FeMoS clusters

Many FeMoS clusters have been prepared in the quest to duplicate the FeMoco center, but none of the chemically synthesized clusters can reactivate the (UW-45 or NifB cofactor-less mutants, perhaps because of their lack of homocitrate, which only recently has been discovered as a key component of FeMoco. Undoubtedly, new FeMoS clusters containing homocitrate will be prepared, and... 

The Mo-Fe component has M, in the range 200,000-270,000, and is tetrameric (02 2)- Each Mo atom forms part of a polynuclear cluster containing Fe, and homocitrate (/f-2-hydroxy-l,2,4-butanetri-carboxylic acid) the cluster takes the form of a distorted octahedron in which the Mo is coordinated by three S atoms, three Fe atoms and three O, N or C atoms all the available evidence indicates that these Fe-Mo coordination centers are the sites of N2 binding and reduction. The FeMo coordination center or cluster can be removed from the denatured protein without causing essential changes in its structure it can then be used to restore activity to inactive MoFe protein from mutants unable to synthesize the FeMo cluster. It is therefore also referred to as the FeMo cofactor or FeMoco. 

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. 

These studies of protein-bound heterometallic cubanes have amply demonstrated that the heterometal site is redox active and able to bind small molecules. Although they have yet to be identified as intrinsic components of any protein or enzyme (except as part of the nitrogenase FeMo cofactor cluster (254)), they are clearly attractive candidates for the active sites of redox enzymes. 

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). 

NIS measurements have been performed on the rubredoxin (FeSa) type mutant Rm 2-A from Pyrococcus abyssi [103], on Pyrococcus furiosus rubredoxin [104], on Fe2S2 - and Fe4S4 - proteins and model compounds [105, 106], and on the P-cluster and FeMo-cofactor of nitrogenase [105, 107]. 

Figure 1 Structure of the MoFe7SgN cluster of FeMo-co in Mo nitrogenase...

On the other hand, such approaches to the metalloenzymes described above in Figures 1 and 2 are still under way. Thus, the model clusters reproducing precisely their complex metal-sulfur assemblies in the native form have not yet been isolated. In this section, the studies aiming at the syntheses of the model compounds of two clusters in nitrogenase, FeMo cofactor and P-cluster, will be surveyed. The choice of these clusters as the representatives of the metal-sulfur clusters in metalloenzymes arises from the fact that these are the largest and most complicated metal-sulfur clusters known at present among those observed in natural enzymes. 

Preparation and Reactions of the FeMo Cofactor Model Clusters... 

Figure 4 Mo-Fe-S clusters containing two cubane-type MoFe3S4 cores, which are the extensively studied, synthetic models of FeMo-co...

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. 

Studies on the extracted FeMo cofactor has allowed researchers to answer questions about the intrinsic reactivity associated with free clusters.31 The reference 31... 

As we will see in subsequent chapters, many metalloproteins have their metal centres located in organic cofactors (Lippard and Berg, 1994), such as the tetrapyrrole porphyrins and corrins, or in metal clusters, such as the Fe-S clusters in Fe-S proteins or the FeMo-cofactor of nitrogenase. Here we discuss briefly how metals are incorporated into porphyrins and corrins to form haem and other metallated tetrapyrroles, how Fe-S clusters are synthesized and how copper is inserted into superoxide dismutase. 

Figure 3.11 (a) and (b) the P-cluster of nitrogenase in its reduced and oxidized state and (c) the FeMo-cofactor. The molecules are represented with C green, N blue, O red, S yellow, Fe orange and Mo pink. (From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc.)... 

The Fe-protein, whose molecular structure is shown in Figure 31,40,41 acts as a one-electron donor to the FeMo-protein. This electron-donating ability arises from the propensity of the Fe4S4 cluster to undergo a one-electron oxidation. 

As illustrated in Figure 32, the FeMo-protein (of Azotobacter vinelandii) incorporates the P-cluster, which for sake of simplicity can be thought of as composed of two cuboidal Fe4S4 centres each linked by a common sulfur atom, and the FeMoco (sometimes defined as the M-centre) of composition MoFe7S9. A Fe4S3 subunit and a MoFe3S3 subunit form this latter centre.42... 

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