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

The hyperthermophilic archaeonPyrococcus 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 bothS = 1/2 andS = 3/2 spin states. Addition of excess cyanide ion converts the cluster exclusively to anS = 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 using¹³CN^? and C¹⁵N^? ligands.¹³C and¹⁵N 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 the¹³C hyperfine coupling has a large isotropic component (a_iso(¹³C) ≈ ?30 MHz). This small contact interaction is not due to low spin density of Fe, as⁵⁷Fe ENDOR of the singly and triply labeledPf- Fd 4FeCN isotopologs, [⁵⁷FeFe₃S₄]⁺ and [Fe⁵⁷Fe₃S₄]⁺, show hyperfine coupling characteristic for [Fe₄S₄]⁺ clusters, particularly for the Fe to which cyanide binds. Thus, the low spin density on¹³C 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.     

Mössbauer and infrared spectroscopic studies of novel mixed valence states in cobaltous ferrocyanides and ferricyanides

Novel mixed valence states have been obtained by the treatment of cobaltous ferrocyanides (Co⁺²Fe^II) and ferricyanides (Co⁺²Fe^III) in an ozone flow. The CN stretching bands occur at 2085 cm^–1 for Co⁺²Fe^II and at 2160 cm^–1 for Co⁺²Fe^III. After the ozonization process of Co⁺²Fe^II, an intense band approximately at 2125 cm^–1 is detected. This intermediate band must correspond to a mixed valence state of the type: Fe^II–CN–Co²⁺–NC–Fe^III Mössbauer spectra recorded in situ during the ozonization of Co⁺²Fe^II show the presence of two components: a doublet with isomer shift and quadrupole splitting values close to the cobalti ferricyanide and a very broad line for the mixed valence state. From the Mössbauer and infrared spectra of the aged samples of the Co⁺²Fe^II after ozonization, a relaxation process to the initial state of the samples is observed but the mixed valence state is stable.