Overexpression and reconstitution of a Rieske iron-sulfur protein from the cyanobacterium Synechocystis PCC 6803.
Chloroplast cyt b6f complexes as well as mitochondrial and bacterial cyt bc1 complexes contain a high potential Rieske iron-sulfur protein which is essential for their function. To characterise the isolated Rieske protein from the mesophilic cyanobacterium Synechocystis PCC6803 we cloned the encoding gene into an expression vector and overexpressed the protein in E. coli. In cells overexpressing the protein no typical Rieske type EPR signal was detected neither in membranes nor in inclusion bodies where the majority of the protein was deposited. The inclusion bodies were isolated from the E. coli cells and denaturated with 8 M urea. With a single anion exchange chromatographic step a pure protein could be obtained which was used for further experiments. The NifS like protein IscS was recently reported to mediate the incorporation of iron-sulfur clusters into ferredoxin in vitro. We used the recombinant IscS protein for the incorporation of the cluster into the folded Rieske apoprotein. Spectroscopic characterisation of the resultant protein by CD and EPR spectroscopy showed the presence of a typical Rieske iron-sulfur centre.
Schneider
D
D
Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie, Ruhr-Universität Bochum, D-447800 Bochum, Germany.
Jaschkowitz
K
K
Seidler
A
A
Rögner
M
M
D001483:Base Sequence; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000737:chemistry*; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D017931:DNA Primers; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; C037989:Rieske iron-sulfur protein; D014450:Electron Transport Complex III