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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.

SUBMITTER: Gangeswaran R

PROVIDER: S-EPMC1217449 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Project description:Structural, energetic, and dynamical studies of Azotobacter vinelandii ferredoxin I are presented for native and mutant forms. The protein contains two iron-sulfur clusters, one of which ([3Fe-4S]) is believed to play a central role in the electron-coupled proton transfer. Different charge sets for the [3Fe-4S] cluster in its reduced and oxidized state are calculated with broken symmetry ab initio density functional theory methods and used in molecular dynamics (MD) simulations. The validity of the ab initio calculations is assessed by comparing partially optimized structures of the [3Fe-4S] clusters with x-ray structures. Possible proton transfer pathways between the protein and the iron-sulfur cluster are examined by both MD simulations and ab initio calculations. The MD simulations identify three main-chain hydrogen atoms--HN(13), HN(14), and HN(16)--that are within H-bonding distance of the [3Fe-4S] cluster throughout the MD simulations. They could thus play a role in the proton transfer from the protein to the iron-sulfur cluster. By contrast, the HD2(15) atom of the Asp-15 is seldom close enough to the [3Fe-4S] cluster to transfer a proton. Poisson-Boltzmann calculations indicate that there is a low, but nonzero probability, that Asp-15 is protonated at pH 7; this is a requirement for it to serve as a proton donor. Ab initio calculations with a fragment model for the protein find similar behavior for the transfer of a proton from the OH of the protonated side chain and the main-chain NH of Asp-15. The existence of a stable salt bridge between Asp-15 and Lys-84 in the D15E mutant, versus its absence in the wild-type, has been suggested as the cause of the difference in the rate of proton transfer. Extensive MD simulations were done to test this idea; the results do not support the proposal. The present findings, together with the available data, serve as the basis for an alternative proposal for the mechanism of the coupled electron-proton transfer reaction in ferredoxin I.

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

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