文摘
The rubredoxin from Clostridium pasteurianumcontains a single iron atom bound to thepolypeptide chain by cysteines 6, 9, 39, and 42. The C42A variantof this protein has been prepared bysite-directed mutagenesis and heterologous expression of the gene inEscherichia coli. The mutatedproteinwas found to contain an unexpected chromophore that has beencharacterized by a variety of techniques.UV-visible absorption and resonance Raman spectra were stronglyreminiscent of those of [2Fe-2S]proteins. Mössbauer spectra of the oxidized chromophoreisolated in oxygen-free conditions indicatedlow-temperature diamagnetism resulting from antiferromagneticallycoupled high-spin ferric ions. Analysisof X-ray absorption fine structure spectra yielded an Fe-Fe distanceof 2.68 Å. Colorimetric assays ofiron and inorganic sulfide showed that the two elements are present ina 1:1 ratio. Electrospray-ionizationmass spectra displayed a major component at M = 6190 Da,i.e. the molecular mass of the C42A apoproteinplus two atomic masses of iron and two atomic masses of sulfur.Taken together, these data show thata mere point mutation allows the stabilization of a binuclear[2Fe-2S] cluster in a protein that normallyaccommodates a mononuclear Fe(Scys)4 site.Assembly of a [2Fe-2S] cluster may occur becauserubredoxinassumes a similar fold around its metal center as the [2Fe-2S] Rieskeprotein. Alternatively, a moreextensive structural rearrangement of the polypeptide chain of the C42Arubredoxin variant may beconsidered as well.
