SITE VALENCIES AND SPIN COUPLING IN THE 3FE AND 4FE (S = 1 2) CLUSTERS OF PYROCOCCUS-FURIOSUS FERREDOXIN BY FE-57 ENDOR/

The hyperthermophilic archaeon Pyrococcus furiosus contains a novel fe rredoxin (Pf-Fd) in which, in the native 4Fe form, three of the Fe ion s are coordinated to the protein by cysteinyl thiolato ligands, but th e 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 [Fe3 S4](+) 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 nativ e and cyanide-bound forms all have been investigated by Fe-57 ENDOR sp ectroscopy 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 cre ated by earlier studies, particularly those of Noodleman and co-worker s (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 cha racterize the spin-coupling within both the native and cyanide-bound S = 1/2 forms of Pf-Fd 4Fe-red. This procedure shows that cyanide bindi ng 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 (Cal zolai 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](+) clus ters, whereas the native form may exhibit a previously unidentified sp in-coupling ground state.