RECONSTITUTION OF THE 2FE-2S CENTER AND G=1.89 ELECTRON-PARAMAGNETIC-RESONANCE SIGNAL INTO OVERPRODUCED NOSTOC SP PCC-7906 RIESKE PROTEIN

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynt hetic electron transport cytochrome b(6)f complex in chloroplast and c yanobacterial thylakoid membranes. We have constructed plasmids for ov erproduction in Escherichia coli of fusion, full-length, and truncated forms of the Rieske (PetC) protein from the cyanobacterium Nostoc sp. PCC 7906. A glutathione S-transferase/Rieske fusion protein was used to prepare specific chicken egg-yolk antibodies against the Rieske pro tein. Expression of the nonfusion petC gene in a T7 RNA polymerase pro moter vector produced copious quantities of the full-length Rieske pro tein predominantly as inclusion bodies. The highly enriched, Rieske pr otein from inclusion bodies has been denatured in guanidine hydrochlor ide and refolded and the characteristic 2Fe-2S cluster reconstituted i n vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafil tration through a 30 000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske prep arations showed intense and highly characteristic g(x) = 1.74, g(y) = 1.89, and g(z) = 2.03 ''Rieske-type'' electron paramagnetic resonance signals at 15 K. Two methods of reconstitution yielded Rieske preparat ions in which 20-60% of the protein contained 2Fe-2S clusters as deter mined by EPR spin quantitation. The reconstituted Rieske protein was s oluble and stable at 4 degrees C in buffers containing nonionic deterg ents and showed a redox midpoint potential of +321 mV at pH 7.0 as det ermined by optical circular dichroism (CD) spectroscopy. These data de monstrate the in vitro restoration of a Cys and His liganded 2Fe-2S cl uster and provide the basis for mutational and structural analysis of a PetC Rieske protein of oxygenic photosynthesis.