MoFe proteins centers

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 exhibit complex redox properties. Each tetra-meric a2/32 molecule of MoFe protein contains two P clusters and two FeMoco centers and, as normally isolated in the presence of sodium dithionite, the FeMoco centers are EPR-active, exhibiting an S = spin state with g values near 4.3 and 3.7 and 2.01 (Fig. 6). The P clusters are EPR silent and there is a wealth of evidence (39) using a variety of techniques that indicates that the iron atoms in these clusters are all reduced to the Fe state. 

Removing two electrons from each P cluster renders each MoFe protein molecule oxidized by four electrons. Further oxidation leads to removal of electrons from the FeMoco centers. The potential of this oxidation is both species and pH dependent. At pH 7.9 the for Kpl is 180 mV, whereas for Cpl it is 0 mV and for Avl -95 mV (46). 

Yet further oxidation removes at least one more electron from each P cluster with an +90 mV to yield a protein oxidized by a total of at least eight electrons and with EPR signals from mixed spin states of S = I and S = I (42, 47). The combined integrations of these signals demonstrated that their intensity was equivalent to that of the FeMoco EPR signals in the same preparations. This provided the first evidence (47) that MoFe proteins contained equivalent numbers of FeMoco centers and P clusters and that P clusters contained 8 Fe atoms. Previously it had been considered that the P clusters were fully reduced Fe4S4 clusters and thus that there were two P clusters for every FeMoco center per molecule. 

In general there are few reproducible data on binding of reducible substrates to the isolated MoFe proteins. However, the S = EPR signal from the FeMoco centers of Kpl is pH dependent, the g values changing with a pKa of 8.7 (50). Of course, the proton is a substrate of nitrogenase however, there is no direct evidence for the proton associated with the pKa being bound directly to FeMoco. Nevertheless, this pKa can be perturbed by addition of the analog substrate acety-... 

From the crystal structure of the complex (Fig. 10) it appears that only minimal conformational changes occur within the MoFe protein on complexation, although it is hard to be dogmatic about these when at 3 A resolution. Nevertheless, ENDOR 116) studies on the FeMoco center demonstrated that at least one class of protons in the vicinity of the FeMoco center is altered in the complex relative to the free protein. 

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. 

To successfully describe the structure and function of nitrogenase, it is important to understand the behavior of the metal-sulfur clusters that are a vital part of this complex enzyme. Metal-sulfur clusters are many, varied, and usually involved in redox processes carried out by the protein in which they constitute prosthetic centers. They may be characterized by the number of iron ions in the prosthetic center that is, rubredoxin (Rd) contains one Fe ion, ferredoxins (Fd) contain two or four Fe ions, and aconitase contains three Fe ions.7 In reference 18, Lippard and Berg present a more detailed description of iron-sulfur clusters only the [Fe4S4] cluster typical of that found in nitrogenase s Fe-protein is discussed in some detail here. The P-cluster and M center of MoFe-protein, which are more complex metal-sulfur complexes, are discussed in Sections 6.5.2. and 6.5.3. 

Fe-protein, the unique, highly specific electron donor to MoFe-protein, mediates coupling between ATP hydrolysis and electron transfer to MoFe-protein and also participates in the biosynthesis and insertion of FeMoco into MoFe-protein. Fe-protein contains one ferredoxin-like [Fe4S 4 2 /1+ cluster as its redox center. There is now evidence for an [Fe4S4]° super-reduced state in which four high-spin iron(II) (S= 2) sites are postulated. These were previously discussed in Section 6.3 and illustrated in Table 6.1.16 The [Fe4S4] cluster in this state bridges a dimer of... 

It is now believed that the MoFe-protein s P-cluster contains a [4Fe-3S] cuboid joined to a [4Fe-4S] cuboid, although, as discussed below, it was first reported crystallographically as two [4Fe-4S] clusters.8 Uncertainty existed for sometime as to exact nature of bridging disulfide or sulfide ligand joining the two Fe S clusters but it is now known that the P-cluster does NOT contain a disulfide bond. This is important because the all-ferrous structure [4Fe-4S]° proposed from Mossbauer studies then becomes more possible for the P-cluster s [4Fe-4S] cube. In 1993 Bolin et al.1 proposed a six-coordinate S for the P-cluster s center as in Figure la,b of Thorneley s article.8 This is now believed to be the correct conformation. A central six-coordinate S makes this cluster much harder to synthesize in the laboratory, and this feat has not been accomplished as of the date of this text s publication. Whatever its oxidation state or structure, the P-cluster mediates electron transfer from Fe-protein to the M center of MoFe-protein, and it must be reduced at some point to allow transfer of its electron(s). 

