Proton NMR investigation of the oxidized three-iron clusters in the ferredoxins from the hyperthermophilic archae Pyrococcus furiosus and Thermococcus litoralis.

The 3Fe forms of ferredoxins (Fd) from the hyperthermophilic archaebacteria Pyrococcus furiosus (Pf) and Thermococcus litoralis (Tl) have been investigated by 1H NMR. A combination of one-dimensional nuclear Overhauser and two-dimensional NOESY and bond correlation spectroscopy provides the assignment of the aromatic residues, one conserved valine, and the location of the signals for each of the three cysteines coordinated to the clusters. Dipolar contacts between the Trp 2 and Tyr 46 in Pf Fd and from an invariant phenylalanine to an invariant valine and a cluster cysteine in both Fd confirm a folding pattern for these proteins that is very similar to that of the crystallographically characterized ferredoxin from the mesophile Desulfovibro gigas. The sequence-specific assignment of the buried cysteine near the invariant phenylalanine has been made. The temperature dependence of the contact-shifted cysteinyl residues reveals a distinct 2:1 asymmetry in the magnetic coupling among the three high-spin ferric ions, in that one cysteine exhibits Curie behavior, while the other two cysteines display anti-Curie behavior. These magnetic properties are rationalized qualitatively on the basis of a magnetic coupling scheme where two iron couple to yield an intermediate spin of 2 which couples to the remaining S = 5/2 iron to yield the total cluster spin 1/2. This magnetic asymmetry appears to be a characteristic feature of oxidized 3 Fe clusters. Pf Fd also undergoes a dynamic equilibrium between two alternate forms that differ slightly in the environment of two of the coordinated cysteines. Analysis of the pattern of the contact shifts for the three cysteines in the two ferredoxins suggests that the cysteine coordinated to the unique iron does not have the same sequence origin.