RESOLUTION AND RECONSTITUTION OF SUCCINATE-UBIQUINONE REDUCTASE FROM ESCHERICHIA-COLI

A modified procedure is developed for isolation of highly purified suc cinate-ubiquinone reductase from Escherichia coli NM256 containing a c loned sdh operon in a multicopy plasmid, Succinate-ubiquinone reductas e is solubilized from the membrane by polyoxyethylene-9-lauryl ether a nd purified by DEAE-Sepharose CL-6B column chromatography, The isolate d reductase is resolved into a reconstitutively active, two-subunit su ccinate dehydrogenase and a two-subunit membrane anchoring protein fra ction (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-6 B column chromatography under anaerobic conditions, Isolated succinate dehydrogenase and the SdhC-SdhD fraction alone show no succinate-ubiq uinone reductase activity, However, when a given amount of the SdhC-Sd hD fraction is mixed with varying amounts of succinate dehydrogenase o r vice versa succinate-ubiquinone reductase activity increases as the amount of succinate dehydrogenase or the SdhC-SdhD fraction added incr eases, Maximum reconstitution is obtained when the weight ratio of suc cinate dehydrogenase to the SdhC-SdhD fraction reaches 5.26. This rati o is slightly higher than the calculated value of 3.37, obtained by as suming 1 mol of succinate dehydrogenase reacts with 1 mol of SdhC and SdhD. The isolated SdhC-SdhD fraction contains 35 nmol cytochrome b(55 6)/mg protein, Unlike mitochondrial cytochrome b(560), the cytochrome b(556) is reducible by succinate in the isolated and complex forms, Fu rthermore, cytochrome b(556) in the isolated SdhC-SdhD fraction has ab sorption properties, carbon mon oxide reactivity, and EPR characterist ics similar to those of cytochrome b(556) in intact succinate-ubiquino ne reductase, indicating that its heme environments are not affected b y the presence of succinate dehydrogenase, However, the redox potentia l of cytochrome b(556) in the SdhC-SdhD fraction (22 mV) increases sli ghtly when complexed with succinate dehydrogenase (34 mV). No hybrid s uccinate-ubiquinone reductase is formed from mitochondrial QPs (the me mbrane-anchoring protein fraction of bovine heart mitochondrial succin ate-ubiquinone reductase) and E. coli succinate dehydrogenase or vice versa, However, the cytochrome b(556) in E. coli SdhC-SdhD fraction is reducible by succinate in the presence of mitochondrial succinate deh ydrogenase, and the rate of cytochrome b(556) reduction correlates wit h the reconstitutive activity of the mitochondrial succinate dehydroge nase.