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

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Abstract
Fumarate reductase, purified from the cytoplasmic membrane of E. coli, was cross-linked with the bifunctional reagent dimethylsuberimidate and shown to exist as an .alpha..beta. dimer of polypeptides of MW 69,000 and 25,000 in a 1:1 molar ratio. The protein has an s20,w [sedimentation coefficient] of 7.67S and a D20,w [diffusion coefficient] of 6.5 .times. 10-7 cm2/s. The purified enzyme contained 4-5 mol of nonheme Fe and 4-5 mol of acid labile S, while the visible absorption spectrum showed a broad peak between 400-470 nm owing to the presence of an Fe-S center and 8.alpha.[N-3]histidyl FAD. Fumarate reductase activity was readily inhibited by the SH reagents 5,5''-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoate and iodoacetamide. Using 5,5''-dithiobis-(2-nitrobenzoic acid), SH group modification was followed as a function of enzyme activity. A single cysteine residue was required for activity, and this essential SH 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.