Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus

J Biol Chem. 2004 Mar 26;279(13):12519-28. doi: 10.1074/jbc.M305923200. Epub 2004 Jan 15.


We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Circular Dichroism
  • Cysteine / chemistry*
  • Electron Transport Complex III / chemistry*
  • Escherichia coli / metabolism
  • Ferredoxins / chemistry*
  • Histidine / chemistry*
  • Iron-Sulfur Proteins / chemistry*
  • Ligands
  • Magnetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Oxygen / metabolism
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Sequence Homology, Amino Acid
  • Spectrometry, X-Ray Emission
  • Spectrum Analysis, Raman
  • Sulfolobus / metabolism*
  • Temperature
  • Thermodynamics
  • Ultraviolet Rays


  • Ferredoxins
  • Iron-Sulfur Proteins
  • Ligands
  • Recombinant Proteins
  • Rieske iron-sulfur protein
  • Histidine
  • Electron Transport Complex III
  • Cysteine
  • Oxygen