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We show that the main properties of the EPR spectrum given by the 3Fe clusters of iron-sulfur proteins are well described if the fine structure terms are considered in the calculation of the ḡ-tensor. This model accounts for the particular asymmetry of the spectrum at low temperature and for the g values well below 2 which are observed in the high-field region. A good agreement with the experimental data is only obtained if the three exchange parameters are nearly equal, with values of about −20 cm −1 . This supports our earlier estimations which were derived by an independent method. The implications of these results for the models which have been proposed to describe the 3Fe cluster structure are discussed.

Some metalloproteins which contain [2f-2S] clusters give EPR spectra characterized by g av ≈ 1.91, which is significantly lower than the value g av ≈ 1.96 usually obtained for the ferredoxins. A similar lowering of g av is observed for some [2F-2S] + thiolate synthetic compounds in glass-forming solvents, or in compounds where phenolates replace thiolates as terminal ligands. We show that a good correlation is observed for all the proteins and synthetic compounds characterized by g av ≈ 1.91, when the main components, g 1 , g 2 , g 3 , of the g tensor are plotted as a function of ( g 2 − g 3 ). This correlation supports the existence of a new class of [2F-2S] + clusters. A ligand-field analysis shows that the difference between the two kinds of EPR spectrum reflect essentially two different structures for the ferrous site. This conclucsion is in agreement with the spectroscopic studies performed on the Rieske protein from Thermus thermophilus .

The states of the different spin multiplets which result from the antiferromagnetic interaction in the Fe iii–Fe ii pairs are mixed by the fine structure Hamiltonians relative to each iron atom. The contribution of this effect to the g values of the lowest Kramers doublet is discussed for 2Fe clusters present in different groups of metalloproteins. Special attention is given to iron–sulfur proteins and hemerythrins where models have been developed to discuss the structural implications of the magnetic properties.

The amount of 3Fe clusters in Thermodesulfobacterium commune ferredoxin is strongly dependent upon the presence of oxygen during the purification. An average of one 3Fe cluster per monomer can be found when the purification is not strictly anaerobic. These clusters are converted into |4Fe-4S| clusters by adding dithionite at usual pH and without adjunction of Fe 2+ . The EPR potentiometric titration reveals the existence of several types of 3Fe clusters with negative midpoint potentials differing by more than 100 mV. When the |4Fe-4S| clusters are partially reduced the EPR signal is composed of two different rhombic components. The component with g z = 2.04 could be related to a site implicated in the interconversion processes. In the fully reduced state, the spectrum presents the typical features of two interacting |4Fe-4S| clusters as those observed in two |4Fe-4S| bacterial ferredoxins. From the redox titration curves the midpoint potentials of these clusters are estimated at −395 and −435 mV.

The g tensor of a triad of high spin FeIII ions coupled by antiferromagnetic exchange interactions, is calculated in the case where the fine structure terms are not negligible compared to the exchange parameters. Using some simplifying assumptions concerning the relative orientations of the magnetic axes, we obtain the expressions of the g components in closed form. A simple ‘‘g strain’’ model based on these expressions is used to simulate the EPR spectra given by the 3Fe clusters of four different iron sulfur proteins. In each case, a good simulation is only observed if the three exchange parameters are nearly equal, and of the order of −20 cm−1. These conclusions are in good agreement with our earlier estimations which were obtained by an independent method [J. P. Gayda, P. Bertrand, F‐X. Theodule, and J. J. G. Moura, J. Chem. Phys. 77, 3387 (1982)].

Resume Nous avons mesurele temps de relaxationelectronique spin-reseau T 1 dans la gamme de temperature 4 K – 10 K, par saturation continue du signal R.P.E. des ferredoxinesa3 fer de Desulfovibrio gigas et Azotobacter vinelandii . La comparison des resultats avec ceux obtenus precedemment sur d'autres types de proteines fer-soufre, met enevidence les proprietes de relaxation particulierement rapide du signal R.P.E. dans les ferrodoxinesatrois fer. Ces resultats sont en accord avec les modeles de centre actif qui prevoient l'existence de niveaux excites tres proches.

