MoFe proteins FeMoco

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. 

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-... 

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... 

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. 

It is clear from these data that homocitrate is intimately involved in the mechanism of substrate reduction and that close homologs such as citrate cannot entirely fill this role. Rationalization of this phenomenon is difficult, but comparisons of the reactivity of extracted FeMoco from the MoFe protein from wild type and NifV strains have led to an intellectually satisfying explanation (see Section V,E,2). 

There is now some understanding of the processes involved in insertion of FeMoco into the a-ifii polypeptides (see Section IV,C,3). However, it is clear that there are other processes involved in generating apo-MoFe protein capable of being activated. These processes in-... 

The term apo-MoFe protein will be used here to denote the MoFe protein lacking FeMoco, although this protein is not devoid of prosthetic groups since it has bound P clusters. 

The purified preparations of the apo-MoFe proteins from both organisms included a small additional polypeptide of around 20 kDa. This was shown to be the nifY product in K. pneumoniae (94) and a non-nif protein denoted y in A. vinelandii (95). These proteins are apparently essential for effective reaction with FeMoco and are associated with the MoFe protein polypeptide through its interaction with the Fe protein and MgATP (96, 97) (see Section IV,C,3). These observations demonstrate a third role for the Fe protein in generating a form of apo-MoFe protein that is capable of accepting FeMoco. 

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. 

In the presence of the product ethylene during turnover the MoFe protein exhibits an EPR signal, with g values at 2.12, 1.998, and 1.987 129), which has been demonstrated to arise from FeMoco in an S = z spin state 130). However, direct interaction of the ethylene with the metal-sulfur cluster was not demonstrated. 

As noted previously, FeVaco can be extracted from AcF by the methods used to extract FeMoco from MoFe proteins (777). When FeVaco was combined with the polypeptides of the MoFe proteins from a nifB mutant, an active hybrid protein was created. However, although this protein had the H2 evolution and acetylene reduction... 

Fig. 1. Schematic illustration of the enzyme nitrogenase being composed of the molybdenum-iron (MoFe) protein, an oc2p2 tetramer with two unique iron-sulfur clusters (P-cluster) and two iron-molybdenum cofactors (FeMoco), and the iron protein with one [4Fe-4S]-cluster and two ATP binding sites.

M-centerof MoFe-prelein, FeMoco, 10 A below protein surface of a subuni 1... 

Actual electron transfer to the dinitrogen substrate at the MoFe-protein, with electrons first passing through the MoFe-protein s P-cluster. During this process, dinitrogen is most probably bound to the iron-molybdenum cofactor (FeMoco) of the MoFe-protein.6... 

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. 

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