Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii

Chisnell, John R.; Premakumar, R.; Bishop, Paul E.

doi:10.1128/jb.170.1.27-33.1988

Cited by 264 publications

(212 citation statements)

References 32 publications

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“…In a preceding publication we described the alternatibc nitrogenase from R. capsulatus, in analogy to the 'nitrogenase-3' from A. vinelmdii [4], as an instable and weakly active protein [7]. The component f of this enzyme was therefore found to be EPR-silent and in the form isolated generally unsuitable for physical studies.…”

Section: Resultsmentioning

confidence: 99%

“…It was like a paradigm change [I] when it became evident that three genetically distinct nitrogenase systems exist in bacteria 12-71, the 'classical' Mo-containing nitrogenase [8,9], a vanadium-containing one [2,3], and an 'iron only' nitrogenase [4,5,7] lacking both MO and V. 'Iron only' nitrogenases have so far been isolated and biochemically characterized only in the case of AzotobacIer vineiattdii [4] and Rhodobacter capsularus 171.…”

Section: Introductionmentioning

confidence: 99%

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EPR spectroscopic characterization of an ‘iron only⌉ nitrogenase S = 3/2 spectrum of component 1 isolated from Rhodobacter capsulatus

et al. 1992

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The alternative nitrogenase of Rhdobacrer cqmdms, isolated from a nrHDK deletion mutant, has been purified to near homogeneity and identified as an 'iron only' nitrogenasc. The dithionitc-reduced componenr 1 ('FcFe protein') of this enzyme showed an EPR spectrum consisting of two components: a minor S = l/2 signal at g = I,93 and a very characteristic S = 312 signal of near-stoichiometric intensity at g = 5.44. I'his resonance is very close to the highest possibk g value (g = 5.46) for the coinciding two intradoublet subspectra of an S = 3/2 system of maximal rhombicity (E/D = 0.33). The deviation from axial symmetry (increasing E/D) correlates with the stability. activity and substrate selectivity of the different (MO, V. Fe) nitrogenases.Alternative nitrogenase; FeFe protein; Hetcromctal; Nitrogcnase cofactor; S = 312 EPR spectrum; Rkodobnctrr utps~rla~~~s 1, INTRODUCTIONIt was like a paradigm change [I] when it became evident that three genetically distinct nitrogenase systems exist in bacteria 12-71, the 'classical' Mo-containing nitrogenase [8,9], a vanadium-containing one [2,3], and an 'iron only' nitrogenase [4,5,7] lacking both MO and V. 'Iron only' nitrogenases have so far been isolated and biochemically characterized only in the case of AzotobacIer vineiattdii [4] and Rhodobacter capsularus 171.From the bioinorganic point of view it is of great importance to elucidate the influence of the heterometal center M (MO, V, (Fe)) on the structure of the related protein and/or the corresponding metal cluster (FeMco with the composition Fe,MS, which may be present in different redox states [l Jj, on its electronic structure and especially on the selectivity for substrate reduction, The existence of nitrogenases with diffcren t heterometals within the catalytically active cluster center provides thus the opportunity for a better understanding of the Nz fixation mechanism by comparative biochemical and biophysical studies. Specific structural and spectroscopic properties by which the V-and the Fe-nitrogenase can be differentiated from each other are lacking until now. For the V-nitrogenase, isolated from. A. chroococcur~z [2] and A. sineiandii [3,10,1 I] an S = 312 EPR signal, assigned to the protein-bound cofactor, has The 'FeFe protein' is component 1 of the Fe-nitrogenase from a tzifHDK deletion mutant of R. capsulatus which is unable to synthesize the proteins of the conventional Mo-nitrogenase. In this note, we report an EPR spectrum of the 'FeFe protein' and correlate this spectrum with those of 12,13). Some comparable properties of the three nitrogenase systems including catalytical aspects are discussed, too. MATERIALS AND METHODS Bucrerial srruitz urld growth rondiriortsThe organism used in this study was a n@fDK-strain generated from A. capsukutus BlOS [7]+ The cells were cultivated in 500-mlvacuum bottles under anaerobic, phototrophic conditions with scrine (5 mM) as N-source and lactate (24 mM) as C-sourcess described [7]. The concemrations of some mineral salts in the nutrient solution were decr...

