1H-NMR studies on partially and fully reduced 2(4Fe-4S) ferredoxin from <em>Clostridium pasteurianum</em>.

The ferredoxin from Clostridium pasteurianum, containing two Fe₄S₄ clusters, has been investigated through ¹H-NMR spectroscopy in the reduced and partially oxidized states. The ¹H-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 β-CH₂ cysteinyl residues is available. In addition, observation of the ¹H-NMR signals of the intermediate species with characteristic chemical shift values for each cluster allowed us to assign all the Cys β-CH₂ signals to cluster I or cluster II and to calculate the difference in redox potential between them. Starting from these results, reanalysis of the ¹H-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 ¹H-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. [ABSTRACT FROM AUTHOR]