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Oxidized ferredoxin from Clostridium acidi urici, containing two [Fe4S4I2+ clusters, has been investigated through 1H NOESY and TOCSY spectroscopies. The protons of coordinated cysteines have been identified and assigned to each cluster with use of a procedure based on the assignment of two spatially close @CH2 pairs and on the shift ratios of each PCH2 proton in oxidized, half-reduced, and reduced forms; each cysteine proton has been then sequence-specifically and stereospecifically assigned by looking for dipolar connectivities with amino acid residues in the vicinity of the cluster. By comparing the present data with the available spectra of the analogous protein from Clostridium pasteurianum, the sequence-specific and stereospecific assignments of cysteine protons have been obtained also for the latter protein. The natural abundance 13C signals of the cysteine protons have been also sequencespecifically assigned. By taking advantage of the X-ray structure of a similar protein, the lH and I3C hyperfine shifts have been related to the dihedral angle between the iron-sulfur-0-carbon plane and the sulfur-fl-carbon-@-proton or sulfur-/3-carbon-a-carbon planes. A parametric equation is proposed. The spin delocalization mechanism has been found to be largely dependent on unpaired spin density on the pz orbital of the sulfur atom. Through EXSY spectroscopy, the proton signals of the [Fe&]+ clusters in the reduced protein have been assigned. Their temperature dependence is compared with that of the [Fe&I3+ clusters present in oxidized HiPIPs and discussed in terms of the Heisenberg model for the magnetic exchange coupling within the clusters.

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...