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

EPR spectroscopic characterization of an ‘iron only⌉ nitrogenase S = 3/2 spectrum of component 1 isolated from Rhodobacter capsulatus

et al. 1992

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“…As a second/third option for diazotrophs, it shares many genes with the conventional MoFe nitrogenase system, and expression of the FeFe nitrogenase is completely inhibited by traces of Mo or V (32,41). Gene redundancy in A. vinelandii and the complexities of metal-dependent regulation in this obligate aerobic diazotroph make analysis of the FeFe system difficult.…”

Section: Discussionmentioning

confidence: 99%

“…The alternative FeFe nitrogenase was initially identified in A. vinelandii grown under combined Mo and V limitation about 25 y ago (41). As a second/third option for diazotrophs, it shares many genes with the conventional MoFe nitrogenase system, and expression of the FeFe nitrogenase is completely inhibited by traces of Mo or V (32,41).…”

Section: Discussionmentioning

confidence: 99%

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Yang

Xie

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium-or irondependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

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“…This allows the independent expression of dinitrogenase reductase 2 (vnfH gene product) and dinitrogenase 2 (vnfDGK products). Unlike dinitrogenase 2, dinitrogenase reductase 2 is present not only under diazotrophic conditions in the presence of V but also in the absence of Mo and V, conditions where nitrogenase 3 is present (3,8,25). Dinitrogenase reductase 2 is unlikely to function in a catalytic role under Mo-and V-deficient conditions because purified dinitrogenase reductase 2 does not effectively complement dinitrogenase 3 in in vitro assays (8).…”

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confidence: 99%

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

1991

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Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK). However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the Vcontaining alternative nitrogenase. Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium. In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo. Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo-and V-deficient medium. In this medium, NifH-VnfH-strains containing an anfH--lacZ transcriptional fusion exhibited less than 3% of the ,B-galactosidase activity observed in the wild type with the same fusion. P-Galactosidase activity expressed by VnfHmutants containing the anJH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the samne fusion. Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in Vnft-mutants, dinitrogenase reductase 1 appears to serve this function. Active dinitrogenase reductase 1 or 2 is probably required for this function since a nijM deletion mutant containing the anffl-lacZ fusion was unable to synthesize ,-galactosidase above background levels. An anfA deletion strain containing the anpfH-lacZ fusion exhibited ,B-galactosidase activity at 16% of that of the wild type containing the same fusion. However, in the presence of NH4', the ,B-galactosidase activity expressed by this strain more than doubled. This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4'-and, to a lesser extent, Mo-mediated repression of this transcription.The regulation of the expression of the three nitrogenases in Azotobacter vinelandii is responsive to the presence or absence of ammonium (NH4'), molybdenum (Mo), and vanadium (V) in the culture medium. The synthesis of all three nitrogenases is repressed by NH4'. Nitrogenase 1 is found in cells grown in the presence of Mo and nitrogenase 2 is expressed in the presence of V but in the absence of Mo, whereas nitrogenase 3 is synthesized only in the absence of both Mo and V (4, 9, 13, and references therein). Our knowledge of the molecular basis for nitrogen and metal regulation in A. vinelandii is still rudimentary. The regulatory genes nifA, vnfA, and anfA have been identified, and some of their functions have been described on the basis of the phenotypes of NifA-, VnfA-, and AnfA-mutants (1, 14). The nifA gene product is required for transcription of the structural genes for nitrogenase 1 (1). NifA binds to an upstream activator sequence (6) or indirectly represses the synthesis of nitrogenase 1 in cells grown in Mo-deficient me...

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Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii

Cited by 264 publications

References 32 publications

EPR spectroscopic characterization of an ‘iron only⌉ nitrogenase S = 3/2 spectrum of component 1 isolated from Rhodobacter capsulatus

EPR spectroscopic characterization of an ‘iron only⌉ nitrogenase S = 3/2 spectrum of component 1 isolated from Rhodobacter capsulatus

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii

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