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 cluster exclusively to an S= 1/2 state (g1 = 2.09, g2 = 1.95, g3 = 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 13CN- and C15N- ligands. 13C and 15N ENDOR indicated that a single cyanide ion bound directly, with the cluster showing an unusually small contact interaction (aiso(13C) ∼ -3 MHz, aiso(15C) ∼ 0). This is in contrast to cyanide bound to monomeric low-spin Fe(III)-containing proteins such as transferrin and myoglobin, for which the 13C hyperfine coupling has a large isotropic component (aiso(13C) ≈ -30 MHz). This small contact interaction is not due to low spin density of Fe, as 57Fe ENDOR of the singly and triply labeled Pf-Fd 4Fe-CN isotopologs, [57FeFe3S4]+ and [Fe57Fe3S4]+, show hyperfine coupling characteristic for [Fe4S4]+ clusters, particularly for the Fe to which cyanide binds. Thus, the low spin density on 13C 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.
ASJC Scopus subject areas
- Atomic and Molecular Physics, and Optics