Nitrogenase. III. Nitrogenaseless mutants of Azotobacter vinelandii: Activities, cross-reactions and epr spectra

Shah, Vinod K.; Davis, Lawrence C.; Gordon, Joyce K.; Orme‐Johnson, William H.; Brill, Winston J.

doi:10.1016/0005-2728(73)90269-7

Cited by 128 publications

(93 citation statements)

References 26 publications

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“…However, results below will show that a native form of NifH with the ability to bind MgATP is not necessarily sufficient to allow NifH to function in FeMo-co synthesis. These results also corroborate previously reported results with A157S NifH, wherein the altered form of NifH was shown to bind MgATP but not to hydrolyze the nucleotide or undergo the nucleotide-induced conformational change; yet it could function in FeMo-co synthesis as judged by the activity of dinitrogenase produced in the strain UW91 (30,52). The features of NifH required for apodinitrogenase maturation were examined by the use of site-specifically altered enzymes in in vitro apodinitrogenase activation assays, as described under "Experimental Procedures."…”

Section: Identification Of Features Of Nifh Required For Femo-co Biossupporting

confidence: 92%

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

Rangaraj

Ryle²,

Lanzilotta³

et al. 1999

Journal of Biological Chemistry

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NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127⌬, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented.

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Section: Identification Of Features Of Nifh Required For Femo-co Biossupporting

confidence: 92%

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

Rangaraj

Ryle²,

Lanzilotta³

et al. 1999

Journal of Biological Chemistry

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“…Interestingly, very early reports of the crystallization of the MoFe protein from A. vinelandii showed an extremely similar spectrum which was attributed to the MoFe protein (9,10). This spectrum was not obtained by others (5,33), and the controversy concerning its origin was never resolved (4,6 degradation under anaerobic and aerobic conditions might be due to the presence of different sets of proteases or an inherent difference between the MoFe proteins produced under the two sets of conditions.…”

Section: Resultsmentioning

confidence: 98%

Analysis of Azotobacter vinelandii strains containing defined deletions in the nifD and nifK genes

Tal

Robinson

et al. 1990

J Bacteriol

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Strains of Azotobacter vinelandi which contain defined deletions within the nijD and nijK genes which encode, respectively, the a and 13 subunits of the MoFe protein of nitrogenase were analyzed. When synthesized without its partner, the 1 subunit accumulated as a soluble 14 tetramer. In contrast, when the a subunit was present without its partner, it accumulated primarily as an insoluble aggregate. The solubility of this protein was increased by the presence of a form of the 13 subunit which contained a large internal deletion, such that the a subunit could participate in the assembly of small amounts of an a212 holoprotein. When synthesized alone, the 13 subunit was remarkably stable, even when the protein contained a large internal deletion. The a subunit, however, was much more rapidly degraded than the 13 subunit, both when it was synthesized alone in its native background and when it was synthesized with its 1 subunit partner in a foreign background. Antibodies raised against purified a2132 MoFe protein recognized epitopes only on the nondenatured 1 subunit and not on the nondenatured a subunit. Our findings that all epitopes for the a2P2 tetramer appeared to be on the 1 subunit, that the 1 subunit assembled-into P4 tetramers, and that the a subunit alone was very insoluble, combined with the previous finding that the Fe protein binds to the 1 subunit (A. H. Willing, M. M. Georgiadis, D. C. Rees, and J. B. Howard, J. Biol. Chem. 264:849948503, 1989) all suggest that the 13 subunit has a more surface location than the a subunit in the a212 tetramer.Nitrogenase catalyzes the biological reduction of N2 to NH3. Molybdenum nitrogenases from diverse procaryotes are composed of two separately purified proteins called the iron protein (Fe protein), and the molybdenum-iron protein (MoFe protein), both of which are required for substrate reduction (28,34,36). The Fe protein from one organism can complex with the MoFe protein from another to yield an active enzyme (13, 14).The MoFe proteins are a232 tetramers with molecular weights of 210,000 to 250,000. The a and 1 subunits encoded by the nijfl and ni/K genes, respectively, are similar in size (29,40). The arrangement of the subunits is unknown, but information from neutron small-angle scattering (26), electron microscopy (35, 39), and X-ray diffraction studies (43) points toward an aspherical, probably pseudotetrahedral particle with twofold molecular symmetry. Current models suggest six metal centers within the a2532 tetramer (25,28,34,36), two of which are FeMo cofactor clusters. The FeMo cofactor, at the site of substrate binding and reduction (18), contains about six iron atoms, eight sulfur atoms, and one molybdenum atom arranged in a novel spin-coupled cluster (28,34,36) with one molecule of homocitrate (19). The other four metal centers appear to be [4Fe-4S]-type clusters in an unusual (zero) oxidation state (22, 25) with some noncysteine ligation (11,22,25,40); they are not all identical and appear to occupy two different environments (25).The relationship...

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“…No mutants that contain a nitrogenase with the ability to reduce one substrate but not another have been described. Of the many hundreds of independently isolated Nif-mutants of K. pneumoniae, those that are unable to fix N2 in vivo also are unable to reduce acetylene in vivo (76,184,198,204,211). This is evidence that the same (or overlapping) enzyme sites are responsible for acetylene reduction as well as N2 fixation.…”

Section: Biochemistry Nitrogenasementioning

confidence: 99%

Biochemical Genetics of Nitrogen Fixation

Brill¹

1983

Structure and Function of Plant Genomes

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Nitrogenase. III. Nitrogenaseless mutants of Azotobacter vinelandii: Activities, cross-reactions and epr spectra

Cited by 128 publications

References 26 publications

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

In Vitro Biosynthesis of Iron-Molybdenum Cofactor and Maturation of the nif-encoded Apodinitrogenase

Analysis of Azotobacter vinelandii strains containing defined deletions in the nifD and nifK genes

Biochemical Genetics of Nitrogen Fixation

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