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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...

Copper, zinc superoxide dismutase is a dimeric enzyme, and it has been shown that no cooperativity between the two subunits of the dimer is operative. The substitution of two hydrophobic residues, Phe 50 and Gly 51, with two Glu's at the interface region has disrupted the quaternary structure of the protein, thus producing a soluble monomeric form. However, this monomeric form was found to have an activity lower than that of the native dimeric species (10%). To answer the fundamental question of the role of the quaternary structure in the catalytic process of superoxide dismutase, we have determined the solution structure of the reduced monomeric mutant through NMR spectroscopy. Another fundamental issue with respect to the enzymatic mechanism is the coordination of reduced copper, which is the active center. The three-dimensional solution structure of this 153-residue monomeric form of SOD (16 kDa) has been determined using distance and dihedral angle constraints obtained from 13C, 15N triple-resonance NMR experiments. The solution structure is represented by a family of 36 structures, with a backbone rmsd of 0.81 +/- 0.13 A over residues 3-150 and of 0.56 +/- 0.08 A over residues 3-49 and 70-150. This structure has been compared with the available X-ray structures of reduced SODs as well as with the oxidized form of human and bovine isoenzymes. The structure contains the classical eight-stranded Greek key beta-barrel. In general, the backbone and the metal sites are not affected much by the monomerization, except in the region involved in the subunit-subunit interface in the dimeric protein, where a large disorder is present. Significative changes are observed in the conformation of the electrostatic loop, which forms one side of the active site channel and which is fundamental in determining the optimal electrostatic potential for driving the superoxide anions to the copper site which is the rate-limiting step of the enymatic reaction under nonsaturating conditions. In the present monomer, its conformation is less favorable for the diffusion of the substrate to the reaction site. The structure of the copper center is well-defined; copper(I) is coordinated to three histidines, at variance with copper(II) which is bound to four histidines. The hydrogen atom which binds the histidine nitrogen detached from copper(I) is structurally identified.

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.

Paramagnetic 'H-NMR spectra of Co(II)-substituted Cysll2Asp azurin from Pseudomonas aeruginosa have been analyzed and compared with those of the Co(II) wild-type (WT) protein. Hyperfine-shifted signals (including Aspl 12 /3-CHi signals in the mutant as well as previously unobserved Cysl 12 /I-CH2 signals in WT) from all the metal-coordinated residues have been detected and unambiguously assigned. Notably, the spectra indicate that very little if any unpaired spin density is located on the Metl21 protons in the Cysl 12Asp protein. A computergenerated model of the mutant Co(II) structure consistent with electronic absorption as well as the NMR data includes a Gly45 carbonyl, His46, an unusually coordinated Aspl 12, and Hisll7 in the ligation sphere.

The relative importance of paramagnetism-based constraints (i.e. pseudocontact shifts, residual dipolar couplings and nuclear relaxation enhancements) with respect to classical constraints in solution structure determinations of paramagnetic metalloproteins has been addressed. The protein selected for the study is a calcium binding protein, calbindin D9k, in which one of the two calcium ions is substituted with cerium(III). From 1823 NOEs, 191 dihedral angles, 15 hydrogen bonds, 769 pseudocontact shifts, 64 orientational constraints, 26 longitudinal relaxation rates, plus 969 pseudocontact shifts from other lanthanides, a final family with backbone r.m.s.d. from the average of 0.25 A was obtained. Then, several families of structures were generated either by removing subsets of paramagnetism-based constraints or by removing increasing numbers of NOEs. The results show the relative importance of the various paramagnetism-based constraints and their good complementarity with the diamagnetic ones. Although a resolved structure cannot be obtained with paramagnetism-based constraints only, it is shown that a reasonably well resolved backbone fold can be safely obtained by retaining as few as 29 randomly chosen long-range NOEs using the standard version of the program PSEUDYANA.

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