MoFe-protein

Fig. 4. Requirements, substrates, and products of Mo-nitrogenase catalysis, where I is the MoFe protein II the Fe protein and Pi is inorganic phosphate. The generating system is composed of creatine phosphate and creatine phosphokinase to recycle the inhibitory MgADP produced during catalysis to...

Structure of the MoFe Protein. Extensive spectroscopic studies of the MoEe proteia, the appHcation of cluster extmsion techniques (84,151), x-ray anomalous scattering, and x-ray diffraction (10,135—137,152) have shown that the MoEe proteia contains two types of prosthetic groups, ie, protein-bound metal clusters, each of which contains about 50% of the Ee and content. Sixteen of the 30 Ee atoms and 14—16 of the 32—34... 

Fig. 6. View of the nitrogenase MoFe protein P-cluster pair where ( ) represents Fe, (O) S, and (Q) C as modeled (153). The side chain of one of the...

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...

In late 1992 the first crystal structures of the Fe and MoFe proteins of Mo nitrogenase frora Azotobacter vinelandii were published (1-3). 

The nitrogenase proteins are generally characterized by two letters indicating the species and strains of bacteria and the numerals 1 for the MoFe protein and 2 for the Fe protein. Thus, the Fe protein from Azotobacter vinelandii is Av2 and the MoFe protein from Klebsiella pneumoniae is Kpl. 

As well as donating electrons to the MoFe protein, the Fe protein has at least two and possibly three other functions (see Section IV,C) It is involved in the biosynthesis of the iron molybdenum cofactor, FeMoco it is required for insertion of the FeMoco into the MoFe protein polypeptides and it has been implicated in the regulation of the biosynthesis of the alternative nitrogenases. 

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). 

Figure 3 shows the three-dimensional structure of the MoFe protein from Klebsiella pneumoniae, Kpl, obtained at 1.65-A resolution (7). The overall structure of the polypeptides is frilly consistent with that reported earlier for Avl (3). The a and /8 subunits exhibit similar polypeptide folds with three domains of parallel /3 sheet/a helical type. At the interface between the three domains in the a subunit is a wide shallow cleft with the FeMoco at the bottom of the cleft about 10 A from the solvent. FeMoco is enclosed within the a subunit. The P cluster, however, is buried within the protein at the interface between the a and /8 subunits, being bound by cysteine residues from each subunit. A pseudo-twofold rotation axis passes between the two halves of the P cluster and relates the a and (3 subunits. Each af3 pair of subunits contains one FeMoco and one P cluster and thus appears... 

Fig. 3. The tetrameric structure of the MoFe protein (Kpl) from Klebsiella pneumoniae (7). The two FeMoco clusters and the P clusters are depicted by space-filling models and the polypeptides by ribbons diagrams (MOLSCRIPT (196)). The FeMoco clusters are bound only to the a subunits, whereas the P clusters span the interface of the a and j8 subunits.

FeMoco can be extracted from the MoFe protein into A(-methylfor-mamide (NMF) solution 32) and has been analyzed extensively using a wide range of spectroscopic techniques both bound to the protein and in solution after extraction from it (33). The extracted FeMoco can be combined with the MoFe protein polypeptides, isolated from strains unable to synthesize the cofactor, to generate active protein. The structure of the FeMoco is now agreed 4, 5, 7) as MoFeTSg homocitrate as in Fig. 4. FeMoco is bound to the a subunit through residues Cys 275, to the terminal tetrahedral iron atom, and His 442 to the molybdenum atom (residue numbers refer to A. vinelandii). A number of other residues in its environment are hydrogen bonded to FeMoco and are essential to its activity (see Section V,E,2). The metal... 

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. 

During oxidation of the MoFe protein the P clusters are the first to be oxidized at about -340 mV. This redox potential was first measured (40) using Mossbauer spectroscopy and exhibited a Nemst curve consistent with a two-electron oxidation process. It is possibly low enough for this redox process to be involved in enzyme turnover (see Section V). No additional EPR signal was observed from this oxidized form at this time. However, later a weak signal near g = 12 was detected and was finally confirmed, using parallel mode EPR... 

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. 

C. Substrate Interactions with the MoFe Protein 1. Interactions with Reducible Substrates... 

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-... 

The significance of the observed interactions between MoFe proteins and nucleotides is not easy to determine. However, as noted in Section IV, ATP is involved in the maturation of the MoFe protein and the biosynthesis of FeMoco. It is therefore possible that the nucleotide interactions noted here are associated with the maturation of the MoFe protein and not with its catalytic activity. 

Fe protein polypeptide NifH is also involved in FeMoco and MoFe protein biosynthesis MoFe protein polypeptides... 

Probably involved with NifS in iron-sulfur cluster synthesis Possibly accelerate MoFe protein maturation Unknown function... 

The biosynthesis of the MoFe protein is extremely complex. Here we will first describe the biosynthesis of FeMoco, then that of the apo-MoFe protein, encoded by the nifD and nifK genes and containing the P clusters, and finally we will summarize what is known about the combination of FeMoco with the apo-MoFe protein to form active MoFe protein. 

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