Removal of a cysteine ligand from rubredoxin: assembly of Fe2S2 and Fe(S-Cys)3(OH) centres

Abstract

The electron transfer protein rubredoxin from Clostridium pasteurianum contains an Fe(S-Cys)₄ active site. Mutant proteins C9G, C9A, C42G and C42A, in which cysteine ligands are replaced by non-ligating Gly or Ala residues, have been expressed in Escherichia coli. The C42A protein expresses with a Fe^III ₂S₂ cluster in place. In contrast, the other proteins are isolated in colourless forms, although a Fe^III ₂S₂ cluster may be assembled in the C42G protein via incubation with Fe^III and sulfide. The four mutant proteins were isolated as stable mononuclear Hg^II forms which were converted to unstable mononuclear Fe^III preparations that contain both holo and apo protein. The Fe^III systems were characterized by metal analysis and mass spectrometry and by electronic, electron paramagnetic resonance, X-ray absorption and resonance Raman spectroscopies. The dominant Fe^III form in the C9A preparation is a Fe(S-Cys)₃(OH) centre, similar to that observed previously in the C6S mutant protein. Related centres are present in the proteins NifU and IscU responsible for assembly and repair of iron-sulfur clusters in both prokaryotic and eukaryotic cells. In addition to Fe(S-Cys)₃(OH) centres, the C9G, C42G and C42A preparations contain a second four-coordinate Fe^III form in which a ligand appears to be supplied by the protein chain. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-002-0355-1.