Flavodoxin 1 of Azotobacter vinelandii: Characterization and role in electron donation to purified assimilatory nitrate reductase

Abstract

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. Electrospray mass spectrometry gave M(r) 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.