The succinate dehydrogenase of Escherichia coli. Immunochemical resolution and biophysical characterization of a 4-subunit enzyme complex.

C Condon,R Cammack,D S Patil,P Owen

doi:10.1016/s0021-9258(17)39384-5

Abstract

Using EPR spectroscopy to monitor the integrity of the enzyme, conditions have been established which allow specific immunoprecipitation of the succinate dehydrogenase complex of Escherichia coli. The enzyme complex precipitated from Lubrol PX-solubilized membranes by monospecific antiserum in the presence of a cocktail of protease inhibitors contains four polypeptides of apparent MrS 71,000, 26,000, 17,000, and 15,000. The 71-kDa flavopeptide is readily susceptible to proteolysis, and the enzyme complex shows unusual facile dissociation. Spectroscopic measurements indicate the presence of a [2Fe-2S] cluster (Center 1), a [3Fe-xS] cluster (Center 3), and a b-type cytochrome. In addition, a change in relaxation of Center 1 at low potentials is indicative of Center 2. Midpoint redox potentials of Centers 1-3 for both the membrane-bound and detergent-solubilized enzyme were estimated to be +10 mV, -175 mV, and +65 mV, respectively.

Highlights

From the Departmentof Microbiology, Moyne Institute, TrinityCollege, Dublin 2, Ireland and the $Department of Plant Sciences, King’s College, 68 Half Moon Lane, LondonSE24 SJF, England
The enzyme complex precipitated from Lubrol PX-solubilized membranesby monospecific antiserum in the presence of a cocktail of protease inhibitors contains four polypeptides of apparent Mrs 71,000,26,000, 17,000,and 15,000
Immunoprecipitation of succinate dehydrogenase cation we report on the resolution by these methods of a 4subunit succinate dehydrogenase complex from E. coli containing redox Centers 1-3 and a b-type cytochrome

Summary

IMMUNOCHEMICALRESOLUTIONANDBIOPHYSICALCHARACTERIZATIONOF COMPLEX*

From the Departmentof Microbiology, Moyne Institute, TrinityCollege, Dublin 2, Ireland and the $Department of Plant Sciences, King’s College, 68 Half Moon Lane, LondonSE24 SJF, England. For the mitochondrial enzyme, the presence of EPR signals arising from the normally labile Center 3 membrane preparation (envelopes, Kaback vesicles, or isolated plasma membranes), the temperature (0-37 "C), and pH (pH 6.8 to pH 8.6) of extraction andprecipitation, or modification of procedures for washing (omissionof detergent, protease inhibitors, or sonication) and processing (solubilization in SDS a t 40, 60, or 100 "C) of immucorrelates with reconstitutive ability (2, 3 ). Caused rapid inactivation and precipitation of approximately 50% of succinate dehydrogenase activity on incubation with detergent ex-

RESULTS

Reducing agent

Findings

DISCUSSION