Characterization of lysyl residues of NADH-cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein.
An expression vector for bovine NADH-cytochrome b5 reductase was constructed from two DNA fragments that were derived from beef liver poly(A+) RNA using the polymerase chain reaction. Site-directed mutagenesis of the 3 lysine residues of the reductase, previously implicated in the formation of active-site charge pairs with carboxylate residues of cytochrome b5, was then used to obtain the purified catalytic domains of flavoproteins modified at each of these sites. The observed marked decreases in catalytic efficiencies of substitutions of a negative charge at the normally positively charged residues with the catalytic domain of cytochrome b5 are consistent with their participation in the formation of charge pairs with carboxylate groups of the hemeprotein to optimize rapid electron transfer from the reductase flavin to the heme of the cytochrome.
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
P
P
Strittmatter
J M
JM
Kittler
J E
JE
Coghill
J
J
Ozols
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D003579:Cytochrome Reductases / Q000235:genetics / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005420:Flavoproteins / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics*; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008239:Lysine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011061:Poly A / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger
D005420:Flavoproteins; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D008239:Lysine