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](+), t he 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 th e EPR signal of native reduced protein indicating that the cluster is not irreversibly altered by cyanide. Both the native protein and prote in in the presence of excess cyanide ion (Pf-Fd 4Fe-CN) were investiga ted here using the techniques of electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy. In partic ular, Pf-Fd 4Fe-CN was investigated using (CN-)-C-13 and (CN-)-N-15 li gands. C-13 and N-15 ENDOR indicated that a single cyanide ion bound d irectly with the cluster showing an unusually small contact interactio n (a(iso)(C-13) similar to - 3 MHz, a(iso)(N-15) similar to 0). This i s in contrast to cyanide bound to monomeric low-spin Fe(III)-containin g proteins such as transferrin and myoglobin, for which the C-13 hyper fine coupling has a large isotropic component (a(iso)(C-13) approximat e 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 4 Fe-CN isotopologs, [(FeFe3S4)-Fe-57](+) and [(FeFe3S4)-Fe-57](+), show hyperfine coupling characteristic for [Fe4S4](+) clusters, particular ly 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. Fur ther theoretical work is needed to explain the contrast between the st rong electronic effect of cyanide ion binding with the low spin densit y on the ligand.