Removal of a cysteine ligand from rubredoxin: assembly of Fe(2)S(2) and Fe(S-Cys)(3)(OH) centres.
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)(2)S(2) cluster in place. In contrast, the other proteins are isolated in colourless forms, although a Fe(III)(2)S(2) 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.
Cross
Maddalena
M
School of Chemistry, University of Melbourne, Parkville, Victoria 3010, Australia.
Xiao
Zhiguang
Z
Maes
Estelle M
EM
Czernuszewicz
Roman S
RS
Drew
Simon C
SC
Pilbrow
John R
JR
George
Graham N
GN
Wedd
Anthony G
AG
D019943:Amino Acid Substitution; D003013:Clostridium / Q000737:chemistry; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D007501:Iron / Q000737:chemistry*; D008024:Ligands; D011506:Proteins / Q000737:chemistry; D012416:Rubredoxins / Q000737:chemistry* / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013059:Spectrum Analysis, Raman; D013440:Sulfides / Q000737:chemistry*
D008024:Ligands; D011506:Proteins; D012416:Rubredoxins; D013440:Sulfides; D007501:Iron; D003545:Cysteine