H-1 NMR is used to characterize the solution electronic structure and magnetic properties of the cubane iron-sulfur clusters for wild-type (WT) and the D14C and D14S cluster ligand mutants of the ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus with the goal of identifying the oxidation states of the individual iron ligated by a particular protein ligand. Sequence-specific assignments of the contact-shifted and paramagnetically relaxed protons for all cluster ligands are obtained in the alternate cluster oxidation states, [4Fe:4S](+), [4Fe:4S](2+), each with Cys21 and Cys48 either as free sulfhydryls or in a disulfide bond. Detailed analysis of the oxidized cluster Fds shows that the ground state is S = 0 with similar population of excited S = 1 and 2 states for each of Asp14, Ser14, or Cys14 ligated to the cluster. The reduced cluster D14C Fd exhibits pairs of Cys (Cys11,Cys17) with strong Curie, and pairs of Cys (Cys14, Cys56) with strong anti-Curie temperature dependence diagnostic of ligands to the S = 9/2, 2Fe(2.5+), and S = 4, 2Fe(2.0+) iron pairs, respectively. Moreover, it is observed that the ligand protons of Cys ligated to the mixed-valence pair are much more effectively relaxed than the Cys ligated to the diferrous pair, which is consistent with expectations for the relative spin magnetizations of the iron pair. Substitution of Cys14 by Ser14 leads to changes in contact shift magnitudes and slopes in Curie plots that demonstrate the presence of two electronic states that interchange the sequence positions of the 2Fe(2.5+) and 2Fe(2.0+). Similarly, replacement of Cys14 by Asp14 leads to changes in shift magnitude and Curie slope that indicate the population of two states for which the Asp14 is ligated to a Fe2.5+ and Cys11 is ligated to a Fe2.0+ in each state, while the other two ligands showed effective oxidation states midway between Fe2.5+ and Fe2.0+. Thus the relative stabilization of the mixed-valence pair for the iron ligated by residue 14 is Asp > Ser > Cys. The localization of the mixed-valence pair to the irons ligated to Cys11 and 17 in both D14C- and D14S-Fd is significantly stabilized for the disulfide relative to the free sulfydryl form of Cys21 and Cys48, and likely reflects on a hydrogen bond between the backbones of Cys17 and Cys21. Ligation by cyanide in reduced WT Fd yields a contact shift pattern indicative of the same electronic state as for the Cys14 mutant, with cyanide ligated to one of the 2Fe(2.0+). The conversion of a Fe2.5+ ligated by Asp in WT Fd to a Fe2.0+ upon cyanide displacement of the Asp is consistent with EPR and ENDOR studies (Telser, et al. J. Am. Chem. Sec. 1995, 117, 5133-5140; Telser et al., submitted for publication).