Molecular properties of fumarate reductase isolated from the cytoplasmic membrane of Escherichia coli.

Fumarate reductase, purified from the cytoplasmic membrane of Escherichia coli, has been cross-linked with the bifunctional reagent dimethylsuberimidate and shown to exist as an alpha beta dimer of polypeptides of molecular weights 69,000 and 25,000 in a 1:1 molar ratio. The protein has an s20,w of 7.67S and a D20,w of 6.5 X 10(-7) cm2/s. The purified enzyme contained 4-5 mol of nonheme iron and 4-5 mol of acid labile sulfur while the visible absorption spectrum showed a broad peak between 400 and 470 nm owing to the presence of an Fe-S centre and 8 alpha[N-3]histidyl FAD. Fumarate reductase activity was readily inhibited by the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoate, and iodoacetamide. Using 5,5'-dithiobis-(2-nitrobenzoic acid) sulfhydryl group modification was followed as a function of enzyme activity. A single cysteine residue was shown to be required for activity and this essential sulfhydryl group was located in the 69,000 dalton subunit. The amino acid composition of E. coli fumarate reductase was similar to the succinate dehydrogenases from beef heart mitochondrion and Rhodospirillum rubrum.