Construction and Characterization of Hybrid Component 1 from V-Nitrogenase Containing FeMo Cofactor

Moore, Virginia G.; Tittsworth, R. C.; Hales, Brian J.

doi:10.1021/ja00105a080

Cited by 33 publications

(47 citation statements)

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“…Conflicting results have been reported earlier on whether a FeMoco-containing Av1 V hybrid (in contrast to the FeVcocontaining Av1 V ) is active in terms of N 2 reduction (23,50). Our data clearly show that such a hybrid is indeed able to reduce N 2 and other tested substrates (Table 3).…”

Section: Discussioncontrasting

confidence: 92%

Nitrogenase reactivity with P-cluster variants

Corbett

Fay

et al. 2005

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

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Nitrogenase is a multicomponent metalloenzyme that catalyzes the conversion of atmospheric dinitrogen to ammonia. For decades, it has been generally believed that the [8Fe-7S] P-cluster of nitrogenase component 1 is indispensable for nitrogenase activity. In this study, we identified two catalytically active P-cluster variants by activity assays, metal analysis, and EPR spectroscopic studies. Further, we showed that both P-cluster variants resemble [4Fe-4S]-like centers based on x-ray absorption spectroscopic experiments. We believe that our findings challenge the dogma that the standard P-cluster is the only cluster species capable of supporting substrate reduction at the FeMo cofactor and provide important insights into the general mechanism of nitrogenase catalysis and assembly.MoFe protein ͉ VFe protein N itrogenase catalyzes one of the most remarkable chemical transformations in biological systems, the reduction of atmospheric dinitrogen to a bioavailable form, ammonia. Three classes of nitrogenase systems, namely, the Mo-, V-, and Fe-only nitrogenases, have been identified (for recent reviews see refs. 1-11). f Although encoded by different structural genes, all three nitrogenases are comprised of two essential component metalloproteins, component 1 (MoFe, VFe, or FeFe protein) and component 2 (Fe protein). g The homodimeric Fe protein of the Mo-nitrogenase has one [4Fe-4S] cluster bridged between the two subunits, whereas the ␣ 2 ␤ 2 -tetrameric MoFe protein contains two unique metal clusters per ␣␤-subunit: the [8Fe-7S] P-cluster (14), which is located at the ␣␤-interface, and the [Mo-7Fe-9S-X-homocitrate] h FeMo cofactor (FeMoco), which is situated within the ␣-subunit. ATP-dependent electron transfer is believed to proceed from the [4Fe-4S] cluster of the Fe protein to the P-cluster of the MoFe protein and finally to FeMoco where substrate reduction takes place. The Fe protein of the V-nitrogenase shares a high degree of homology with the Fe protein of the Mo-nitrogenase and, apart from the presence of an additional ␥-subunit, the VFe protein is also believed to be highly homologous to the MoFe protein with respect not only to the structural genes encoding the ␣-and ␤-subunits, but also to the redox centers contained within the protein, designated the P-cluster and the FeV cofactor (FeVco), respectively, in this case (4, 10, 16). The structurally homologous heterometal centers, i.e., FeMoco and FeVco, are also categorically termed cofactors.It is generally believed that the structural integrity of the P-cluster is indispensable for nitrogenase reactivity. However, the exact mechanism by which the P-cluster carries out its function in substrate reduction and, in particular, the oxidation states and structural conformations of the P-cluster involved in this process, remain largely a puzzle (17). Two types of cofactordeficient MoFe or VFe protein variants, generated by nifH or nifB deletion, i respectively, have been used to study the features of P-clusters without the interference of the cofactor centers...

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Section: Discussioncontrasting

confidence: 92%

Nitrogenase reactivity with P-cluster variants

Corbett

Fay

et al. 2005

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

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“…[9–10] A species with a rhombic EPR spectrum ( g -values of 2.09, 1.98, and 1.93) called lo-CO forms under a much lower [CO] [11] and is proposed to contain only one bound CO. [9–10] A third species and EPR signal, both called hi(5)-CO, have also been identified. [11c, 11d] Not all of these EPR signals are generated when variant Mo N 2 ases are turned over under CO, [12] and the integrated EPR intensities rarely exceed 40% of the active site species. [11d] Presumably, EPR-silent species with bound CO must co-exist with the EPR-active species.…”

Section: Introductionmentioning

confidence: 99%

IR‐Monitored Photolysis of CO‐Inhibited Nitrogenase: A Major EPR‐Silent Species with Coupled Terminal CO Ligands

