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 ferredo xin (Pf-Fd) in which, in the native 4Fe form, three of the Fe ions are coor dinated to the protein by cysteinyl thiolato ligands, but the fourth Fe is coordinated by an aspartyl carboxylato ligand ([Fe4S4(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 ind icated by [Fe4S4](+)(red)) Pf-Fd 4Fe-red, [Fe4S4](+)(red) core, in frozen s olution 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 gamma-irradia tion produces in high yield the reduced state of the cluster that is trappe d in the structure of the oxidized parent cluster, and thus has a cluster c ore denoted by [Fe4S4](+)(ox). The [Fe4S4](+)(ox) form also exhibits non th ermally converting S = 3/2 and 1/2 components in the same proportion as see n for [Fe4S4](+)(red). The EPR signal of the S = 3/2 component that results from cryoreduction ([Fe4S4](+)(ox2)) is indistinguishable, within experime ntal variability, from that seen in the ambient-temperature, chemically red uced protein ([Fe4S4](+)(red2)), and the signals of the two S = 1/2 compone nts ([Fe4S4](+)(ox1) and [Fe4S4](+)(red1), respectively) closely resemble e ach other, although they are not identical. Previous NMR studies at ambient temperature showed evidence for only one species in fluid solution for bot h Pf-Fd 4Fe-ox and 4Fe-red. Taken together, the NMR and EPR results indicat e that fluid solutions of either oxidized or reduced Pf-Fd contain only one conformer, but that frozen solutions of each contain two distinct conforme rs, with each one of the pair of oxidized protein forms having a correspond ing reduced form. A shift in the coordination mode of the aspartyl carboxyl ato ligand is proposed to account for this conformational flexibility.