FeMo cofactor

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...

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

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]. 

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... 

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... 

Felting fabric shrinkage, 26 390 Felting shrinkage, 24 622-623 FeMo-cofactors, 17 302, 303, 308, 309, 310, 315. See also Iron entries Molybdenum entries Femtosecond lasers, 14 620 Fenac, 13 315... 

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.)... 

Barney, B.M., Lukoyanov, D., Yang, T.-C., Dean, D.R., Hoffman, B.M. and Seefeldt, L.C. (2006) A methyldiazene (NH=N-CH3)-derived species bound to the nitrogenase active-site FeMo cofactor implications for mechanism, Proc. Natl. Acad. Sci. U.S.A., 103, 17113-17118. 

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