Nitrogenases third

Fe component property Mo nitrogenase V nitrogenase Third nitrogenase... 

Although, as indicated in Fig. 12, there is clear genetic evidence for a third nitrogenase in Azotobacter vinelandii, the initial preparations of this enzyme had low activity and contained small quantities of molybdenum as well as iron, and thus the activity might have been... 

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. 

Part 1 of the nitrogenase protein contains another interconnected group of Fe-S atoms, this one with eight iron atoms and seven sulfur atoms. This [8Fe-7S] group collects electrons and transmits them to the binding center. Part 2 of nitrogenase contains a third Fe-S group, this one made up of four iron atoms and four sulfur atoms. This part of the enzyme also binds two molecules of ATP. 

The next two entries to Table 3 are cited for completeness. Nitrogenase is treated in Chapter 7 and CO dehydrogenase in Chapter 9. Nitrogenase contains a very complex iron-sulfur cluster that includes another metal, molybdenum or vanadium. The crystal structure of the Mo variant has been determined. There is a third variant, alternative nitrogenase [92], whose cluster apparently does not contain any heterometal. That cluster would thus be a perfect candidate for our definition of a redox-catalytic iron-sulfur cluster. Unfortunately, this third nitrogenase has thus far been characterized to a much lesser extent than the other two forms. For all nitrogenases holds that the binding of N2 to the cluster has not been established [53] therefore, formally these enzymes have not yet been positively identified as redox iron-sulfur catalysts. 

Mutant strains of A. chroococcum carrying deletions of both Mo and V nitrogenase structural genes are unable to grow on N2 ill). In contrast, comparable strains of A. vinelandii can grow provided neither Mo nor V is added to the growth medium (12). This ability is due to the presence of the third nitrogenase in A. vinelandii. 

The presence of an open reading frame between vnfD and vnfK led to the identification of an additional small subunit type (8, encoded by vnfG) of the VFe protein it has the hexameric structure 012 2 2 ill). A homologous gene exists between anfD and anfK (13a) and encodes a small subunit of the third nitrogenase (R. Eady and R. Pau, unpublished). 

The structural genes of the Fe proteins of the V nitrogenase of A. chroococcum and A. vinelandii and of the third nitrogenase of A. vine-landii have been cloned and sequenced 13a, 14,22). Comparison of the derived amino acid sequences shows the Fe proteins of Mo and V nitro-genases to be very similar (91% identical) and that of the third nitrogenase to be 61% identical. All sequences show the spacing of the five invariant Cys residues, a conserved Arg residue near position 100, and a consensus nucleotide-binding sequence. 

Fig, 1. Schematic representation of the a and /3 polypeptides of the MoFe protein, the VFe protein, and the third nitrogenase. The vertical lines represent the position of the five invariant Cys residues of the a subunit and the three invariant Cys residues of the /3 subunit. The flanking amino acid sequences are shown to indicate the degree of sequence homology around these Cys residues. The wide horizontal lines represent the polypeptide chains of the subunits of the three nitrogenases (see Ref 25). 

MoFe protein in K. pneumoniae VFe proteins Third nitrogenase of A. vinelandii ... 

The components of the third nitrogenase, analogous to the MoFe and VFe proteins (dinitrogenase-3) have been purified and partially characterized (5). The third nitrogenase can be isolated in two active forms with different subunit configurations, ai/ 2 and a2(32 there is a 8 subunit present at uncertain stoichiometry encoded by anfG (R. Eady and R. Pau, unpublished). 

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