Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis.
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.
Schröder
I
I
Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90024.
Gunsalus
R P
RP
Ackrell
B A
BA
Cochran
B
B
Cecchini
G
G
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010071:Oxaloacetates / Q000378:metabolism; D012395:Rose Bengal / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013385:Succinate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics
D010071:Oxaloacetates; D012395:Rose Bengal; D013385:Succinate Dehydrogenase; D005033:Ethylmaleimide