Assembly of a [2Fe-2S]2+ cluster in a molecular variant of Clostridium pasteurianum rubredoxin.
The rubredoxin from Clostridium pasteurianum contains a single iron atom bound to the polypeptide chain by cysteines 6, 9, 39, and 42. The C42A variant of this protein has been prepared by site-directed mutagenesis and heterologous expression of the gene in Escherichia coli. The mutated protein was found to contain an unexpected chromophore that has been characterized by a variety of techniques. UV-visible absorption and resonance Raman spectra were strongly reminiscent of those of [2Fe-2S] proteins. Mössbauer spectra of the oxidized chromophore isolated in oxygen-free conditions indicated low-temperature diamagnetism resulting from antiferromagnetically coupled high-spin ferric ions. Analysis of X-ray absorption fine structure spectra yielded an Fe-Fe distance of 2.68 A. Colorimetric assays of iron and inorganic sulfide showed that the two elements are present in a 1:1 ratio. Electrospray-ionization mass spectra displayed a major component at M = 6190 Da, i.e. the molecular mass of the C42A apoprotein plus two atomic masses of iron and two atomic masses of sulfur. Taken together, these data show that a mere point mutation allows the stabilization of a binuclear [2Fe-2S] cluster in a protein that normally accommodates a mononuclear Fe(Scys)4 site. Assembly of a [2Fe-2S] cluster may occur because rubredoxin assumes a similar fold around its metal center as the [2Fe-2S] Rieske protein. Alternatively, a more extensive structural rearrangement of the polypeptide chain of the C42A rubredoxin variant may be considered as well.
Meyer
J
J
Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 38054 Grenoble, France. jac@ebron.ceng.cea.fr
Gagnon
J
J
Gaillard
J
J
Lutz
M
M
Achim
C
C
Münck
E
E
Pétillot
Y
Y
Colangelo
C M
CM
Scott
R A
RA
D001665:Binding Sites / Q000235:genetics; D003013:Clostridium; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D005583:Fourier Analysis; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012416:Rubredoxins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012694:Serine / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer; D013059:Spectrum Analysis, Raman; D013329:Structure-Activity Relationship; D013455:Sulfur / Q000378:metabolism*
D008024:Ligands; D011994:Recombinant Proteins; D012416:Rubredoxins; D012694:Serine; D013455:Sulfur; D007501:Iron; D003545:Cysteine