Cofactor clusters

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 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. 

Proposed cofactor cluster in A. vinelandii FeMo protein. 

See Figure 5.4 for the atom labeling scheme. The metrics are averaged to C3, symmetry forall clusters for the FeMo-cofactor, all four independent cofactor clusters in the crystal structure are averaged. Listed uncertainties are standard deviations from the mean. 

A substantial fraction of the named enzymes are oxido-reductases, responsible for shuttling electrons along metabolic pathways that reduce carbon dioxide to sugar (in the case of plants), or reduce oxygen to water (in the case of mammals). The oxido-reductases that drive these processes involve a small set of redox active cofactors , that is, small chemical groups that gain or lose electrons. These cofactors include iron porjDhyrins, iron-sulfur clusters and copper complexes as well as organic species that are ET active. 

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. 

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). 

Homocitrate is bound to the molybdenum atom by its 2-carboxy and 2-hydroxy groups and projects down from the molybdenum atom of the cofactor toward the P clusters. This end of FeMoco is surrounded by several water molecules (5, 7), which has led to the suggestion that homocitrate might be involved in proton donation to the active site for substrate reduction. In contrast, the cysteine-ligated end of FeMoco is virtually anhydrous. 

The elucidation of the crystal structures of two high-spin EPR proteins has shown that the proposals for novel Fe-S clusters are not without substance. Two, rather than one novel Fe-S cluster, were shown to be present in nitrogenase, the key enzyme in the biotic fixation of molecular nitrogen 4, 5). Thus the FeMoco-cofactor comprises two metal clusters of composition [4Fe-3S] and [lMo-3Fe-3S] bridged by three inorganic sulfur atoms, and this is some 14 A distant from the P-cluster, which is essentially two [4Fe-4S] cubane moieties sharing a corner. The elucidation of the crystal structure of the Fepr protein (6) provides the second example of a high-spin EPR protein that contains yet another unprecedented Fe-S cluster. 

D. gigas AOR was the first Mo-pterin-containing protein whose 3D structure was solved. From D. desulfuricans, a homologous AOR (MOD) was purified, characterized, and crystallized. Both proteins are homodimers with-100 kDa subunits and contain one Mo-pterin site (MCD-cofactor) and two [2Fe-2S] clusters. Flavin moieties are not found. The visible absorption spectrum of the proteins (absorption wavelengths, extinction coefficients, and optical ratios at characteristic wavelengths) are similar to those observed for the deflavo-forms of... 

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