The P-cluster, located at the interface of MoFe-protein s a- and (3-subunits, is believed to function as the electron transfer mediator between Fe-protein and the N2 reduction site at the M center. The P-cluster is contained within a hydrophobic environment and located approximately 10 A below the MoFe-protein surface. Three cysteine side chains from each subunit bind to iron ions in the P-cluster. The cluster is now known to exist in Pox and PN forms in active enzyme, both with stoichiometry FegS7. The PN form, with its octahedrally coordinated central sulfur, has the structure shown in Figure 6.6. As can be seen in Table 6.3, the PN form contains all ferrous irons, corresponding to the P (5 = 0) state, whereas the Pox form corresponds to the P2+ (5=3 or 4) form. 

Figure 6.8 The M center (FeMoco) of MoFe- protein as reprinted from Figure 2A of reference 29b. "Y" atom assigned as sulfur in reference 24. (Reprinted with permission from Kim, J. Rees, D. C. Science 1992, 257, 1677-1682. Copyright 1992, American Association for the Advancement of Science.)...

Figure 6.9 Schematic diagram of the M center of MoFe-protein.

Concurrently with the X-ray crystallographic studies, extended X-ray absorption fine structure (EXAFS) studies confirmed many of the bond distances proposed for nitrogenase s FeMoco cluster. The EXAFS data of reference 25 indicate short Fe-Fe distances of 2.61, 2.58, and 2.54 A for M+, M (resting state), and M forms, respectively. The authors believe that the short M center bond lengths indicate Fe-Fe bonds in this cluster. In another study using dithionite-reduced MoFe-protein Fe-S, Fe-Fe, Fe-Mo distances of 2.32, 2.64, and 2.73 A, respectively, were found in the 1 to 3 A region and Fe-Fe, Fe-S and Fe-Fe distances of 3.8, 4.3, and 4.7 A, respectively, were found in the 3 to 5 A region.30... 

Fe4(p3-S)3(p2-S)3, a 10-atom cluster present in the nitrogenase M center. Fragment condensation was used by the authors of reference 37 to synthesize [MoFe6S6(CO)i6 2 from the fragments Fe2S2(CO)612 and [Mo(CO)4I3] . None of the clusters mentioned so far mimic either the total structure or any function of those present in MoFe-protein. 

Figure 3.28 The FeMo cofactor, M center, of nitrogenase s MoFe protein.

The P clusters of nitrogenase. The enzyme nitrogenase consists of two proteins the Fe protein (m.w. 55,000), which contains a single 4Fe-4S center, and the more complex MoFe protein (m.w. 220,000) (48,49). The minimum functional unit of the latter appears to be the half molecule, an asymmetric dimer containing 1 Mo, 14-16 Fe, and 16-18 sulfides. Application of a vast array of spectroscopic methods to the MoFe protein in a variety of oxidation states has led to the conclusion that it contains two types of metal-sulfur cluster in a 2 1 ratio unusual Fe S units termed P clusters, and the protein-bound form of the FeMo-cofactor (50). 

D = -0.7 cm", E/D = 0.26 for the S = 3/2 center in dithionite-reduced AvV (B. J. Hales, unpublished results). MCD magnetization data for dithionite-reduced Avl and Avl (8) indicate that the EPR-detectable S = 3/2 paramagnets are also responsible for the temperature-dependent MCD transitions. Therefore, by analogy with the MoFe protein it seems probable that the S = 3/2 paramagnetic chromophore in dithionite-reduced Avl is a V-Fe-S cluster. However, the EPR and MCD studies indicate that this cluster has magnetic and electronic properties distinct from that of the Mo-Fe-S clusters in conventional Mo nitrogenases. 

One type of the constituent metallocenters in the MoFe protein has the properties of a somewhat independent structural entity. This component, referred to as the FeMo cofactor (FeMo-co), was first identified by Shah and Brill (1977) as the stable metallocluster extracted from acid-denatured MoFe protein. The FeMo-co was able to fully activate a defective protein in the extracts of mutant strain UW45, a protein which subsequently was shown to contain the P clusters but not the EPR-active center. The isolated cofactor accounted for the total S = t system observed by EPR and Mdssbauer spectroscopies of the holo-MoFe protein (Rawlings et al., 1978). Elemental analysis indicated a composition of Mo Fee-8 Se-g for the cofactor, which, if there are two FeMo-co s per a2 2> accounts for all the molybdenum and approximately half the iron in active enzyme (Nelson etai, 1983). Although FeMo-co has been extensively studied [reviewed in Burgess (1990)] the structure remains enigmatic. To date, all attempts to crystallize the cofactor have failed. This is possibly due to the instability and resultant heterogeneity of the cofactor when removed from the protein. Also, there is a paucity of appropriate models for spectral comparison (see Coucouvanis, 1991, for a recent discussion). Final resolution of this elusive structure may require its determination as a component of the holoprotein. 

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