Distinct structural features of the .alpha. and .beta. subunits of nitrogenase molybdenum-iron protein of Clostridium pasteurianum: an analysis of amino acid sequences

Wang, Shu Zhen; Chen, Jiann Shin; Johnson, John L.

doi:10.1021/bi00408a021

Cited by 28 publications

(22 citation statements)

References 55 publications

(70 reference statements)

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“…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.…”

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confidence: 99%

“…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).…”

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confidence: 99%

“…The relationships of the metal centers of the MoFe protein to each other and to the polypeptides are unknown, but much information has been deduced from sequence comparisons (40) and site-directed mutagenesis studies (11). Amino acid sequence information is now available for the at and Comparisons show that the a subunit (nifD) contains 5 conserved cysteine residues while the P subunit contains 3 (23,40), giving 16 for the holoprotein.…”

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confidence: 99%

“…Amino acid sequence information is now available for the at and Comparisons show that the a subunit (nifD) contains 5 conserved cysteine residues while the P subunit contains 3 (23,40), giving 16 for the holoprotein. Site-directed mutagenesis has shown that these residues are important for enzyme activity (11).…”

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confidence: 99%

“…The remaining two conserved cysteine residues in the a subunit are proposed to be involved in FeMo cofactor ligation (3,11). The location of the clusters relative to the surface of the protein is not known, although the FeMo cofactor has been proposed to be buried (28,31) while the [4Fe-4S]-type clusters may be located near the surface or at subunit interfaces (40). The clusters could also be bridged between subunits, as in the Fe protein (17).…”

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confidence: 99%

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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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confidence: 99%