Flavodoxins synthesized by Azotobacter vinelandii strain UW 136 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 chroococcum [Bagby, Barker, Hill, Eady and Thorneley (1991) Biochem. J. 277, 313–319]. Electrospray mass spectrometry gave Mr 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 (Δ = -136.3 p.p.m. and -135.48 p.p.m.), AvFld 1 had a signal at Δ = -142.1 p.p.m. and AvFld 2 at Δ = -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.
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July 1996
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Research Article|
July 01 1996
Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase
Rathi GANGESWARAN;
Rathi GANGESWARAN
*
1The Nitrogen Fixation Laboratory, John Innes Centre, Colney, Norwich NR4 7HU, Norfolk, U.K.
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Robert R. EADY
Robert R. EADY
†
2The Nitrogen Fixation Laboratory, John Innes Centre, Colney, Norwich NR4 7HU, Norfolk, U.K.
†To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
February 27 1996
Accepted:
March 05 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (1): 103–108.
Article history
Received:
February 27 1996
Accepted:
March 05 1996
Citation
Rathi GANGESWARAN, Robert R. EADY; Flavodoxin 1 of Azotobacter vinelandii: characterization and role in electron donation to purified assimilatory nitrate reductase. Biochem J 1 July 1996; 317 (1): 103–108. doi: https://doi.org/10.1042/bj3170103
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