Monothiol glutaredoxins play a crucial role in iron-sulfur (Fe/S) protein biogenesis. Essentially all of them can coordinate a [2Fe-2S] cluster and have been proposed to mediate the transfer of clusters from scaffold proteins to target apo proteins, possibly by acting as cluster transfer proteins. The molecular basis of cluster transfer from monothiol glutaredoxins to target proteins is a fundamental, but still unresolved, aspect to be defined in Fe/S protein biogenesis. In mitochondria monothiol glutaredoxin 5 (GRX5) is involved in the maturation of all cellular Fe/S proteins and participates in cellular iron regulation. Here we show that the structural plasticity of the dimeric state of the [2Fe-2S] bound form of human GRX5 (holo hGRX5) is the crucial factor that allows an efficient cluster transfer to the partner proteins human ISCA1 and ISCA2 by a specific protein-protein recognition mechanism. Holo hGRX5 works as a metallochaperone preventing the [2Fe-2S] cluster to be released in solution in the presence of physiological concentrations of glutathione and forming a transient, cluster-mediated protein-protein intermediate with two physiological protein partners receiving the [2Fe-2S] cluster. The cluster transfer mechanism defined here may extend to other mitochondrial [2Fe-2S] target proteins.Fe/S protein maturation | [2Fe-2S] cluster transfer mechanism | monothiol Grxs | NMR G lutaredoxins (Grxs) and glutathione (GSH) are universally distributed among all organisms, and they have been shown to play a fundamental role in iron-sulfur (Fe/S) protein biogenesis (1-5). Specifically, the [2Fe-2S]-bound forms of monothiol Grxs and a [2Fe-2S]-glutathione complex are the species suggested to be responsible for trafficking [2Fe-2S] clusters within the cell (6-9). The current working model is that in the cell monothiol Grxs receive a [2Fe-2S] cluster from the scaffold protein ISCU (where de novo synthesis of the [2Fe-2S] cluster occurs) and transfer it to specific targeting proteins, which then facilitate Fe/S cluster insertion into the final acceptor apo protein (7, 10, 11). Another possible cluster transfer mechanism, which has been proposed (8), hypothesizes the cellular presence of a [2Fe-2S](GS) 4 complex, which could transiently store [2Fe-2S] clusters, facilitate cluster exchange with the cellular Fe/S cluster biosynthesis machineries, and regulate the biosynthesis of Fe/S clusters. However, a drawback of the latter model is that all of the Fe/S cellular trafficking processes will result to be protein-independent and therefore highly unspecific, thus potentially inflicting severe cellular damage.The mitochondrial, monothiol glutaredoxin 5 protein (GRX5) belongs to the core part of the mitochondrial Fe/S cluster (ISC) assembly system (10, 12, 13), is required in the maturation of all cellular [2Fe-2S] and [4Fe-4S] proteins (11), and participates in cellular iron regulation (14). Human GRX5 in vitro binds a [2Fe-2S] cluster (15) and yeast GRX5, which in vivo and in vitro binds a [2Fe-2S] cluster (11), has been...

The generation of [4Fe-4S] clusters in mitochondria critically depends, in both yeast and human cells, on two A-type ISC proteins (in mammals named ISCA1 and ISCA2), which perform a nonredundant functional role forming in vivo a heterocomplex. The molecular function of ISCA1 and ISCA2 proteins, i.e., how these proteins help in generating [4Fe-4S] clusters, is still unknown. In this work we have structurally characterized the Fe/S cluster binding properties of human ISCA2 and investigated in vitro whether and how a [4Fe-4S] cluster is assembled when human ISCA1 and ISCA2 interact with the physiological [2Fe-2S](2+) cluster-donor human GRX5. We found that (i) ISCA2 binds either [2Fe-2S] or [4Fe-4S] cluster in a dimeric state, and (ii) two molecules of [2Fe-2S](2+) GRX5 donate their cluster to a heterodimeric ISCA1/ISCA2 complex. This complex acts as an "assembler" of [4Fe-4S] clusters; i.e., the two GRX5-donated [2Fe-2S](2+) clusters generate a [4Fe-4S](2+) cluster. The formation of the same [4Fe-4S](2+) cluster-bound heterodimeric species is also observed by having first one [2Fe-2S](2+) cluster transferred from GRX5 to each individual ISCA1 and ISCA2 proteins to form [2Fe-2S](2+) ISCA2 and [2Fe-2S](2+) ISCA1, and then mixing them together. These findings imply that such heterodimeric complex is the functional unit in mitochondria receiving [2Fe-2S] clusters from hGRX5 and assembling [4Fe-4S] clusters before their transfer to the final target apo proteins.

Human anamorsin was implicated in cytosolic iron-sulfur (Fe/S) protein biogenesis. Here, the structural and metal-binding properties of anamorsin and its interaction with Mia40, a well-known oxidoreductase involved in protein trapping in the mitochondrial intermembrane space (IMS), were characterized. We show that (1), anamorsin contains two structurally independent domains connected by an unfolded linker; (2), the C-terminal domain binds a [2Fe-2S] cluster through a previously unknown cysteine binding motif in Fe/S proteins; (3), Mia40 specifically introduces two disulfide bonds in a twin CX(2)C motif of the C-terminal domain; (4), anamorsin and Mia40 interact through an intermolecular disulfide-bonded intermediate; and (5), anamorsin is imported into mitochondria. Hence, anamorsin is the first identified Fe/S protein imported into the IMS, raising the possibility that it plays a role in cytosolic Fe/S cluster biogenesis also once trapped in the IMS.