title: Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase.
abstract: Flavodoxins synthesized by Azotobacter vinelandii strain UW 36 during growth on nitrate as nitrogen source were separated by FPLC on a Mono Q column into two species, flavodoxin 1 (AvFld 1) and flavodoxin 2 (AvFld 2). Both proteins migrated as single bands on SDS/PAGE. AvFld 1 was approx. 5-fold more abundant than AvFld 2 in the unresolved flavodoxin mixture. N-terminal amino acid analysis showed the sequence of AvFld 2 to correspond to the nif F gene product, an electron donor to nitrogenase. The sequences also show that these species corresponded to the flavodoxins Fld A and Fld B isolated from N2-grown cultures of the closely related organism Azotobacter throococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem.J.277, 313-319]. Electrospray mass spectrometry gave M, values for the polypeptides of 19430 +/- 3 and 19533 +/- 5 respectively. 31P-NMR measurements showed that in addition to the phosphate associated with the FMN (delta = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at delta = -142.1 p.p.m. and AvFld 2 at delta = -138.59 p.p.m. present in substoichiometric amounts with FMN. These appeared to arise from unstable species since they were readily lost on further manipulation of the proteins. The mid-point potentials of the semiquinone hydroquinone redox couples were -330 mV and -493 mV for AvFld 1 and AvFld 2 respectively, but only AvFld 1 was competent in donating electrons to the purified assimilatory nitrate reductase of A. vinelandii to catalyse the reduction of nitrate to nitrite. Flavodoxin isolated from NH4(+)-grown cells (Fld 3) also functioned as electron donor at half the rate of AvFld 1, but ferredoxin 1 from A. chroococcum did not.
authors.affiliation: Nitrogen Fixation Laboratory, John Innes Centre, Colney, Norwich, Norfolk, UK.
authors.forename: R
authors.initials: R
authors.lastname: Gangeswaran
authors.forename: R R
authors.initials: RR
authors.lastname: Eady
mesh_terms: D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology / Q000378:metabolism*; D004579:Electron Transport; D005418:Flavodoxin / Q000378:metabolism*; D006873:Hydroquinones / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D011199:Potentiometry; D017421:Sequence Analysis
chemical_list: D005418:Flavodoxin; D006873:Hydroquinones; D009566:Nitrates; C100447:flavodoxin 1; D009565:Nitrate Reductases; D050901:Nitrate Reductase; C031927:hydroquinone