Effects of mutations in aspartate 14 on the spectroscopic properties of the [Fe3S4](+,0) clusters in Pyrococcus furiosus ferredoxin

The properties of [Fe3S4](+.0) clusters in wild-type and mutant forms of Pf Fd with Asp, Ser, Cys, Val, His, Asn, and Tyr residues occupying position 14, i.e., proximal to the three mu(2)-S atoms of the cluster, have been inv estigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD), and resonance Raman (RR) spectroscopies. Two distinct t ypes of [Fe3S4] clusters are identified on the basis of the breadth of the S = 1/2 [Fe3S4](+) EPR resonances and the marked differences in the VTMCD s pectra of the S = 2 [Fe3S4](0) clusters. On the basis of the available NMR data for [Fe3S4](+.0) clusters in ferredoxins, the distinctive properties o f these two types of [Fe3S4] clusters are interpreted in terms of different locations of the more strongly coupled pair of irons in the oxidized clust ers and the valence-delocalized pair in the reduced clusters. Near-IR VTMCD measurements indicate the presence of S = 9/2 valence-delocalized pairs in both types of [Fe3S4](0) clusters, and the spin-dependent delocalization e nergies associated with the Fe-Fe interactions were determined to be simila r to 4300 cm(-1) in both cases. We conclude that the nature of the residue at position 14 in Pyrococcus furiosus ferredoxin is an important determinan t of the location of the reducible pair of irons in a [Fe3S4](+.0) cluster, and the redox properties of the wild-type and mutant ferredoxins are discu ssed in light of these new results.