Reconstitution of the 2Fe-2S center and g = 1.89 electron paramagnetic resonance signal into overproduced Nostoc sp. PCC 7906 Rieske protein

Biochemistry
B HoltonT Kallas

Abstract

The Rieske 2Fe-2S protein is a distinguishing subunit of the photosynthetic electron transport cytochrome b6f complex in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overproduction 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 protein. Expression of the nonfusion petC gene in a T7 RNA polymerase promoter vector produced copious quantities of the full-length Rieske protein predominantly as inclusion bodies. The highly enriched, Rieske protein from inclusion bodies has been denatured in guanidine hydrochloride and refolded and the characteristic 2Fe-2S cluster reconstituted in vitro by incubation with iron and sulfide under reducing conditions. Purification by chromatography on Whatman DE52 cellulose and ultrafiltration through a 30000 molecular weight cutoff membrane yielded pure and predominantly monomeric Rieske protein. Reconstituted Rieske preparations showed intense and highly characteristic gx = 1.74, gy = 1.89, and gz = 2.03 "Rieske-type" electron paramagnetic resonanc...Continue Reading

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Citations

May 6, 2003·Protein Expression and Purification·Beata GubernatorAndrzej Szczepaniak
Sep 21, 2012·Acta Crystallographica. Section D, Biological Crystallography·Sebastian VeitKunio Miki
Mar 12, 2004·Annual Review of Plant Physiology and Plant Molecular Biology·Sabeeha Merchant, Beth Welty Dreyfuss
Feb 21, 2008·Biometals : an International Journal on the Role of Metal Ions in Biology, Biochemistry, and Medicine·S BoxhammerChristian L Schmidt
Oct 29, 2009·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Hugo M BotelhoCláudio M Gomes
Feb 7, 2006·Biochimica Et Biophysica Acta·Beata GubernatorAndrzej Szczepaniak
Apr 27, 1999·Biochimica Et Biophysica Acta·F A WollmanR Nechushtai
Jan 15, 2002·The Journal of Biological Chemistry·Dirk SchneiderMatthias Rögner

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