Cyanide Binding to the Novel 4Fe Ferredoxin from Pyrococcus furiosus: Investigation by EPR and ENDOR Spectroscopy

The hyperthermophilic archaeon Pyrococcus furiosus contains a 4-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 bothS = ¹/₂andS = ³/₂spin states. Addition of excess cyanide ion converts the cluster exclusively to anS = ¹/₂state (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-CN) were investigated here using the techniques of electron paramagnetic resonance (EPR) and electron—nuclear double-resonance (ENDOR) spectroscopy. No evidence for a strongly coupled solvent-derived hydrogen (¹H or ²H) from an OH^-or H₂O ligand in either spin state of the [Fe₄S₄]⁺cluster was observed, contrary to an earlier report. Rather, ^1,2H ENDOR characteristic of 4Fe-Fd's was seen for both native Pf-Fd and Pf-Fd-CN. Pf-Fd-CN was further 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 (a_iso(¹³C) ∼ -3 MHz, a_iso(¹⁵N) ~ 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 (a_iso(¹³C) ≈ — 30 MHz). The full ¹³C and ¹⁵N hyperfine tensors were determined by computer simulation of the ENDOR spectra. The A(¹³C) is rotated by ~40° about g₂. The g and A(¹³C) tensor information is combined with recently reported single-crystal EPR studies on [Fe₄S₄]^+,3+model compounds and leads to a simple geometrical picture of cyanide binding to Pf-Fd, in which CN^-replaces the Asp-14 ligand and binds in an orientation similar to that of the Cys residue found in an ordinary 4Fe-4S ferredoxin. This reversible binding of an exogenous ligand may have implications for the catalytic activity of Fe-S enzymes.