Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus

Yakunin, Alexander F.; Gennaro, Giuseppa; Hallenbeck, Patrick C.

doi:10.1128/jb.175.21.6775-6780.1993

Cited by 34 publications

(25 citation statements)

References 40 publications

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“…Similar results were obtained with nifH-and rpoNlacZ fusions (carried, respectively, on pPHU266 and pPHU289 [12], pRK replicons). These results confirm and amplify our previously reported immunoblot results (34). The level of expression of the nifF-lacZ fusion was 15 to 65% of the expression level of the nifH-lacZ fusion, whereas the quantity of NifF in wild-type cells is about 100-fold lower than that of NifH.…”

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confidence: 81%

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Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus

et al. 1996

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The Rhodobacter capsulatus nifF gene and upstream sequence were cloned by using a probe based on the N-terminal sequence of NifF. nifF was found to not be contained in the previously described nif regions I, II, and III. Comparison of the deduced amino acid sequence showed that it is highly similar to NifF from Azotobacter vinelandii and NifF from Klebsiella pneumoniae. Analysis of translational fusions demonstrated that the regulation of transcription was the same as previously reported at the protein level. Insertional mutagenesis showed that NifF contributes significantly to nitrogenase activity under normal nitrogen-fixing conditions and that it is absolutely required for nitrogen fixation under iron limitation.

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confidence: 81%

“…4A). Thus, nifF is highly expressed under iron limitation, again agreeing with our previously reported NifF immunoblot data (34).…”

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Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus

et al. 1996

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“…Deletion of the nitrogenase structural genes did not affect DNP photoreduction (Table 1). NifF is a flavodoxin required for electron transfer to nitrogenase under iron deprivation (27). The molecular properties of this protein resemble those of NPR (3), but nifF mutants showed an NPR ϩ phenotype (Table 1).…”

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Role for draTG and rnf Genes in Reduction of 2,4-Dinitrophenol by Rhodobacter capsulatus

et al. 2001

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The phototrophic bacterium Rhodobacter capsulatus is able to reduce 2,4-dinitrophenol (DNP) to 2-amino-4-nitrophenol enzymatically and thus can grow in the presence of this uncoupler. DNP reduction was switched off by glutamine or ammonium, but this short-term regulation did not take place in a draTG deletion mutant. Nevertheless, the target of DraTG does not seem to be the nitrophenol reductase itself since the ammonium shock did not inactivate the enzyme. In addition to this short-term regulation, ammonium or glutamine repressed the DNP reduction system. Mutants of R. capsulatus affected in ntrC or rpoN exhibited a 10-fold decrease in nitroreductase activity in vitro but almost no DNP activity in vivo. In addition, mutants affected in rnfA or rnfC, which are also under NtrC control and encode components involved in electron transfer to nitrogenase, were unable to metabolize DNP. These results indicate that NtrC regulates dinitrophenol reduction in R. capsulatus, either directly or indirectly, by controlling expression of the Rnf proteins. Therefore, the Rnf complex seems to supply electrons for both nitrogen fixation and DNP reduction.The industrial production and abusive use of dyes, explosives, herbicides, pesticides, and drugs result in the release of nitroaromatic compounds into the environment (26). These xenobiotic compounds are resistant to oxygenolytic reactions since the nitroaromatic ring is rendered impervious to electrophilic attack, especially in the case of polynitroaromatics. Therefore, microorganisms have developed reductive pathways that facilitate the metabolism of these recalcitrant compounds. The process may began with reduction of the aromatic ring (5, 18) or with reduction of the nitro group to the corresponding amino or hydroxylamino derivatives that can be assimilated upon release of ammonium and hydroxyaromatic adducts (11,24).Under light and anaerobiosis, Rhodobacter capsulatus cometabolizes the uncoupler 2,4-dinitrophenol (DNP) by reducing it to 2-amino-4-nitrophenol, which is almost stoichiometrically accumulated in the medium (2). The reaction is catalyzed by a cytosolic and homodimeric Flavin mononucleotide-linked, 54-kDa nitrophenol reductase (NPR) (3). Once DNP is consumed, the cells began to grow by fixing the dinitrogen dissolved in the medium.The reduction of DNP in R. capsulatus is repressed by ammonium since the process does not takes place in ammoniumgrown cells in the presence of chloramphenicol (2). To assess if the reduction of DNP is activated in N 2 -fixing cultures, cells cultured as previously described (2) with glutamate (nitrogenase-derepressing conditions), ammonium (negative control), and glutamate plus DNP (positive control) as nitrogen sources were transferred to media with DNP. DNP consumption and NPR activity were determined as published elsewhere (3). As expected, the maximal rate of DNP photoreduction was observed in cells previously cultured with DNP ( Fig. 1), showing a NPR activity of 3.2 Ϯ 0.5 mU mg Ϫ1 . The consumption of DNP by these cells was indepe...

