Conformational properties of membrane-bound fumarate reductase of Escherichia coli

Anaerobically grown cells of Escherichia coli harboring the plasmid pFRD63 overproduce fumarate reductase, a membrane-bound complex localized in the inner membrane of the cell, where this enzyme represents at least 90% of the total membrane proteins (B. D. Lemire, J. J. Robinson, and J. H. Weiner (1982) J. Bacteriol. 152, 1126-1131). Preparations of inner membrane fractions suspended in 40% sucrose are optically clear, allowing optical spectroscopic measurements. Circular dichroism spectra showed that between pH 6 and 11 the secondary structure of the enzyme is at least 55% in α helix and that above pH 11 the structure abruptly changes to a β-like conformation. The same phenomenon is observed in samples solubilized in the nonionic detergent C₁₂E₉. Absorption spectra of the enzyme either membrane bound or solubilized in detergents or exposed to alkaline pH showed that the accessibility of the active site to solvent components is modulated by the interaction of the protein with the membrane. Solubilization of the membrane-bound enzyme with 1% Triton X-100 or C₁₂E₉ produced a decrease in ellipticity and in enzymatic activity.