Fumarate Reductase Mutants of Escherichia coli That Lack Covalently Bound Flavin

作者：Michaël Blaut, Katherine Whittaker, A Valdovinos, Brian A.C. Ackrell, Robert P. Gunsalus, Gary Cecchini · 发表于：Journal of Biological Chemistry · 年份：1989 · DOI：10.1016/s0021-9258(18)80039-4 · 被引用次数：106 · 研究领域：Metal-Catalyzed Oxygenation Mechanisms、Vitamin K Research Studies、Pesticide and Herbicide Environmental Studies

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The catalytic domain of fumarate reductase consists of the FrdA subunit, which contains the active site, and a FAD prosthetic group covalently attached to His44, plus the FrdB subunit which contains at least two of the three nonidentical iron-sulfur clusters of the enzyme. To examine the role of covalently bound FAD in enzyme activity and electron transfer during anaerobic cell growth, site-directed mutagenesis was used to alter His44 of the FrdA subunit to a Ser, Cys, or Tyr residue. The resulting mutant enzyme complexes that were synthesized associated normally with the cytoplasmic membrane, but had decreased ability (greater than 70%) to reduce fumarate with reduced benzyl viologen, an artificial electron donor of low redox potential (Em = -359 mV; Clark, W. M. (1972) Oxidation-Reduction Potentials of Organic Systems, Robert E. Kreiger Publishing Co., Melbourne, FL). Even lower activities were measured when the higher potential, natural electron donor menaquinol was used, which, however, correlated with the slower growth rates of the different mutant complexes. In contrast to the normal enzyme, the mutant enzyme complexes were unable to oxidize succinate. Substitution of Arg for His44 produced a totally inactive enzyme complex that permitted no cell growth on nonfermentable su...