title: Resolution and reconstitution of succinate-ubiquinone reductase from Escherichia coli.
abstract: A modified procedure is developed for isolation of highly purified succinate-ubiquinone reductase from Escherichia coli NM256 containing a cloned sdh operon in a multicopy plasmid. Succinate-ubiquinone reductase is solubilized from the membrane by polyoxyethylene-9-lauryl ether and purified by DEAE-Sepharose CL-6B column chromatography. The isolated reductase is resolved into a reconstitutively active, two-subunit succinate dehydrogenase and a two-subunit membrane anchoring protein fraction (the SdhC-SdhD fraction) by alkaline (pH 10.2) treatment of the reductase in the presence of 1 M urea, followed by DEAE-Sepharose CL-6B column chromatography under anaerobic conditions. Isolated succinate dehydrogenase and the SdhC-SdhD fraction alone show no succinate-ubiquinone reductase activity. However, when a given amount of the SdhC-SdhD fraction is mixed with varying amounts of succinate dehydrogenase or vice versa succinate-ubiquinone reductase activity increases as the amount of succinate dehydrogenase or the SdhC-SdhD fraction added increases. Maximum reconstitution is obtained when the weight ratio of succinate dehydrogenase to the SdhC-SdhD fraction reaches 5.26. This ratio is slightly higher than the calculated value of 3.37, obtained by assuming 1 mol of succinate dehydrogenase reacts with 1 mol of SdhC and SdhD. The isolated SdhC-SdhD fraction contains 35 nmol cytochrome b556/mg protein. Unlike mitochondrial cytochrome b560, the cytochrome b556 is reducible by succinate in the isolated and complex forms. Furthermore, cytochrome b556 in the isolated SdhC-SdhD fraction has absorption properties, carbon monoxide reactivity, and EPR characteristics similar to those of cytochrome b556 in intact succinate-ubiquinone reductase, indicating that its heme environments are not affected by the presence of succinate dehydrogenase. However, the redox potential of cytochrome b556 in the SdhC-SdhD fraction (22 mV) increases slightly when complexed with succinate dehydrogenase (34 mV). No hybrid succinate-ubiquinone reductase is formed from mitochondrial QPs (the membrane-anchoring protein fraction of bovine heart mitochondrial succinate-ubiquinone reductase) and E. coli succinate dehydrogenase or vice versa. However, the cytochrome b556 in E. coli SdhC-SdhD fraction is reducible by succinate in the presence of mitochondrial succinate dehydrogenase, and the rate of cytochrome b556 reduction correlates with the reconstitutive activity of the mitochondrial succinate dehydrogenase.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.forename: L
authors.initials: L
authors.lastname: Yu
authors.forename: C A
authors.initials: CA
authors.lastname: Yu
mesh_terms: D000818:Animals; D002417:Cattle; D003573:Cytochrome b Group / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D042963:Electron Transport Complex II; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D013054:Spectrophotometry, Atomic; D013385:Succinate Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D003573:Cytochrome b Group; D009097:Multienzyme Complexes; C018256:cytochrome b556; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase