Fe-Protein Structure

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 

The two protein subunits found in the holoenzyme, each containing an iron-sulfur cluster, fold into interconnected a-helices and p-pleated sheet that, at their interface, ligate the 4Fe-4S cluster through four cysteine residues, two from each 

In reference 21, the Fe-protein X-ray structures of A. vinelandii, Av2, PDB code 2NIP, at 2.2-A resolution is compared to that of C. pasteurianum, Cp2, PDB code 1CP2, at resolution 1.93 A as well as to Fe-protein aa sequences and 

---

Figure 1 Schematic representation of the Fe-protein (60 kD, 2 dimer) and the MoFe-protein (250 kD, a2fh. tetramer) of the Mo-nitrogenase. Outline of the electron transfer path through the metal clusters. Structure of the Fe4S4 cluster of the Fe-protein. Structure of the P-cluster (in two oxidation states) and of the FeMo-cofactor in the MoFe-Protein. Limiting stoichiometry for the function of Mo-nitrogenase...

Kinetic studies indicate that nucleotide hydrolysis precedes electron transfer in the Fe-protein-MoFe-protein complex (12, 13, 114). Although the detailed structural consequences of nucleotide binding on the Fe-protein structure have not been established, it is likely that... 

RL Dunbrack Jr, DL Gerloff, M Bower, X Chen, O Lichtarge, FE Cohen. Meeting review The second meeting on the critical assessment of techniques for protein structure prediction (CASP2), Asilomar, CA, Dec 13-16, 1996. Folding Des 2 R27-R42, 1997. 

Fig. 2. The structure of the Fe protein (Av2) from Azotobacter vinelandii, after Geor-giadis et al. (1). The dimeric polypeptide is depicted by a ribbon diagram and the Fe4S4 cluster and ADP by space-filling models (MOLSCRIPT (196)). The Fe4S4 cluster is at the top of the molecule, bound equally to the two identical subunits, Emd the ADP molecule spans the interface between the subunits with MoO apparently binding in place of the terminal phosphate of ATP.

Studies (113) on Fe protein with an Asp39Asn mutation in the nucleotide binding site indicate that the conformational change observed in the crystal structure of the complex is dependent on electron treuis-fer from the Fe protein to the MoFe protein. 

The solutions of the structures of the Fe protein, the MoFe proteins, and the putative transition-state complex of these two proteins with ADP and A1F represent major advances in our understanding of ni-trogenase, but still many questions remain. 

When one of the Fe-coordinating Ns of the porphyrin is made inequivalent to the others, for example, by pulling on it, or by putting a protein structure around the cofactor, then the molecular x axis and y axis become inequivalent, and the axial EPR spectrum turns into the rhombic spectrum in trace d with derivative trace e (see also Table 5.4). There are now three features in the spectrum a peak, a zero crossing, and a negative peak, and their field positions closely (exactly for zero linewidth) correspond to those of the g-values, gx, gy, and gz. Finally, in trace f of Figure 5.4, which is the experimental X-band spectrum of cytochrome c, it can be seen that not only the g-value (peak position) but also the linewidth is frequently found to be anisotropic. This extra complication will be discussed extensively in Chapter 9. 

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. 

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. 

---