Desulfovibrio desulfuricans Norway tetraheme cytochrome c3 and (4Fe-4S) cluster containing ferredoxin I are physiological electron-transfer partners. To obtain structural informations on the complex formation, a covalent cytochrome c3-ferredoxin I complex was used. EPR investigations suggest that the covalent cytochrome c3-ferredoxin I complex is a stable analogue of the native one and bring experimental support to an intramolecular electron exchange in cytochrome c3. The reduction of cross-linked ferredoxin I under a hydrogen atmosphere in the presence of hydrogenase necessitates an intramolecular electron exchange between the hemes of cytochrome c3. A spectrophotometric redox titration indicates that the redox potentials of cytochrome c3 are weakly affected by the complex formation.

The two semi-met (FeIII, FeII) forms of hemerythrin prepared by either oxidation of the deoxy form or reduction of the met form, exhibit rather different EPR spectra. It is shown that a very weak difference in the rhombic distortion of the ferrous site is sufficient to account for this large shift of the g values. It is proposed that the important departure from g = 2.00 and the large anisotropy of the g tensor reflect directly the octahedral coordination of the ferrous ion. Such a coordination could then be present in other proteins which contain binuclear clusters characterized by similar EPR spectra.

In the photosystem I of thylakoid membranes, the photoinduced electron transfer involves three iron-sulfur centers, A, B, and X. Among them, center X is characterized by very unusual spectroscopic and redox properties. Recent arguments have been presented in favor of a [2Fe-2S] structure for the clusters implicated in this center, but the number of these clusters is still a controversial question. By using an original EPR method, based on the differences in the relaxation properties of A, B, and X, we have determined the stoichiometry for the iron-sulfur clusters in photosystem I. Our measurements indicate that center X is composed of a single iron-sulfur cluster per P700. The possible implications of this result for the polypeptide composition of the core reaction center are discussed.

Esta investigación se publicó originalmente en el Journal of Biological Chemistry. Abou-Hamdan, A.; Ceccaldi, P.; Lebrette, Hugo; Gutiérrez-Sanz, Óscar ; Richaud, P.; Cournac, L.; Guigliarelli, Bruno; López de Lacey, Antonio ; Léger, Christophe; Volbeda, A.; Burlat, Bénédicte; Dementin, Sébastien. A threonine stabilizes the NiC and NiR catalytic intermediates of [NiFe]-hydrogenase. J. Biol. Chem 2015; 290(13): 8550-8558. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc., The heterodimeric [NiFe] hydrogenase from Desulfovibrio fructosovorans catalyzes the reversible oxidation of H2 into protons and electrons. The catalytic intermediates have been attributed to forms of the active site (NiSI, NiR, and NiC) detected using spectroscopic methods under potentiometric but non-catalytic conditions. Here, we produced variants by replacing the conserved Thr-18 residue in the small subunit with Ser, Val, Gln, Gly, or Asp, and we analyzed the effects of these mutations on the kinetic (H2 oxidation, H2 production, and H/D exchange), spectroscopic (IR, EPR), and structural properties of the enzyme. The mutations disrupt the H-bond network in the crystals and have a strong effect on H2 oxidation and H2 production turnover rates. However, the absence of correlation between activity and rate of H/D exchange in the series of variants suggests that the alcoholic group of Thr-18 is not necessarily a proton relay. Instead, the correlation between H2 oxidation and production activity and the detection of the NiC species in reduced samples confirms that NiC is a catalytic intermediate and suggests that Thr-18 is important to stabilize the local protein structure of the active site ensuring fast NiSI-NiC-NiR interconversions during H2 oxidation/production., This work was supported by the CNRS, the Agence Nationale de la Recherche (HYLIOX and HEROS projects: ANR-07-BIOE-010 and ANR-14-CE05- 0010), the Aix-Marseille Université, and the City of Marseilles. This work was also supported by grants from the CNRS and the Région Provence-Alpes Côte d’Azur (to A. A.-H.), the Spanish MINECO CTQ2012-32448 project (A. L. D. L.), the Spanish FPI program (to O. G.-S.), the Commissariat à l’Energie Atomique et aux Energies Alternatives (CEA), and the CNRS (to H. L. and A. V.)

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