Yan

Pelmenschikov

Dapper

et al. 2012

Chemistry A European J

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We have used Fourier transform infrared spectroscopy (FT-IR) to observe the photolysis and recombination of a novel EPR-silent CO-inhibited form of α-H195Q nitrogenase from Azotobacter vinelandii. Photolysis at 4 K yields a strong negative IR difference band at 1938 cm−1, along with a weaker negative feature at 1911 cm−1. These bands and the associated chemical species have both been assigned the label ‘Hi-3’. A positive band at 1921 cm−1 is assigned to the ‘Lo-3’ photoproduct. By using an isotopic mixture of 12C16O and 13C18O, we show that the Hi-3 bands arise from coupling of two similar CO oscillators with one uncoupled frequency at ~1917 cm−1. Although in previous studies Lo-3 was not observed to recombine, by extending the observation range to 200–240 K we found that recombination to Hi-3 does indeed occur, with an activation energy of ~6.5 kJ mol−1. The frequencies of the Hi-3 bands suggest terminal CO ligation. We tested this hypothesis with DFT calculations on models with terminal CO ligands on Fe2 and Fe6 of the FeMo-cofactor. An S = 0 model with both CO ligands in exo positions predicts symmetric and asymmetric stretches at 1938 and 1909 cm−1 respectively, with relative band intensities of ~3.5:1, in good agreement with experiment. From the observed IR intensities, we find that Hi-3 is present at a concentration about equal to that of the EPR-active Hi-1 species. The relevance of Hi-3 to the nitrogenase catalytic mechanism and its recently discovered Fischer-Tropsch chemistry is discussed.

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“…Therefore, A. vinelandii BG54 cells must have accumulated V nitrogenase with FeMo cofactor inserted in the presence of molybdenum. A hybrid A. vinelandii VFe protein containing FeMo cofactor has been reconstituted in vitro [31]. This VFe (FeMo co) protein had a mixed EPR signal, which combined both MoFe protein and VFe protein signals and had similar electron allocation to the MoFe protein in nitrogen reduction and to the VFe protein in C 2 H 2 reduction [31].…”

Section: Resultsmentioning

confidence: 99%

“…A hybrid A. vinelandii VFe protein containing FeMo cofactor has been reconstituted in vitro [31]. This VFe (FeMo co) protein had a mixed EPR signal, which combined both MoFe protein and VFe protein signals and had similar electron allocation to the MoFe protein in nitrogen reduction and to the VFe protein in C 2 H 2 reduction [31]. However, this V nitrogenase containing FeMo cofactor was not as efficient in nitrogen fixation as the Mo nitrogenase in vitro.…”

Section: Resultsmentioning

confidence: 99%

“…The sequence comparisons of the products of the structural genes show that amino acid residues that serve as ligands for FeMo cofactor in the MoFe protein are conserved in all three nitrogenases [22]. Moreover, a new species of V nitrogenase containing FeMo cofactor was reconstituted in vitro and found to be functional [31]. Here we report the isolation and characterization of an A. vinelandii mutant which supports diazotrophic growth due to the synthesis of V nitrogenase in the presence of Mo.…”

Section: Introductionmentioning

confidence: 89%

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Activation of vanadium nitrogenase expression in Azotobacter vinelandii DJ54 revertant in the presence of molybdenum

2000

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Azotobacter vinelandii carries three different and genetically distinct nitrogenase systems on its chromosome. Expression of all three nitrogenases is repressed by high concentrations of fixed nitrogen. Expression of individual nitrogenase systems is under the control of specific metal availability. We have isolated a novel type of A. vinelandii DJ54 revertant, designated A. vinelandii BG54, which carries a defined deletion in the nifH gene and is capable of diazotrophic growth in the presence of molybdenum. Inactivation of nifDK has no effect on growth of this mutant strain in nitrogen-free medium suggesting that products of the nif system are not involved in supporting diazotrophic growth of A. vinelandii BG54. Similar to the wild type, A. vinelandii BG54 is also sensitive to 1 mM tungsten. Tn5-B21 mutagenesis to inactivate the genes specific to individual systems revealed that the structural genes for vnf nitrogenase are required for diazotrophic growth of A. vinelandii BG54. Analysis of promoter activity of different nif systems revealed that the vnf promoter is activated in A. vinelandii BG54 in the presence of molybdenum. Based on these data we conclude that A. vinelandii BG54 strain utilizes vnf nitrogenase proteins to support its diazotrophic growth. ß

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Construction and Characterization of Hybrid Component 1 from V-Nitrogenase Containing FeMo Cofactor

Cited by 33 publications

References 0 publications

Nitrogenase reactivity with P-cluster variants

Nitrogenase reactivity with P-cluster variants

IR‐Monitored Photolysis of CO‐Inhibited Nitrogenase: A Major EPR‐Silent Species with Coupled Terminal CO Ligands

Activation of vanadium nitrogenase expression in Azotobacter vinelandii DJ54 revertant in the presence of molybdenum

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