Reconstitution of quinone reduction and characterization of Escherichia coli fumarate reductase activity.
Resolution of the fumarate reductase complex (ABCD) of Escherichia coli into reconstitutively active enzyme (AB) and a detergent preparation containing peptides C and D resulted in loss of quinone reductase activity, but the phenazine methosulfate or fumarate reductase activity of the enzyme was unaffected. An essential role for peptides C and D in quinone reduction was confirmed by restoration of this activity on recombination of the respective preparations. Neither peptide C nor peptide D by itself proved capable of permitting quinone reduction and membrane binding by the enzyme when E. coli cells were transformed with plasmids coding for the enzyme and the particular peptides. Transformation of a plasmid coding for all subunits resulted in a 30-fold increase in membrane-bound complex, which exhibited, however, turnover numbers for succinate oxidation and fumarate reduction that were intermediate between the high values characteristic of chromosomally produced complex and the relatively low values found for the isolated complex. It is also shown that preparations of the isolated complex and membrane-bound form of the enzyme, as obtained from anaerobically grown cells, are in the deactivated state owing to the presence of tightly bound oxalacetate and thus must be activated prior to assay.
D000332:Aerobiosis; D001426:Bacterial Proteins / Q000378:metabolism* / Q000502:physiology*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism*; D010071:Oxaloacetates / Q000378:metabolism; D010957:Plasmids; D055503:Protein Multimerization; D011808:Quinone Reductases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013385:Succinate Dehydrogenase / Q000378:metabolism*; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid
D001426:Bacterial Proteins; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D010071:Oxaloacetates; D011994:Recombinant Proteins; D013386:Succinates; D019802:Succinic Acid; C033727:fumarate reductase complex; D013385:Succinate Dehydrogenase; D011808:Quinone Reductases