at least thirteen known proteins, among which there are human ORAOV1 and YAE1. A hetero-complex formed by the two latter proteins facilitates Fe-S cluster insertion in the human ABC protein ABCE1 within a chain of binding events that are still not well understood. In the present work, ORAOV1 and the YAE1-ORAOV1 complex were produced and their structural and cluster binding properties spectroscopically investigated. It resulted that both ORAOV1 and the YAE1-ORAOV1 complex are characterized by well-structured alpha-helical regions and by unstructured, flexible regions, and are both able to bind a [4Fe-4S]2+ cluster. Bioinformatics and site-directed mutagenesis studies indicated that ORAOV1, and not YAE1, is the protein involved in [4Fe-4S]2+ cluster binding in the hetero-complex. ORAOV1 has indeed a conserved cluster-binding motif able to coordinate a [4Fe-4S] cluster. Overall, our data suggested that the YAE1-ORAOV1 complex might actively participate in the Fe-S cluster insertion into ABCE1 thanks to the [4Fe-4S]2+ cluster binding properties of ORAOV1.