Overexpression and reconstitution of a Rieske iron-sulfur protein from thecyanobacterium Synechocystis PCC 6803

Chloroplast cyt b(6)f complexes as well as mitochondrial and bacterial cyt bc(1) complexes contain a high potential Rieske iron-sulfur protein which i s essential for their function. To characterise the isolated Rieske protein from the mesophilic cyanobacterium Synechocystis PCC6803 we cloned the enc oding gene into an expression vector and overexpressed the protein in E. co li. In cells overexpressing the protein no typical Rieske type EPR signal w as 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 c hromatographic step a pure protein could be obtained which was used for fur ther experiments. The NifS like protein IscS was recently reported to media te the incorporation of iron-sulfur clusters into ferredoxin in vitro. We u sed the recombinant IscS protein for the incorporation of the cluster into the folded Rieske apoprotein. Spectroscopic characterisation of the resulta nt protein by CD and EPR spectroscopy showed the presence of a typical Ries ke iron-sulfur centre.