Isolation of Two Forms of the Nitrogenase VFe Protein from <i>Azotobacter vinelandii</i>

Blanchard, Carol Z.; Hales, Brian J.

doi:10.1021/bi951429j

Cited by 59 publications

(92 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…No Mo (<0.01 mol Mo per mol protein) was detected in this preparation. The V:Fe ratio also indicates that there was no loss of metal-containing subunits during purification as previously reported (30). A limited amount of VnfH (specific activity of 1000 nmol H 2 .…”

Section: Experimental Procedures Cell Growth and Protein Purificationsupporting

confidence: 78%

Azotobacter vinelandiiVanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

2006

View full text Add to dashboard Cite

The nitrogenase-catalyzed reduction of both cyanide and azide results in the production of "excess NH 3 ", which is an amount of NH 3 over and above that expected to be formed from the wellrecognized reactions. Several suggestions have been made as to the possible sources of "excess NH 3 ", but previous attempts to characterize these reactions have met with either limited (or no) success or controversy. Because V-nitrogenase has a propensity to release partially reduced intermediates, e.g., N 2 H 4 during N 2 reduction, it was selected to probe the reduction of cyanide and azide. Sensitive assay procedures were developed and employed to monitor the production of either HCHO or CH 3 OH (its further two-electron-reduced product) from HCN. Like Monitrogenase, V-nitrogenase suffered electron-flux inhibition by CN − (but was much less sensitive than Mo-nitrogenase) but, unlike Mo-nitrogenase, MgATP hydrolysis was also inhibited by CN − . V-nitrogenase also released more of the four-electron-reduced intermediate, CH 3 NH 2 , than did Mo-nitrogenase. At high NaCN concentrations, V-nitrogenase directed a significant percentage of electron flux into "excess NH 3 " and, under these conditions, substantial amounts of HCHO, but no CH 3 OH, were detected for the first time. With azide, in contrast to Mo-nitrogenase, both total electron flux and MgATP hydrolysis with V-nitrogenase were inhibited. V-nitrogenase, unlike Mo-nitrogenase, showed no preference between the two-electron reduction to N 2 -plus-NH 3 and the six-electron reduction to N 2 H 4 -plus-NH 3 . V-nitrogenase formed more "excess NH 3 ", but reduction of the N 2 produced by the two-electron reduction of N 3 − was not its source. Rather, it was formed directly by the eight-electron reduction of N 3 − . Unlike Mo-nitrogenase, CO could not completely eliminate either cyanide or azide reduction by V-nitrogenase. CO did, however, eliminate the inhibition of both electron flux and MgATP hydrolysis by CN − , but not that caused by azide. These different responses to CO suggest different sites or modes of interaction for these two substrates with V-nitrogenase. † Support from the National Institutes of Health (Grant #DK 37255 to W.E.N.) is gratefully acknowledged. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2015 January 16. Azotobacter vinelandii is a strictly aerobic N 2 -fixing organism that harbors three related, but genetically distinct, nitrogenases. Which one of these enzymes is operational at any particular time depends on which metal ions are present (1). For more than 50 years, the requirement of molybdenum for nitrogenase was thought to be mandatory (2), even though vanadium was demonstrated early on to be almost as stimulatory as Mo to the growth of some bacteria on N 2 as the sole nitrogen source (3). Despite these and other early experiments, the essentiality of Mo became entrenched. However, it is now clear that Mo is not essential for biological nitrogen fixation. Progress through the 1980's clearly identi...

show abstract

Section: Experimental Procedures Cell Growth and Protein Purificationsupporting

confidence: 78%

Azotobacter vinelandiiVanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

2006

View full text Add to dashboard Cite

show abstract

“…This observation, in combination with results from quantitative analysis of the SDS͞PAGE and N-terminal amino acid sequencing (data not shown), indicates a subunit composition of ␣ 2 ␤ 2 for ⌬nifH Av1Ј and ␣␤ 2 k for ⌬nifB Av1 V (Table 2). Additional small subunits, encoded by nafY and vnfG, have been reported to be associated with various species of Av1 and Av1 V , respectively (23,27,45,46). However, no additional subunits are detectable in ⌬nifH Av1Ј or ⌬nifB Av1 V based on SDS͞PAGE (Fig.…”

