Abstract
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 enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes. Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents. Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase. In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function. A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids. Evidence is also presented that suggests a second arginine residue may form part of the active site. Potential catalytic and substrate-binding roles for arginine are discussed. The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst. The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232. The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.
Highlights
From the Departmentof Microbiology and Molecular Genetics and the Molecular Biology Institute, University of California, Los Angeles, California 90024
Menaquinol-fumarate oxidoreductaseof Escherichia electron acceptor [1].The enzymecomplex is composed of coli is a four-subunit membrane-bound complex that four nonidentical peptides forminga membrane-extrinsic catcatalyzes the final step in anaerobic respiration whenalytic domain anda membrane-intrinsic hydrophobic domain
Centers 1 and 3 have the present study, a combination of site-directed mu- been localized to the FrdB subunit whereas Cente2r requires tagenesis and chemical modification techniques have the presenceof the FrdA peptide, and tmhuays be coordinated been used to investigate the role(s) of the conserved by amino acid residue(s) from both FrdA and FrdB [7, 8]
Summary
The conserved amino acids His-232, Cys-247, and Arg-248 of FrdA that are likely to be part of the active site of the enzyme [16,17,18,19,20,21,22,23,24,25,26,27] were chosen as targets for site-directed mutagenesis to determine threole of each aminoacid in catalytic kan insertion.' Phage Ml3KWIwas constructed by inserting the2.0- activity. This fragment contains1124 bp of the 3' region of frdA. Site-directedMutagenesis-Site-directed mutagenesis was performed as previously described [8,33]. Substitution of Arg-248 of FrdA perazineethanesulfonic acid; kb, kilobase(s)
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