Heterometallic [AgFe(3)S (4)] ferredoxin variants: synthesis, characterization, and the first crystal structure of an engineered heterometallic iron-sulfur protein

J Biol Inorg Chem. 2013 Feb;18(2):261-276. doi: 10.1007/s00775-012-0971-3. Epub 2013 Jan 8.


Heterometallic [AgFe(3)S(4)] iron-sulfur clusters assembled in wild-type Pyrococcus furiosus ferredoxin and two variants, D14C and D14H, are characterized. The crystal structure of the [AgFe(3)S(4)] D14C variant shows that the silver(I) ion is indeed part of the cluster and is coordinated to the thiolate group of residue 14. Cyclic voltammetry shows one redox pair with a reduction potential of +220 mV versus the standard hydrogen electrode which is assigned to the [AgFe(3)S(4)](2+/+) couple. The oxidized form of the [AgFe(3)S(4)] D14C variant is stable in the presence of dioxygen, whereas the oxidized forms of the [AgFe(3)S(4)] wild type and D14H variants convert to the [Fe(3)S(4)] ferredoxin form. The monovalent d (10) silver(I) ion stabilizes the [Fe(3)S(4)](+/0) cluster fragment, as opposed to divalent d (10) metal ions, resulting in more than 0.4 V difference in reduction potentials between the silver(I) and, e.g., zinc(II) heterometallic [MFe(3)S(4)] ferredoxins. The trend in reduction potentials for the variants containing the [AgFe(3)S(4)] cluster is wild type ≤ D14C < D14H and shows the same trend as reported for the variants containing the [Fe(3)S(4)] cluster, but is different from the D14C < D14H < wild type trend reported for the [Fe(4)S(4)] ferredoxin. The similarity in the reduction potential trend for the variants containing the heterometallic [AgFe(3)S(4)] cluster and the [Fe(3)S(4)] cluster can be rationalized in terms of the electrostatic influence of the residue 14 side chains, rather than the dissociation constant of this residue, as is the case for [Fe(4)S(4)] ferredoxins. The trends in reduction potentials are in line with there being no electronic coupling between the silver(I) ion and the Fe(3)S(4) fragment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Anisotropy
  • Archaeal Proteins / biosynthesis
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Coordination Complexes / chemistry*
  • Crystallography, X-Ray
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli
  • Ferredoxins / biosynthesis
  • Ferredoxins / chemistry*
  • Ferredoxins / genetics
  • Iron / chemistry*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oxidation-Reduction
  • Protein Engineering
  • Protein Stability
  • Pyrococcus furiosus*
  • Silver / chemistry*


  • Archaeal Proteins
  • Coordination Complexes
  • Ferredoxins
  • Silver
  • Iron

Associated data

  • PDB/4DHV