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 fer redoxin (Pf-Fd) that differs from most other 4Fe-Fd's in that its [Fe4 S4] cluster is anchored to protein by only three cysteinyl residues. P f-Fd also is of interest because in its reduced form, [Fe4S4](+) the c luster exhibits both S = 1/2 and S = 3/2 spin states. Addition of exce ss cyanide ion converts the cluster exclusively to an S = 1/2 state (g l = 2.09, g(2) = 1.95, gs = 1.92); however, dialysis restores the EPR signal of native reduced protein, indicating that the cluster is not i rreversibly 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 elec tron-nuclear double-resonance (ENDOR) spectroscopy. No evidence for a strongly coupled solvent-derived hydrogen (H-1 or H-2) from an OH- or H2O ligand in either spin state of the [Fe4S4](+) cluster was observed , contrary to an earlier report. Rather, H-1,H-2 ENDOR characteristic of 4Fe-Fd's was seen for both native Pf-Fd and Pf-Fd-CN. Pf-Fd-CN was further 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 t he 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 t o cyanide bound to monomeric low-spin Fe(III)-containing proteins such as transferrin and myoglobin, for which the C-13 hyperfine coupling h as a large isotropic component (a(iso)(C-13) approximate to -30 MHz). The full C-13 and N-15 hyperfine tensors were determined by computer s imulation of the ENDOR spectra. The A(C-13) is rotated by similar to 4 0 degrees about g(2). The g and A(C-13) tenser information is combined with recently reported single-crystal EPR studies on [Fe4S4](+,3+) mo del compounds and leads to a simple geometrical picture of cyanide bin ding 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 4 Fe-4S ferredoxin. This reversible binding of an exogenous ligand may h ave implications for the catalytic activity of Fe-S enzymes.