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“…In cyanobacteria, the reductants for nitrogenase are rather provided by a specific [2Fe-2S] ferredoxin (Schrautemeier and Bohme, 1985;Bohme and Haselkorn, 1988). Rkodobacter capsulatus is remarkable for the abundance of electron carriers it contains, including six ferredoxins (Jouanneau et al, 1995a) and one flavodoxin (Yakunin et al, 1993). Ferrodexin I (FdI) encoded by fdxN is supposedly the natural electron carrier to nitrogenase in R. capsulatus (Hallenbeck et al, 1982;Yakunin and Gogotov, 1983;Schatt et al, 1989;Saeki et al, 1991 ;Schmehl et al, 1993;Jouanneau et al, 1995b).…”

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Identification of Residues of Rhodobacter capsulatus Ferredoxin I Important for Its Interaction with Nitrogenase

Naud

Meyer

David

et al. 1996

European Journal of Biochemistry

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In Rhodobacter capsulatus, ferredoxin I (FdI) serves as natural electron donor to nitrogenase. In order to probe amino acid residues possibly involved in the interaction with dinitrogenase reductase, FdI was subjected to site-specific mutagenesis. A three-dimensional structure of Fdl was designed by computer modelling and used for selecting target residues. Mutant ferredoxins bearing substitutions of surface residues, as well as a variant having a Met2-Tyr replacement in the vicinity of one cluster, have been constructed. All FdI variants were expressed to similar levels both in Escherichia coli and in a FdIdeleted mutant of the natural host. Once purified, the mutant ferredoxins exhibited molecular and spectroscopic properties almost identical to wild-type FdI. Determination of the reduction potential of FdI by cyclic voltammetry gave an EA of -510 mV (pH 7.6) for both clusters, which is one of the lowest values reported for a 2[4Fe-4S] ferredoxin. Only the [Tyr2]FdI variant showed a significant difference in redox potential (AE', = -15 mV). Based on in vitro assays, a [Glu27, Glu281FdI double mutant exhibited a twofold decrease in the electron transfer rate to dinitrogenase reductase while the affinity of this mutant for the enzyme was barely affected. On the other hand, an Asp36-.His substitution resulted in a sevenfold increase of the apparent K,,, for dinitrogenase reductase. Unlike FdI and the other mutant ferredoxins, the [His36]FdI variant also failed to form a cross-linked complex with dinitrogenase reductase upon incubation with a carbodiimide. It is concluded that Asp36 in FdI probably participates in the interaction between the two protein partners. Nevertheless, all the FdI mutants proved competent in restoring a wild-type phenotype when expressed in a FdI-deleted mutant background, indicating that none of the studied residues was absolutely critical for electron transfer to nitrogenase.

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Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus

Cited by 34 publications

References 40 publications

Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus

Cloning, characterization, and regulation of nifF from Rhodobacter capsulatus

Role for draTG and rnf Genes in Reduction of 2,4-Dinitrophenol by Rhodobacter capsulatus

Identification of Residues of Rhodobacter capsulatus Ferredoxin I Important for Its Interaction with Nitrogenase

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