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

Joshua Telser, Roman Davydov, Chul Hwan Kim, Michael W.W. Adams, Brian M. Hoffman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)3550-3553
Number of pages4
JournalInorganic chemistry
Issue number15
StatePublished - Dec 1 1999

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Inorganic Chemistry


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