Site valencies and spin coupling in the 3Fe and 4Fe (S = 1/2) clusters of Pyrococcus furiosus ferredoxin by 57Fe ENDOR

The hyperthermophilic archaeon Pyrococcus furiosus contains a novel ferredoxin (Pf-Fd) in which, in the native 4Fe form, three of the Fe ions are coordinated to the protein by cysteinyl thiolato ligands, but the fourth, labile, Fe is coordinated by an aspartyl carboxalato ligand. Addition of excess cyanide ion to the reduced 4Fe protein, Pf-Fd 4Fe-red, yields a species with EPR properties that differ greatly from the native cluster form (Pf-Fd 4Fe-CN). The unique Fe can be removed, to form a 3Fe cluster, which in turn can be. reconstituted to regenerate the 4Fe form. The lability of this fourth Fe allows the preparation of a series of Fe-57 cluster isotopologs: Pf-Fd 3Fe-ox (S = 1/2) as [Fe3S4](+) and [(Fe3S4)-Fe-57](+); Pf-Fd 4Fe-red as [Fe4S4](+), [(FeFe3S4)-Fe-57](+), and [(FeFe3S4)-Fe-57](+) as well as the corresponding 57Fe isotopologs of PS-Fd 4Fe-CN. The 3Fe and 4Fe cluster-containing native and cyanide-bound forms all have been investigated by Fe-57 ENDOR spectroscopy at cryogenic temperatures. The ground state of Pf-Fd 3Fe-ox is similar to that seen for other 3Fe Fd's but shows evidence of less symmetrical intracluster spin-coupling. The theoretical framework created by earlier studies, particularly those of Noodleman and co-workers (Mouesca, J.-M.; Noodleman, L.; Case, D. A.; Lamotte, B. Inorg. Chem. 1995, 34, 4347-4359), allows us to use Fe-57 ENDOR data alone to characterize the spin-coupling within both the native and cyanide-bound S = 1/2 forms of Pf-Fd 4Fe-red. This procedure shows that cyanide binding to the unique Fe ion causes it to undergo a "valency switch" from Fe2.5+ to Fe2+. The interchange also is detected by NMR analysis (Calzolai et al. J. Am. Chem. Sec. 1997, 119, 9341-9350). We find that the cyanide-bound cluster has a spin state common to many [Fe4S4](+) clusters, whereas the native form may exhibit a previously unidentified spin-coupling ground state.