The terminal electron-transfer enzyme fumarate reductase ofEscherichia coli is a complex iron-sulfur flavoenzyme composed of four nonidentical subunits organized into two domains: FrdA and -B (a membrane-extrinsic catalytic domain) and FrdC and -D (a transmembrane anchor domain). We have identified a mutation within the membrane-intrinsic domain that alters the electron transfer properties of the iron-sulfur and flavin redox centers of the catalytic domain. Functional electron flow from the quinone analog 2,3-dimethyl-1,4-naphthoquinone or from the electron transport chain is impaired. However, the mutant enzyme can be reduced normally by single-electron donors such as the dye benzyl viologen.The mutant phenotype results from a single A -* G transition changing His-82, within the second transmembrane a-helix of the FrdC anchor sequence, to an arginine. The mutation, physically located within the anchor domain, is manifested by altered catalytic properties, indicating that the intrinsic and extrinsic domains are conformationally connected. These resuilts confirm the important role of the anchor subunits in functional electron transport and have implications for communication between intrinsic and extrinsic domains of membrane proteins.Fumarate reductase is a complex, membrane-bound iron-sulfur flavoenzyme that serves as the terminal electrontransfer enzyme when Escherichia coli is grown anaerobically with fumarate as the electron acceptor (1). The enzyme is composed of two distinct domains: a membrane-extrinsic catalytic domain comprised of the FrdA and -B polypeptides of 69 and 27 kDa, respectively, and a membrane-intrinsic domain consisting of the FrdC and -D subunits of 15 and 13 kDa, respectively (2). Two forms of the enzyme can be isolated: a tetrameric holoenzyme, composed of equimolar amounts of each subunit, and a catalytic dimer, composed of the FrdA and -B subunits (3, 4). By analogy with the E. coli succinate dehydrogenase, the active site of fumarate reductase is located in FrdA, the flavin-containing subunit (5). The iron-sulfur centers are located in the FrdB subunit (6) and the FrdC and -D polypeptides do not contain any known redox centers. The FrdC and -D polypeptides not only anchor the catalytic subunits to the membrane surface (4) but also induce an optimal conformation in the catalytic dimer as reflected by increased stability and modulated turnover ofthe holoenzyme (7). The enzyme is believed to accept reducing equivalents (electrons) from a reduced b-type cytochrome buried within the membrane. However, the pathways for electrons within the electron transport chain and within the enzyme itself remain to be firmly established.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.Previously characterized mutants ofthefrd operon have been localized to thefrdA gene (8). We now report the isolation and analysis of a mutant within the Fr...
Recombinant plasmids which carried portions of the Escherichia coli frd operon were constructed and their expression examined by in vivo complementation of E. coli MI1443. This strain lacked a chromosomal frd operon and was unable to grow anaerobically on glycerol and fumarate. Introduction of all four fumarate reductase subunits into E. coli MI1443 was essential for the restoration of growth. The FRD A, FRD B dimer (but neither subunit alone) was active in the benzyl viologen oxidase assay. Both FRD C and FRD D were required for membrane association of fumarate reductase and for the oxidation of reduced quinone analogues. Introduction into E. coli MI1443 of the frdABC and frdD genes on two separate plasmid vectors failed to restore anaerobic growth on glycerol and fumarate. Thus separation of the DNA coding for the FRD C and FRD D proteins affected the ability of fumarate reductase to assemble into a functional complex.
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