Nitrogenase components

A preparation of the third nitrogenase from A. vinelandii, isolated from a molybdenum-tolerant strain but lacking the structural genes for the molybdenum and vanadium nitrogenases, was discovered to contain FeMoco 194). The 8 subunit encoded by anfG was identified in this preparation, which contained 24 Fe atoms and 1 Mo atom per mol. EPR spectroscopy and extraction of the cofactor identified it as FeMoco. The hybrid enzyme could reduce N2 to ammonia and reduced acetylene to ethylene and ethane. The rate of formation of ethane was nonlinear and the ethane ethylene ratio was strongly dependent on the ratio of nitrogenase components. 

Recently, Brill and co-workers (43, 44) have isolated mutant strains of Azotobacter vinelandii which produce an inactive nitrogenase component. This component can be reactivated by treatment with the neutralized acid-hydrolysis products of other nitrogenases (which themselves become inactive on such a treatment) but not apparently with any other molybdenum enzymes. This may either reflect a difference between the cofactor in nitrogenase and other molybdenum enzymes or may be caused by the reconstitution conditions used which may not have been sufficiently varied to allow for different molybdenum oxidation states to be attained. In any event, the chemical characterization and authentication of the molybdenum cofactor should reveal some of the intimate details of the molybdenum site. 

Both the reductase and the nitrogenase components of the complex are iron-sulfur proteins, in which iron is bonded to the sulfur atom of a cysteine residue and to inorganic sulfide. The reductase (also called the iron protein or the Fe protein) is a dimer of identical 30-kd subunits bridged by a 4Fe-4S cluster (Figure 24.3). 

The nitrogenase component is an a 2 P 2 tetramer (240 kd), in which the a and P subunits are homologous to each other and structurally quite similar (Figure 24.4). Electrons enter at the P clusters, which are located at the a-P interface. 

These clusters are each composed of eight iron atoms and seven sulfide ions. In the reduced form, each cluster takes the form of two 4Fe-3S partial cubes linked by a central sulfide ion. Each cluster is linked to the protein through six cysteinate residues. Electrons flow from the P cluster to the FeMo cofactor, a very unusual redox center. Because molybdenum is present in this cluster, the nitrogenase component is also called the molybdenum-iron protein (MoFe protein). The FeMo cofactor consists of two M-3Fe-3S clusters, in which molybdenum occupies the M site in one cluster and iron occupies it in the other. The two clusters are joined by three sulfide ions. The FeMo cofactor is also coordinated to a homocitrate moiety and to the a subunit through one histidine residue and one cysteinate residue. This cofactor is distinct from the molybdenum-containing cofactor found in sulfite oxidase and apparently all other molybdenum-containing enzymes except nitrogenase. 

Positive DNA hybridization with DNA of some 50 species of N2-fixing bacteria to a probe of the structural genes of Mo nitrogenase indicates that the structure of this nitrogenase has been highly conserved. Consistent with this, the physicochemical properties of purified Mo nitrogenase components have been shown to be very similar they have been extensively reviewed and will only be summarized here (see Refs. 17-20 for primary references). 

Describe the nitrogenase complex and explain the roles of its reductase and nitrogenase components. Note the function of the FeMo-cofactor. 

Specific activity refers to nmoles of acetylene reduced per mg protein per minute at 30 C in the presence of saturating amounts of the other protein. Superscript numbers indicate fractions 1 (MoFe protein) or 2 (Fe protein). The nitrogenase components firom several other organisms including Mycobacterium flavum (Biggins et al., 1971), Azospirillum brasilense (Ludden et al., 1978), and Anabaena cylindrica (Tsai and Mortenson, 1978) have been separated and partly purified. 

A positive assertion regarding the roles of the two nitrogenase components... 

Fig. 13. The effect of nitrogenase component ratio and ATP concentration on the Hj/N ratio using purified Acl and Ac2. The Ac2 concentration was constant at 9.2 /u.g/ml (0.14 /nW). ATP...

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. However, Hu et al identified two cataly-tically active P-cluster variants by ESR spectroscopic studies, and found that both P-cluster variants resemble [4Fe-4S]-like centers based on XAS experiments. 

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