Investigation of the unusual electronic structure of pyrococcus furiosus 4Fe ferredoxin by EPR spectroscopy of protein reduced at ambient and cryogenic temperatures

The hyperthermophilic archaeon Pyrococcus furiosus contains a novel ferredoxin (Pf-Fd) in which, in the native 4Fe form, three of the Fe ions are coordinated to the protein by cysteinyl thiolato ligands, but the fourth Fe is coordinated by an aspartyl carboxylato ligand ([Fe₄S ₄(cys)3(asp)]2-,3-). Chemical reduction at ambient temperature of the oxidized 4Fe form (Pf-Fd 4Fe-ox, S = 0 ground state, with the cluster core indicated by [Fe₄S₄]²⁺_ox) produces a reduced 4Fe form (Pf-Fd 4Fe-red, with the cluster core indicated by [Fe ₄S₄]⁺_red). Pf-Fd 4Fe-red, [Fe ₄S₄]⁺_red core, in frozen solution exhibits S = 1/2 and 3/2 electronic states that are not in thermal equilibrium. The two spin states thus represent alternate ground states of the reduced cluster (cluster cores indicated by [Fe₄S₄] ⁺_red1 and [Fe₄S₄]⁺ _red2, respectively), rather than a ground and excited spin state. Low-temperature (77 K) reduction of 4Fe-ox in frozen solution by γ-irradiation produces in high yield the reduced state of the cluster that is trapped in the structure of the oxidized parent cluster, and thus has a cluster core denoted by Fe₄S₄]⁺_ox. The Fe₄S₄]⁺_ox form also exhibits non thermally converting S = 3/2 and 1/2 components in the same proportion as seen for [Fe₄S₄]⁺_red. The EPR signal of the S = 3/2 component that results from cryoreduction ([Fe₄S ₄]⁺_ox2) is indistinguishable, within experimental variability, from that seen in the ambient-temperature, chemically reduced protein ([Fe₄S₄]⁺_red2), and the signals of the two S = 1/2 components ([Fe₄S₄] ⁺_ox1 and [Fe₄S₄]⁺ _red1, respectively) closely resemble each other, although they are not identical. Previous NMR studies at ambient temperature showed evidence for only one species in fluid solution for both Pf-Fd 4Fe-ox and 4Fe-red. Taken together, the NMR and EPR results indicate that fluid solutions of either oxidized or reduced Pf-Fd contain only one conformer, but that frozen solutions of each contain two distinct conformers, with each one of the pair of oxidized protein forms having a corresponding reduced form. A shift in the coordination mode of the aspartyl carboxylato ligand is proposed to account for this conformational flexibility.