Resolution and Reconstitution of Succinate-Ubiquinone Reductase from Escherichia coli

Xudong Yang,Linda Yu,Chang-An Yu

doi:10.1074/jbc.272.15.9683

Xudong Yang, Linda Yu + Show 1 more

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https://doi.org/10.1074/jbc.272.15.9683

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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.

Highlights

In E. coli, the genes for the Fp and Ip subunits and for the membrane anchoring proteins are located in one operon, which is transcribed in the following sequence: sdhC, sdhD, sdhA (Fp), sdhB (Ip) [6, 7]
E. coli succinate-ubiquinone reductase is structurally and functionally similar to the mitochondrial enzyme, cytochrome b556 in the E. coli enzyme is fully reducible by succinate [2], whereas that in mitochondria is not [11], indicating that the role of cytochrome b may differ in these two enzyme systems
Isolation and Properties of E. coli Succinate-Ubiquinone Reductase— a one-step purification procedure has been reported [2] for isolation of succinate-ubiquinone reductase from an E. coli strain containing the sdh operon on a pGS133 plasmid, the detergent used in this method, Lubrol PX, is no longer commercially available

Summary

Introduction

In E. coli, the genes for the Fp and Ip subunits and for the membrane anchoring proteins are located in one operon (sdh), which is transcribed in the following sequence: sdhC, sdhD, sdhA (Fp), sdhB (Ip) [6, 7]. The difference in succinate reducibility of cytochrome b in these two reductases, together with the fact that E. coli SdhD bears no sequence similarity with QPs3 of beef mitochondria, raises the possibility that interactions between succinate dehydrogenase and membrane anchoring subunits in these two systems are different. We developed a method to resolve E. coli succinate-ubiquinone reductase into reconstitutively active succinate dehydrogenase and membrane-anchoring protein fraction (the SdhC-SdhD fraction).

Results

Conclusion