MoFe-cofactor

Figure 17.10 Structure of the MoFe-cofactor highlighting Fe atoms 2, 3, 6 and 7 with the MoFe-protein ligands Cysa273 and Hisa442 and the side-chains of Hisal95 and Vala70. (From Barney et al., 2006. Copyright (2006) National Academy of Sciences, USA.)...

The MoFe protein of the enzyme contains two different types of metal clusters an FegS cluster called the P-cluster and a MoFe 89 homocitrate cluster known as the M-center or MoFe-cofactor. The latter cluster is beheved to be the site of substrate (N2) reduction. The cofactor cluster is paramagnetic in the as-isolated form of the protein with 5 = 3/2 while the P-clusters are diamagnetic. Inflections for the resting-state cofactor cluster occur at = 4.6, 3.7 and 2.0. These inflections (Figure 14) are associated with an 5 = 3/2 signal with an apparent rhombicity (Figure 13) of E/D = 0.05. 

The MoFe cofactor of the nitrogenase MoFe-protein component... 

In the nitrogenase enzyme isolated from Azotobacter vinelandii (e.g., see PDB IMIN, 2MIN, and 3MIN entries), it appears that there are three sites required for the process given by Equation 12.1 to be consummated. One site is called the 4Fe-4S cluster, a second site is referred to as the P-cluster, and the third site is the MoFe cofactor (sometimes called the M-center ) or MoFe protein where, some have argued, the actual reduction occurs. 

Figure 12.4. A representation of the MoFe cofactor of the nitrogenase enzyme isolated from A. vinelandii. It is argued that the cysteine residue on the one end and the imidazole on the other (which also bears a homocitrate bound to molybdenum) immobilize this in the active site where the reduction of nitrogen actually occurs.

The VFe protein also has the equivalent of P-cluster pairs which have similar properties to those found in the MoFe protein (159). No information is available on whether P-cluster pairs exist in the FeFe protein, but because of the relatively high sequence identity and the similar genetic basis of its biosynthesis, the occurrence seems highly likely. The catalytic role assigned to the P-cluster pair involves accepting electrons from the Fe protein for storage and future deUvery to the substrate via the FeMo-cofactor centers. As of this writing (ca early 1995), this role has yet to be proved. 

Fig. 7. View of the FeMo-cofactor prosthetic group of the nitrogenase MoFe protein with some of the surrounding amino acid residues where ( ) represents the molybdenum coordinated to a-His-442 and homocitrate (at the top), ( ) represents the iron, interspersed with the sulfur (O) and carbon...

As well as donating electrons to the MoFe protein, the Fe protein has at least two and possibly three other functions (see Section IV,C) It is involved in the biosynthesis of the iron molybdenum cofactor, FeMoco it is required for insertion of the FeMoco into the MoFe protein polypeptides and it has been implicated in the regulation of the biosynthesis of the alternative nitrogenases. 

The MoFe proteins are all a2 2 tetramers of 220-240 kDa, the a and (3 subunits being encoded by the nifD and K genes, respectively. The proteins can be described as dimers of a(3 dimers. They contain two unique metallosulfur clusters the MoFeTSg homocitrate, FeMo-cofactors (FeMoco), and the FesSy, P clusters. Neither of these two types of cluster has been observed elsewhere in biology, nor have they been synthesized chemically. Each molecule of fully active MoFe protein contains two of each type of cluster 2-7). 

FeMoco can be extracted from the MoFe protein into A(-methylfor-mamide (NMF) solution 32) and has been analyzed extensively using a wide range of spectroscopic techniques both bound to the protein and in solution after extraction from it (33). The extracted FeMoco can be combined with the MoFe protein polypeptides, isolated from strains unable to synthesize the cofactor, to generate active protein. The structure of the FeMoco is now agreed 4, 5, 7) as MoFeTSg homocitrate as in Fig. 4. FeMoco is bound to the a subunit through residues Cys 275, to the terminal tetrahedral iron atom, and His 442 to the molybdenum atom (residue numbers refer to A. vinelandii). A number of other residues in its environment are hydrogen bonded to FeMoco and are essential to its activity (see Section V,E,2). The metal... 

The iron K-edge EXAFS measurements on AVI " 182) and the extracted FeVaco from AcF 183) show Fe-S and Fe-Fe interactions at 2.32 and 2.64 A, with a longer Fe-Fe distance of 3.7 A very similar again to the EXAFS data on FeMoco. These data emphasize the structural similarities between the cofactor centers of the MoFe and VFe proteins. 

Fig. 1. Schematic illustration of the enzyme nitrogenase being composed of the molybdenum-iron (MoFe) protein, an oc2p2 tetramer with two unique iron-sulfur clusters (P-cluster) and two iron-molybdenum cofactors (FeMoco), and the iron protein with one [4Fe-4S]-cluster and two ATP binding sites.

Actual electron transfer to the dinitrogen substrate at the MoFe-protein, with electrons first passing through the MoFe-protein s P-cluster. During this process, dinitrogen is most probably bound to the iron-molybdenum cofactor (FeMoco) of the MoFe-protein.6... 

The mechanism and sequence of events that control delivery of protons and electrons to the FeMo cofactor during substrate reduction is not well understood in its particulars.8 It is believed that conformational change in MoFe-protein is necessary for electron transfer from the P-cluster to the M center (FeMoco) and that ATP hydrolysis and P release occurring on the Fe-protein drive the process. Hypothetically, P-clusters provide a reservoir of reducing equivalents that are transferred to substrate bound at FeMoco. Electrons are transferred one at a time from Fe-protein but the P-cluster and M center have electron buffering capacity, allowing successive two-electron transfers to, and protonations of, bound substrates.8 Neither component protein will reduce any substrate in the absence of its catalytic partner. Also, apoprotein (with any or all metal-sulfur clusters removed) will not reduce dinitrogen. 

The MoFe-protein s environment is tuning the cofactor FeMoco to maximize N2 reduction and minimize H2 production. 

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