Investigation by EPR and ENDOR spectroscopy of the novel 4Fe ferredoxin from Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus contains a four-Fe ferredoxin (Pf-Fd) that differs from most other 4Fe-Fd's in that its [Fe4S4] cluster is anchored to protein by only three cysteinyl residues. Pf-Fd also is of interest because in its reduced form, [Fe4S4](+), the cluster exhibits both S = 1/2 and S = 3/2 spin states. Addition of excess cyanide ion converts the duster exclusively to an S = 1/2 state (g(1) = 2.09, g(2) = 1.95, g(3) = 1.92), however dialysis restores the EPR signal of native reduced protein indicating that the cluster is not irreversibly altered by cyanide. Both the native protein and protein in the presence of excess cyanide ion (Pf-Fd 4Fe-CN) were investigated here using the techniques of electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy. In particular, Pf-Fd 4Fe-CN was investigated using (CN-)-C-13 and (CN-)-N-15 ligands. C-13 and N-15 ENDOR indicated that a single cyanide ion bound directly with the cluster showing an unusually small contact interaction (a(iso)(C-13) similar to - 3 MHz, a(iso)(N-15) similar to 0). This is in contrast to cyanide bound to monomeric low-spin Fe(III)-containing proteins such as transferrin and myoglobin, for which the C-13 hyperfine coupling has a large isotropic component (a(iso)(C-13) approximate to - 30 MHz). This small contact interaction is not due to low spin density of Fe, as Fe-57 ENDOR of the singly and triply labeled Pf-Fd 4Fe-CN isotopologs, [(FeFe3S4)-Fe-57](+) and [(FeFe3S4)-Fe-57](+), show hyperfine coupling characteristic for [Fe4S4](+) clusters, particularly for the Fe to which cyanide binds. Thus, the low spin density on C-13 is not due to low spin density on the Fe ion to which it binds. Further theoretical work is needed to explain the contrast between the strong electronic effect of cyanide ion binding with the low spin density on the ligand.