Expression and characterization of the heme-binding domain of Chlorella nitrate reductase.
A recombinant protein corresponding to the putative heme-binding domain of assimilatory NADH:nitrate reductase from Chlorella vulgaris has been expressed and purified from transformed Escherichia coli BL21 cells. The recombinant protein, exhibited a subunit molecular mass of approximately 10 kDa with a N-terminal sequence beginning with the residues PAGA in agreement with that predicted by cDNA analysis. The UV-visible spectrum of the protein confirmed the incorporation of heme with maxima at 413 nm and 423, 528, and 557 nm for the oxidized and reduced forms, respectively. Circular dichroism spectra indicated the environment of the heme chromophore was very similar to that of the native enzyme. Potentiometric titrations of the recombinant heme domain yielded a midpoint potential of +16 mV (n = 1, pH 7), substantially higher than the values of -160 mV obtained for the native enzyme and -28 mV obtained for a previously expressed recombinant heme domain that contained part of the Mo-pterin domain. These results indicate that portions of the amino acid sequence that are involved in the formation of the Mo-pterin domain of Chlorella nitrate reductase influence the redox potential of the heme prosthetic group.
Cannons
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AC
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612.
Barber
M J
MJ
Solomonson
L P
LP
D001483:Base Sequence; D001665:Binding Sites; D002708:Chlorella / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011199:Potentiometry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013053:Spectrophotometry
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006418:Heme; D009565:Nitrate Reductases; D050901:Nitrate Reductase