Section: Resultsmentioning

confidence: 95%

“…In contrast to the reddish-brown color of ⌬nifB Av1, which j The ␣-subunit of Av1 V is known to migrate faster than the ␤-subunit on an SDS͞PAGE, despite a larger molecular weight of the ␣-subunit (16,27). Faster migration of the ␣-subunit has also been observed in the case of the VFe protein of Azotobacter chroococcum, designated Ac1 V (44).…”

Section: Resultsmentioning

confidence: 99%

“…Both ⌬nifH Av1Ј and ⌬nifB Av1 V contain catalytically active P-cluster variants presumably composed of [4Fe-4S]-like centers that are clearly distinct from the normal P-clusters. Interestingly, the S ϭ 1͞2 EPR signal exhibited by the P-cluster variant in ⌬nifB Av1 V is also observed in WT Av1 V and thought to be associated with the activity of the protein, indicating that the same P-cluster variant is present in WT Av1 V and may represent a physiologically relevant species during the process of nitrogenase assembly and͞or turnover (4,27). The S ϭ 1͞2 EPR signal of ⌬nifH Av1Ј is also observed in ⌬nifH Av1, the protein species isolated from the same nifH-deletion A. vinelandii strain without preincubation with Fe protein and MgATP (17).…”

Section: Discussionmentioning

confidence: 97%

See 1 more Smart Citation

Nitrogenase reactivity with P-cluster variants

Corbett

Fay

et al. 2005

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

View full text Add to dashboard Cite

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...

show abstract

“…The three forms of VNFG observed in species A (Fig. 2A, lane 1 both forms but was not quantitated (12).…”

Section: Characterization Of Electrophoretically Distinct Species Of mentioning

confidence: 93%

Characterization of VNFG, the δ Subunit of the vnf-Encoded Apodinitrogenase from Azotobacter vinelandii

Chatterjee

Ludden

Shah³

1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

The vnf-encoded apodinitrogenase (apodinitrogenase 2) from Azotobacter vinelandii is an ␣ 2 ␤ 2 ␦ 2 hexamer. The ␦ subunit (the VNFG protein) has been characterized in order to further delineate its function in the nitrogenase 2 enzyme system. Two species of VNFG were observed in cell-free extracts resolved on anoxic native gels; one is composed of VNFG associated with the VNFDK polypeptides, and the other is a homodimer of the VNFG protein.Both species of VNFG are observed in extracts of A. vinelandii strains that accumulate dinitrogenase 2, whereas extracts of strains impaired in the biosynthetic pathway of the iron-vanadium cofactor (FeV-co) that accumulate apodinitrogenase 2 (a catalytically inactive form of dinitrogenase 2 that lacks FeV-co) exhibit only the VNFG dimer on native gels. FeV-co and nucleotide are required for the stable association of VNFG with the VNFDK polypeptides; this stable association can be correlated with the formation of active dinitrogenase 2. The iron-molybdenum cofactor was unable to replace FeV-co in promoting the stable association of VNFG with VN-FDK. FeV-co specifically associates with the VNFG dimer in vitro to form a complex of unknown stoichiometry; combination of this VNFG-FeV-co species with apodinitrogenase 2 results in its reconstitution to dinitrogenase 2. The results presented here suggest that VNFG is required for processing apodinitrogenase 2 to functional dinitrogenase 2.

show abstract

Isolation of Two Forms of the Nitrogenase VFe Protein from Azotobacter vinelandii

Cited by 59 publications

References 30 publications

Azotobacter vinelandiiVanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

Azotobacter vinelandiiVanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

Nitrogenase reactivity with P-cluster variants

Characterization of VNFG, the δ Subunit of the vnf-Encoded Apodinitrogenase from Azotobacter vinelandii

Contact Info

Product

Resources

About