Purification and characterization of a NADP+/NADPH-specific flavoprotein that is overexpressed in FdI- strains of Azotobacter vinelandii.
Earlier studies have established that mutant strains of Azotobacter vinelandii that do not synthesize ferredoxin I (AvFdI) overexpress another protein designated Protein X (Morgan, T. V., Lundell, P. J., and Burgess, B. K. (1988) J. Biol. Chem. 263, 1370-1375). This protein has now been purified using two-dimensional gel electrophoresis as an assay. The purified protein is a monomer with M(r) approximately 29,000 which degrades slowly to a specific M(r) approximately 22,000 form when stored in solution. The native protein is bright yellow and contains noncovalently attached FAD that is reduced by either dithionite or NADPH without formation of a stable semiquinone. Titration with NADP+/NADPH gives an E0' value of approximately -327 mV versus SHE. Because this E0' is so close to that of the NADP+/NADPH couple it is not clear if Protein X is an NADPH oxidase or an NADP+ reductase in vivo. Comparison of the NH2-terminal sequence and other properties of Protein X with those of other proteins, suggests that it is likely to be related to the Escherichia coli ferredoxin NADP+ reductase (the fpr gene product), and affinity chromatography shows that Protein X binds specifically to AvFdI.
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
J M
JM
Isas
B K
BK
Burgess
D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D005288:Ferredoxins / Q000096:biosynthesis / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005418:Flavodoxin / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000302:isolation & purification*; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry
D005288:Ferredoxins; D005418:Flavodoxin; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D009249:NADP