Coordination of the [2Fe-2S] cluster in wild type and molecular variants of Clostridium pasteurianum ferredoxin, investigated by ESEEM spectroscopy.
The [2Fe-2S] ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56, and 60. This pattern is unique, and a combination of site-directed mutagenesis and spectroscopy is therefore being implemented to identify the ligands of the [2Fe-2S] cluster. The possible involvement of ligands other than cysteine in some molecular variants of this ferredoxin has been considered, histidines being likely candidates. Therefore, the three histidine residues in positions 6, 7, and 90 of the amino acid sequence have been individually and collectively replaced by alanine or valine. The mutated ferredoxins have been purified and were all found to contain [2Fe-2S] clusters of which the UV-visible absorption spectra were identical to that of the wild-type protein. The H6A/H7A/ H90A triply mutated ferredoxin was further characterized by EPR and by ESEEM spectroscopy and was found to differ only marginally from the wild-type protein. The ESEEM spectra of wild-type ferredoxin displayed weak 14N hyperfine interactions at the three principal g-factors of the [2Fe-2S] center. The estimated 14N coupling constants (Aiso = 0.6 MHz; e2qQ approximately 3.3 MHz) indicate that the ESEEM effect is most likely due to 14N from the polypeptide backbone. 2H2O ESEEM spectra showed that the [2Fe-2S] cluster is accessible for exchange with solvent deuterons. ESEEM spectra of the previously characterized C24A and C14A/C24A variants have been recorded and were also found to be very similar to those of the wild-type protein. There was no evidence for coordination of the [2Fe-2S] cluster by [14N]histidine or other 14N nuclei, in either wild-type or mutant forms of the ferredoxin. By these criteria, the environment of the [2Fe-2S] center is not distinguishable from those in plant-type ferredoxins. Non-cysteinyl coordination most probably occurs only in the C14A/C24A variant, which contains no more than three cysteine residues. The data shown here indicate that the fourth ligand of the [2Fe-2S] cluster is neither a histidine residue nor another nitrogenous ligand. The possibility of oxygenic coordination for this molecular variant is discussed.
Shergill
J K
JK
Centre for the Study of Metals in Biology and Medicine, King's College, University of London, U.K. j.shergill@umds.ac.uk
Golinelli
M P
MP
Cammack
R
R
Meyer
J
J
D003013:Clostridium / Q000737:chemistry*; D003545:Cysteine / Q000235:genetics; D017666:Deuterium Oxide; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D014867:Water
D005288:Ferredoxins; D011994:Recombinant Proteins; D014867:Water; D006639:Histidine; D017666:Deuterium Oxide; D003545:Cysteine