Redox thermodynamics of mutant forms of the rubredoxin from Clostridiumpasteurianum: identification of a stable Fe-III(S-Cys)(3)(OH) centre in the C6S mutant

Redox thermodynamic data provide a detailed insight into control of the reduction potential E-o' of the [Fe(S-Cys)(4)] site in rubredoxin. Mutant forms were studied in which specific structural changes were made in both the primary and secondary coordination spheres. Those changes have been probed by resonance Raman spectroscopy. The decrease of similar to 200mV in E-o' observed for the [Fe(S-Cys)(3)(O-Ser)](-/2-) couples in the surface ligand mutants C9S and S42S is essentially enthalpic in origin and associated with the substitution of ligand thiolate by ligand elate. However, the pH dependence of the potentials below characteristic pK(a)(red)approximate to7 is an entropic contribution, plausibly associated with increased conformational flexibility induced by a longer Fe-II-O(H)-Ser bond in the reduced form. The presence of a second surface Ser ligand in the new double mutant protein C9S/C42S affects the enthalpic term primarily for pH>pK(a)(red)greater than or equal to9.3, but for pHpK(a)similar to9: [Fe-III(S-Cys)(3)(OH)](-) + e(-) --> [Fe-II(S-Cys)(3)(OH>)](2-) pH [Fe-II(S-Cys)(3)(OH2)](-)

Removal of a cysteine ligand from rubredoxin: assembly of Fe2S2 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 (Fe2S2)-S-III cluster in place. In contrast, the other proteins are isolated in colourless forms, although a (Fe2S2)-S-III 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/s00775-0020355-1