Overexpression, purification, and crystallization of the membrane-bound fumarate reductase from Escherichia coli

Quinol-fumarate reductase (QFR) from Escherichia coli is a membrane-bound f our-subunit respiratory protein that shares many physical and catalytic pro perties with succinate-quinone oxidoreductase (EC 1.3.99.1) commonly referr ed to as Complex II. The E. coli QFR has been overexpressed using plasmid v ectors so that more than 50% of the cytoplasmic membrane fraction is compos ed of the four-subunit enzyme complex. The growth characteristics required for optimal levels of expression with minimal degradation by host cell prot eases and oxidation factors were determined for the strains harboring the r ecombinant plasmid. The enzyme is extracted from the enriched membrane frac tion using the nonionic detergent Thesit (polyoxyethylene(9)dodecyl ether) in a monodisperse form and then purified by a combination of anion-exchange , perfusion, and gel filtration chromatography. The purified enzyme is high ly active and contains all types of redox cofactors expected to be associat ed with the enzyme. Crystallization screening of the purified QFR by vapor diffusion resulted in the formation of crystals within 24 h using a sodium citrate buffer and polyethylene glycol precipitant. The crystals contain th e complete four-subunit QFR complex, diffract to 3.3 Angstrom resolution, a nd were found to be in space group P2(1)2(1)2(1) with unit cell dimensions a 96.6 Angstrom b = 138.1 Angstrom, and c = 215.3 Angstrom. The purificatio n and crystallization procedures are highly reproducible and the general pr ocedure may prove useful for Complex IIs from other sources. (C) 2000 Acade mic Press.