Oxidative Titration of the Nitrogenase VFe Protein from <i>Azotobacter vinelandii</i>: An Example of Redox-Gated Electron Flow

Tittsworth, R. C.; Hales, Brian J.

doi:10.1021/bi951430i

Cited by 35 publications

(51 citation statements)

References 32 publications

(49 reference statements)

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“…The minority species may represent a damaged version of the majority species. Analogous signals have been seen in the vnf-dinitrogenases of A. chroococcum (31) and A. vinelandii (32) and have been suggested to represent damaged FeV-co in those proteins. Therefore, the spectroscopic properties of the S ϭ 3/2 species are consistent with an [Fe-V-S] cluster with similar properties to both FeV-co (proposed to be [VFe 7 S 9 ]) and a [VFe 3 S 4 ] cluster.…”

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confidence: 54%

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Rüttimann-Johnson

Staples

Rangaraj

et al. 1999

Journal of Biological Chemistry

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The vnf-encoded nitrogenase from Azotobacter vinelandii contains an iron-vanadium cofactor (FeV-co) in its active site. Little is known about the synthesis pathway of FeV-co, other than that some of the gene products required are also involved in the synthesis of the ironmolybdenum cofactor (FeMo-co) of the widely studied molybdenum-dinitrogenase. We have found that VnfX, the gene product of one of the genes contained in the vnf-regulon, accumulates iron and vanadium in a novel V-Fe cluster during synthesis of FeV-co. The electron paramagnetic resonance (EPR) and metal analyses of the V-Fe cluster accumulated on VnfX are consistent with a VFe 7-8 S x precursor of FeV-co. The EPR spectrum of VnfX with the V-Fe cluster bound strongly resembles that of isolated FeV-co and a model VFe 3 S 4 compound. The V-Fe cluster accumulating on VnfX does not contain homocitrate. No accumulation of V-Fe cluster on VnfX was observed in strains with deletions in genes known to be involved in the early steps of FeV-co synthesis, suggesting that it corresponds to a precursor of FeV-co. VnfX purified from a nifB strain incapable of FeV-co synthesis has a different electrophoretic mobility in native anoxic gels than does VnfX, which has the V-Fe cluster bound. NifB-co, the Fe and S precursor of FeMo-co (and presumably FeV-co), binds to VnfX purified from the nifB strain, producing a shift in its electrophoretic mobility on anoxic native gels. The data suggest that a precursor of FeV-co that contains vanadium and iron accumulates on VnfX, and thus, VnfX is involved in the synthesis of FeV-co.

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confidence: 54%

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Rüttimann-Johnson

Staples

Rangaraj

et al. 1999

Journal of Biological Chemistry

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“…This possibility is supported by the observation that the P-cluster in MoFe protein can undergo a two-electron oxidation process that concurrently opens up half of the [Fe 8 S 7 ] cage (6). Further, in the resting state, the P-cluster in VFe protein gives rise to signals that are analogous to its MoFe protein counterpart in the turnover state (18), suggesting a somewhat interconvertible nature of these P-clusters in oxidation state and/or structure. More importantly, this observation points to a plausible redox and/or structural change of the P-cluster that may be mechanisticallyrelevant to catalysis.…”

