The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein

Tal, Sara; Chun, Teresa W.; Gavini, Nara; Burgess, Barbara K.

doi:10.1016/s0021-9258(18)99273-2

Cited by 49 publications

(25 citation statements)

References 20 publications

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“…Immunoblot analysis showed that polypeptides cross-reacting with NifDK, as well as the NifD and NifK specific antibodies, comigrated (Figure a). As NifDK is known to migrate differently depending on the clusters present − (e.g., FeMo-co present at holo-NifDK increases its migration compared to the P-cluster containing, but FeMo-co-less, apo-NifDK), the purified A. vinelandii holo- and apo-NifDK proteins were included as comparisons. Yeast NifD and NifK migrated closely, but not identical to any of the two A. vinelandii NifDK forms, indicating that some conformational or biochemical discrepancies exist between the proteins expressed in the two hosts and suggesting that some of the critical components of NifDK could be missing in the yeast expressed protein.…”

Section: Results and Discussionmentioning

confidence: 99%

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

et al. 2017

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Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.

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Section: Results and Discussionmentioning

confidence: 99%

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

et al. 2017

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“…Protein Characterization. Iron (Van DeBogart & Belnert, 1967) and molybdenum (Clark & Axley, 1955) determinations, one-dimensional and two-dimensional gel electrophoresis (Robinson et al, 1989), and Western analysis (Tal et al, 1991) were carried out as described elsewhere. Anaerobic native gel electrophoresis was performed by a modification of published procedures (Ornstein, 1964;Davis, 1964), whereby all buffers were degassed and the gel was prerun with 2 mM Na2S2C>4 under Ar for 15 min before loading the samples.…”

Section: Methodsmentioning

confidence: 99%

“…others, NifH (Robinson et al, 1989) and NifY (White et al, 1992;Homer et al, 1993), appear to be required for the insertion of preformed FeMo cofactor into the MoFe protein polypeptides. Strains with mutations in the nifB (Shah & Brill, 1977), N, E (Shah & Brill, 1977; Tal et al, 1992), or H (Robinson et al, 1989;Tal et al, 1991;Robinson et al, 1987) genes accumulate FeMo cofactor-deficient MoFe proteins that can be activated in vitro by addition of isolated FeMo cofactor in 7V-methylformamide. Recent studies have established, however that the FeMo cofactor-deficient MoFe proteins synthesized by Nif B_, N-, and Estrains are like each other but are unlike the protein synthesized by a Nif Hstrain (Tal et al, 1991).…”

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Purification and characterization of a FeMo cofactor-deficient MoFe protein

Gavini

Ma²,

Watt³

et al. 1994

Biochemistry

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Previous studies have shown that the nifH gene product is required for FeMo cofactor biosynthesis and insertion and that a delta nifH strain of Azotobacter vinelandii designated DJ54 accumulates a FeMo cofactor-deficient MoFe protein that is distinct from the FeMo cofactor-deficient protein synthesis by Nif B-, N-, or E- strains [Tal, S., Chun, T., Gavini, N., & Burgess, B. K. (1991) J. Biol. Chem. 266, 10654-10657]. Here we report the purification and activation of the MoFe protein from DJ54. The purified protein is an alpha 2 beta 2 tetramer that is indistinguishable from the wild-type MoFe protein by the criteria of SDS-polyacrylamide gel electrophoresis, native gel electrophoresis, and two-dimensional gel electrophoresis. It binds normally to its redox partner, the Fe protein, by the criterion of chemical cross-linking. It does not contain FeMo cofactor and does not catalyze significant C2H2 reduction or reduction-independent MgATP hydrolysis. It can, however, be activated with FeMo cofactor following the addition of the Fe protein and MgATP when an additional required component(s) is supplied by cell-free extracts from a delta nifD strain of A. vinelandii. The purified DJ54 MoFe protein does contain P-clusters by the criteria of metal analysis, CD spectroscopy, cluster extrusion, and electrochemical reduction of the POX state. In the presence of dithionite it exhibits an axial S = 1/2 EPR signal that integrates to 0.1-0.3 spin per alpha 2 beta 2 tetramer.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Along with decreased activity, modification of the proton transfer pathway can stabilize states that are otherwise elusive, including some with unusual IR signatures now assigned as H hyd . , The H hyd state is thought to feature a reduced [4Fe-4S] H + subcluster and a homovalent Fe p (II)Fe d (II) binuclear [2Fe] H site, with a hydride H h bound in the apical position on Fe d …”

Section: Introductionmentioning

confidence: 99%

“…7,17 The H hyd state is thought to feature a reduced [4Fe-4S] H + subcluster and a homovalent Fe p (II)Fe d (II) binuclear [2Fe] H site, with a hydride H h bound in the apical position on Fe d . 20 In previous work, we used nuclear resonance vibrational spectroscopy (NRVS) to examine the [2 57 Fe-ODT] H -CrHydA1 H hyd species. 21 NRVS is a synchrotron-based X-ray technique that involves observation of vibrational sidebands that occur in combination with nuclear transitions.…”

Section: ■ Introductionmentioning

confidence: 99%

Reaction Coordinate Leading to H₂ Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory

et al. 2017

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[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (Hhyd) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57Fe Nuclear Resonance Vibrational Spectroscopy (NRVS) and Density Functional Theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, with the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that Hhyd is the catalytic state one step prior H2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H2 bond formation by [FeFe]-hydrogenases.

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The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein

Cited by 49 publications

References 20 publications

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

Purification and characterization of a FeMo cofactor-deficient MoFe protein

Reaction Coordinate Leading to H₂ Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory

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The delta nifB (or delta nifE) FeMo cofactor-deficient MoFe protein is different from the delta nifH protein

Cited by 49 publications

References 20 publications

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

Purification and characterization of a FeMo cofactor-deficient MoFe protein

Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory

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Reaction Coordinate Leading to H₂ Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory