Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli

Abstract

Fumarate reductase was purified 100-fold to 95% homogeneity from the cytoplasmic membrane of E. coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic-lipophilic balance (HLB) number near 13 and the enzyme was routinely solubilized with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme, whether membrane bound, in Triton extracts or purified, had an apparent Km near 0.42 mM. Two peptides with MW of 70,000 and 24,000, present in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native MW of 100,000 was calculated for fumarate reductase by Sephacryl S-200 gel filtration in the presence of sodium cholate. This indicates that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.