Aerobic inactivation of fumarate reductase from Escherichia coli by mutation of the [3Fe-4S]-quinone binding domain

Abstract

Fumarate reductase from Escherichia coli functions both as an anaerobic fumarate reductase and as an aerobic succinate dehydrogenase. A site-directed mutation of E. coli fumarate reductase in which FrdB Pro-159 was replaced with a glutamine or histidine residue was constructed and overexpressed in a strain of E. coli lacking a functional copy of the fumarate reductase or succinate dehydrogenase complex. The consequences of these mutations on bacterial growth, assembly of the enzyme complex, and enzymatic activity were investigated. Both mutations were found to have no effect on anaerobic bacterial growth or on the ability of the enzyme to reduce fumarate compared with the wild-type enzyme. The FrdB Pro-159-to-histidine substitution was normal in its ability to oxidize succinate. In contrast, however, the FrdB Pro-159-to-Gln substitution was found to inhibit aerobic growth of E. coli under conditions requiring a functional succinate dehydrogenase, and furthermore, the aerobic activity of the enzyme was severely inhibited upon incubation in the presence of its substrate, succinate. This inactivation could be prevented by incubating the mutant enzyme complex in an anaerobic environment, separating the catalytic subunits of the fumarate reductase complex from their membrane anchors, or blocking the transfer of electrons from the enzyme to quinones. The results of these studies suggest that the succinate-induced inactivation occurs by the production of hydroxyl radicals generated by a Fenton-type reaction following introduction of this mutation into the [3Fe-4S] binding domain. Additional evidence shows that the substrate-induced inactivation requires quinones, which are the membrane-bound electron acceptors and donors for the succinate dehydrogenase and fumarate reductase activities. These data suggest that the [3Fe-4S] cluster is intimately associated with one of the quinone binding sites found in fumarate reductase and succinate dehydrogenase.During anaerobic respiration in Escherichia coli, the fumarate reductase (FRD) complex catalyzes the fumarate-dependent oxidation of menaquinol, thereby allowing production of a transmembrane proton gradient that can be used by the organism to support metabolism (19). The FRD complex is composed of four nonidentical subunits, FrdA, FrdB, FrdC, and FrdD, arranged into two domains: the FrdAB catalytic domain and the FrdCD membrane anchor domain (2, 22). FrdA (66 kDa) contains covalently bound flavin adenine dinucleotide and the active site of the enzyme (38), and the 27-kDa FrdB subunit contains the three distinct iron-sulfur centers present in the enzyme (23). The iron-sulfur clusters, center 1 ([2Fe-2S] ), are similar in structure and function in all membranebound succinate dehydrogenases (SDHs) and FRDs examined to date (2). The FrdC (15 kDa) and FrdD (13 kDa) subunits are essential for attachment of the catalytic subunits to the inner surface of the cytoplasmic membrane (22) and are also essential for electron transfer reactions involving menaquinol and ubiquinone (8).The p...

Characterization by electron paramagnetic resonance of the role of the Escherichia coli nitrate reductase (NarGHI) iron-sulfur clusters in electron transfer to nitrate and identification of a semiquinone radical intermediate

“…This suggests that there may be a quinol binding site (at which a semiquinol radical can be stabilized) between the hemes of NarI and the [Fe-S] clusters of NarH. It has recently been shown that in E. coli fumarate reductase, the [3Fe-4S] cluster is closely or functionally associated with a menaquinol binding site (13). In E. coli dimethyl sulfoxide reductase (DmsABC), mutants in which one of the four [4Fe-4S] clusters of the wild-type enzyme is converted to a [3Fe-4S] cluster have been generated (38).…”

Interaction of a menaquinol binding site with the [3Fe‐4S] cluster of Escherichia coli fumarate reductase

“…Likewise, the microwave power dependencies of the HOQNOposed to be associated with it [26], we recorded EPR spectra at 12 K of oxidized membranes enriched in wild-type FrdABCD treated and untreated membranes are essentially identical (Fig. 2 b).…”

“…6 shows HOQNO-binding DISCUSSION titrations for membranes from the three strains used here at a It has been previously shown that the aerobic inactivation of protein concentration of 0.32 mg ml Ϫ1 . Both the background FrdA[Gln159]BCD is almost eliminated by the MKH 2 analog membranes (HB101/pBR322) and membranes containing HOQNO [26]. The Pro159→Gln mutation is within the FR3 [Arg82]FrdC (HB101/pFRD300) bind negligible concentrations [3Fe-4S] cluster binding motif of FrdB [11,13]; this prompted of HOQNO compared to the membranes containing overexus to test if there is a conformational link between FR3 and the pressed wild-type FrdABCD (HB101/pFRD84), indicating that proposed dissociable Q B site of the enzyme.…”

“…hypothesis that MKH 2 binding at a dissociable site takes place in close juxtaposition to FR3 [26], and that His82 of FrdC has Calculation of the number of HOQNO-binding sites in FrdABCD. The lack of detectable HOQNO binding in the back-an important role in this binding site [14,15,22].…”

A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: genetic and biophysical characterization

“…The tetranuclear center 2 and trinuclear center 3 are supposed to be ligated by cysteine residues arranged in two clusters near the C terminus. Very likely, the trinuclear center 3 is directly involved in electron transfer to quinones, which was demonstrated by site-directed mutagenesis studies of E. coli QFR and Bacillus subtilis SQR (10,20,37).…”