Cyanide Binding to the Novel 4Fe Ferredoxin from Pyrococcus furiosus: Investigation by EPR and ENDOR Spectroscopy

Richard C. Conover, Michael K. Johnson, Joshua Telser, Brian M. Hoffman, Eugene T. Smith, Michael W.W. Adams*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


The hyperthermophilic archaeon Pyrococcus furiosus contains a 4-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 = 1/2andS = 3/2spin states. Addition of excess cyanide ion converts the cluster exclusively to anS = 1/2state (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-CN) were investigated here using the techniques of electron paramagnetic resonance (EPR) and electron—nuclear double-resonance (ENDOR) spectroscopy. No evidence for a strongly coupled solvent-derived hydrogen (1H or 2H) from an OH-or H2O ligand in either spin state of the [Fe4S4]+cluster was observed, contrary to an earlier report. Rather, 1,2H ENDOR characteristic of 4Fe-Fd's was seen for both native Pf-Fd and Pf-Fd-CN. Pf-Fd-CN was further investigated using 13CN-and C15N-ligands. 13C and 15N ENDOR indicated that a single cyanide ion bound directly, with the cluster showing an unusually small contact interaction (aiso(13C) ∼ -3 MHz, aiso(15N) ~ 0). This is in contrast to cyanide bound to monomeric low-spin Fe(III)-containing proteins such as transferrin and myoglobin, for which the 13C hyperfine coupling has a large isotropic component (aiso(13C) ≈ — 30 MHz). The full 13C and 15N hyperfine tensors were determined by computer simulation of the ENDOR spectra. The A(13C) is rotated by ~40° about g2. The g and A(13C) tensor information is combined with recently reported single-crystal EPR studies on [Fe4S4]+,3+model compounds and leads to a simple geometrical picture of cyanide binding to Pf-Fd, in which CN-replaces the Asp-14 ligand and binds in an orientation similar to that of the Cys residue found in an ordinary 4Fe-4S ferredoxin. This reversible binding of an exogenous ligand may have implications for the catalytic activity of Fe-S enzymes.

Original languageEnglish (US)
Pages (from-to)5133-5140
Number of pages8
JournalJournal of the American Chemical Society
Issue number18
StatePublished - May 1995

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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