The ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated through 'H-NMR spectroscopy in the reduced and partially oxidized states. The 'H-NMR spectrum of fully reduced ferredoxin, obtained by addition of stoichiometric amounts of dithionite, has been characterized. One-and two-dimensional NMR saturation transfer experiments on partially reduced samples have allowed the isotropically shifted signals of the reduced form to be correlated to those of the oxidized form, for which the complete assignment of the P-CH2 cysteinyl residues is available. In addition, observation of the 'H-NMR signals of the intermediate species with characteristic chemical shift values for each cluster allowed us to assign all the Cys P-CH2 signals to cluster I or cluster I1 and to calculate the difference in redox potential between them. Starting from these results, reanalysis of the 'H-NMR features of the two clusters in the oxidized form showed that they are strikingly similar, supporting the idea of a high degree of internal symmetry between them, in agreement with crystallographic results on an homologous ferredoxin. On the other hand, the 'H-NMR properties of the two clusters in the reduced form deviate considerably from each other, suggesting that reduction of the clusters brings about different structural changes and loss of internal symmetry. A theoretical approach is reported to account for the isotropic shifts and the temperature dependence of the NMR signals of the reduced protein.The self-assembling of iron-sulfur clusters of variable composition, starting from solutions of iron salts in the presence of thiolate ligands, is a well known peculiarity of iron chemistry [l, 21. A distinctive property of these clusters is that they show more than one oxidation state. Evolution selected these inorganic moieties as the tunable electron sink incorporated in proteins to build up specific electron transfer chains in biological systems [3 -61.

Bovine erythrocyte superoxide dismutase (SOD) in which the native zinc(I1) is substituted by cobalt(I1) has been investigated through 'H NMR spectroscopy. Owing to the magnetic-exchange coupling between cobalt(I1) and copper(II), proton signals of the histidine residues coordinated to both cobalt(I1) and copper(I1) have been observed. The signals are relatively narrow, Le., of the same quality as those of the copper-deprived cobalt(I1) SOD. Assignment of histidine NH signals is obtained through deuterium exchange, and a tentative assignment of the other signals is proposed on the basis of T I and T2 measurements. Anions like N3-, NCO-, and NCS-cause large variations in the position of the histidine proton signals. The same kind of variations, although to a smaller extent, occur at pH 9, probably due to the binding of the OH-anion. The spectra have been interpreted in terms of one histidine (possibly His 44) being removed from coordination by the anion ligand. The electronic and CD spectra of Cu2C02SOD and its derivatives as compared to those of native SOD derivatives indicate that the two systems behave in a quite similar fashion.Bovine erythrocyte superoxide dismutase (SOD hereafter) is an enzyme containing zinc(I1) and copper(I1) ions, the latter in the site at which the catalytic reaction occurs.' the US. Army, through its European Research Office (Contract

Zinc is an essential element-one that is necessary for the occurrence of reactions that are required in the metabolic processes of living organisms. It is the second most abundant transition or post-transition metal. It is transported by proteins (macroglobulin, transferrin, and albumin), stored in a protein (thionein), and bound to proteins. It is generally bound to histidines, carboxylate-containing residues, and cysteines. In a crude way, zinc can be classified according to its degree of direct involvement in the catalytic mechanism. If so, it may coordinate a substrate molecule and activate it for the required reaction. Independently of this classification zinc acts as a Lewis acid; i.e., it accepts lone pairs by donor groups; its Lewis acid properties are primarily important when it acts as catalyst.

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Oxidized ferredoxin from Clostridium pasteurianum, containing two Fe& clusters, has been investigated using 2D 'H NMR spectroscopy at 600MHz. 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to thefl-CH, protons of the eight metal coordinated cysteines. Geminal connectivities of Cys jl-CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments. A few additional signals could be assigned to the corresponding a-CH protons. The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in pammagnetic metalloproteins is stressed.Ferredoxin; Iron-sulfur cluster; 2D NMR; Metalloprotein INTRODIJCTIONThe 2(Fe,S,) ferredoxin from Clostridium pasteurianum is a low molecular weight iron sulfur protein [l-3]; in the oxidized state each cluster formally contains two iron (III) and two iron (II) ions [4]. Owing to antiferromagnetic exchange coupling the ground state is S=O [4,5]. At room temperature some paramagnetism arises from occupancy of the excited states; the overall magnetic moment at 298 K is =4.1 BM per cluster 161. magnetic compounds have been reported, in particular for systems containing low spin iron (III), for which the nuclear relaxation rates are only slightly increased with respect to diamagnetic systems [13-171.2D 'H NMR experiments have been developed and extensively utilized for the determination of the solution structure of diamagnetic compounds [7,8]. In these systems the 2D pulse sequences take place in a time negligible with respect to proton relaxation times; magnetization transfer occurs in a time shorter or of the same order of magnitude. On the other hand, paramagnetic systems are characterized by drastic enhancements of nuclear relaxation rates [9,10]; so it often happens that the spin system reaches equilibrium without transferring detectable amounts of magnetization from one spin set to another. This fact has heavily hindered the application of 2D NMR techniqtit; ;o paramagnetic systems [I 1,121. In this frame, we here report a 2D NOESY and COSY study of the oxidized 2(Fe&) ferredoxin from Ciostridium pasteurianum. We demonstrate that, by an accurate choice of the experimental parameters, 2D NMR experiments can be fruitfully applied to the investigation of paramagnetic iron-sulfur clusters and can provide valuable information for firm assignments of the isotropically shifted lines. MATERIALS AND METHODSCfosrridium pasfeuriurwm was grown and ferredoxin isolated and purified according to the method of Rabinowitz [l&19]. The purity of the sample was checked by absorption spectroscopy by monitoring the AJA,, absorbance ratio. For 'H NMR experiments the protein was dissolved in 50 mM NaPi buffer, pH 8.0. Deuteration of the sample was achieved by utilizing an ultrafiltration Amicon cell, equipped with a YMl membrane. At least five changes of deuterated buffer were performed to ensure satisfactory solvent exchange. The pH values are reported as uncorrected pH meter ...

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