Section: Discussionmentioning

confidence: 87%

Unique features of the nitrogenase VFe protein from Azotobacter vinelandii

Lee

Ribbe

2009

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

109

150

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Nitrogenase is an essential metalloenzyme that catalyzes the biological conversion of dinitrogen (N2) to ammonia (NH3). The vanadium (V)-nitrogenase is very similar to the ''conventional'' molybdenum (Mo)-nitrogenase, yet it holds unique properties of its own that may provide useful insights into the general mechanism of nitrogenase catalysis. So far, characterization of the vanadium iron (VFe) protein of Azotobacter vinelandii V-nitrogenase has been focused on 2 incomplete forms of this protein: ␣␤2 and ␣2␤2, both of which contain the small ␦-subunit in minor amounts. Although these studies provided important information about the V-dependent nitrogenase system, they were hampered by the heterogeneity of the protein samples. Here, we report the isolation and characterization of a homogeneous, His-tagged form of VFe protein from A. vinelandii. This VFe protein has a previouslyunsuspected, ␣2␤2␦4-heterooctameric composition. Further, it contains a P-cluster that is electronically and, perhaps, structurally different from the P-cluster of molybdenum iron (MoFe) protein.More importantly, it is catalytically distinct from the MoFe protein, particularly with regard to the mechanism of H2 evolution. A detailed EPR investigation of the origins and interplays of FeV cofactor-and P-cluster-associated signals is presented herein, which lays the foundation for future kinetic and structural analysis of the VFe protein. Nitrogenase is a complex metalloenzyme that catalyzes the biological conversion of dinitrogen (N 2 ) to ammonia (NH 3 ). Although the molybdenum (Mo)-dependent nitrogenase has been recognized as the ''conventional'' nitrogenase for the past 70 years, the vanadium (V)-nitrogenase was discovered only some 30 years ago as an ''alternative'' nitrogenase that is expressed under Mo-deficient conditions (1-4)*. Both nitrogenases are binary enzyme systems. The Mo-nitrogenase is composed of (i) nifH-encoded iron (Fe) protein, an ␣ 2 -homodimer bridged by a [Fe 4 S 4 ] cluster; and (ii) nifDK-encoded MoFe protein, an ␣ 2 ␤ 2 -heterotetramer containing, per ␣␤-dimer, a P-cluster ([Fe 8 S 7 ]) that is located at the ␣/␤-subunit interface and a FeMo cofactor (FeMoco) ([MoFe 7 S 9 X-homocitrate], where X ϭ C, O or N) that is buried within the ␣-subunit (6, 7). Catalysis by this nitrogenase involves repeated association/ dissociation between Fe and MoFe proteins and ATP hydrolysisdriven, interprotein electron transfer from the [Fe 4 S 4 ] cluster of the Fe protein to the P-cluster, and finally, the FeMoco site of the MoFe protein, where substrate reduction occurs (1). Like its Mo-counterpart, V-nitrogenase is a 2-component system comprising (i) vnfH-encoded Fe protein and (ii) vnfDGK-encoded vanadium-iron (VFe) protein (2). The Fe protein of Vnitrogenase, a homodimer containing a [Fe 4 S 4 ] cluster, is very similar to the Fe protein of Mo-nitrogenase. The VFe protein, however, differs from the ␣ 2 ␤ 2 -tetrameric MoFe protein in that it contains an additional ␦-subunit (encoded by vnfG) along with the ␣-and ␤-subunits (enc...

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“…Stepwise oxidation of Av1′ A shows [see Tittsworth and Hales (1996)] an unusual redox gating midway through the titration that is not observed during the oxidation of either Av1′ B or Av1.…”

Section: Discussionmentioning

confidence: 99%

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

Blanchard

Hales

1996

Biochemistry

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When Q-Sepharose was used in the purification of the V nitrogenase proteins from Azotobacter vinelandii, an increase in resolution was observed that resulted in a separation of the nitrogenase component 1 protein (Av1') into two forms, labeled Av1'A and Av1'B. Even though both forms possessed the same enzymatic behavior, Av1'A exhibited a lower specific activity and migrated during gel filtration with an apparent lower molecular weight than Av1'B. Furthermore, SDS-polyacrylamide gel electrophoresis showed different relative compositions of the two major subunits of both forms, with Av1'A possessing a trimer (alpha beta 2) pattern compared to the more typical tetramer (alpha 2 beta 2) pattern found for Av1'B. Metal analysis indicated a V-to-Fe ratio of 1:19 for Av1'A and 1:15 (or 2:30) for Av1'B, while acid-labile sulfide analysis showed that Av1'A possessed about half as much sulfide as Av1'B. EPR spectroscopy revealed that both proteins retained the S = 3/2 and S = 1/2 signals observed in earlier isolations, with an additional S = 1/2 signal present in the spectrum of protein A. These results suggest that Av1'A is an incomplete form of the VFe protein, containing only one cofactor and one P cluster with an additional [Fe4-S4]-like cluster. The presence of a V storage protein in A. vinelandii was also investigated. Although no V storage protein was found, two V-binding proteins were observed.

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Oxidative Titration of the Nitrogenase VFe Protein from Azotobacter vinelandii: An Example of Redox-Gated Electron Flow

Cited by 35 publications

References 32 publications

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

A Vanadium and Iron Cluster Accumulates on VnfX during Iron-Vanadium-cofactor Synthesis for the Vanadium Nitrogenase inAzotobacter vinelandii

Unique features of the nitrogenase VFe protein from Azotobacter vinelandii

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

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