MoFe protein crystals

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

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. 

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. 

However, when the X-ray crystal structure of the MoFe protein was examined, it was clear that homocitrate could not directly hydrogen bond to the histidine, since the carboxylate group and imidazole are stacked parallel to each other in the crystal. Nevertheless, as noted in the previous section, studies on model complexes have suggested that homocitrate can become monodentate during nitrogenase turnover, with the molybdenum carboxylate bond breaking to open up a vacant site at molybdenum suitable for binding N2. 

The 1.6-A resolution X-ray crystal structure of Klebsiella pneumoniae MoFe-protein, called Kpl, was solved in 1999 using the coordinates of the 2.2-A Avl... 

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. 

Figure 6.5 Precession photographs of the hk0 plane in native (a) and heavy-atom (b) crystals of the MoFe protein from nitrogenase. Corresponding underlined pairs in the native and heavy-atom patterns show reversed relative intensities. Photos courtesy of Professor Jeffrey Bolin.

The X-ray crystal structures of several conformations of the Fe protein, either alone or in complex with the MoFe protein, have been solved. " Figure 2 shows the MgADP-bound conformational state of the Fe protein of A. vinelandii,where each subunit folds as a single aZ/S-type domain and the subunits together symmetrically ligate the surface-exposed [4Fe-4S] cluster through two cysteines from each subunit. Two MgADP molecules are... 

The X-ray crystal structures of MoFe protein, alone or in complex with the Fe protein, have been reported for A. vinelandii,C. pasteurianum, and K. pneumoniae, ... 

As mentioned above, a FeMo cofactor-deficient MoFe protein was obtained from a nifB deletion strain of A. vinelandii, and its X-ray crystal stmcture has been resolved at 2.3 A.Like the wild-type A. vinelandii MoFe protein (see Section 2.2), this MoFe protein (designated NifB MoFe protein) is an a2Pi tetramer consisting of a pair of ap dimers that are related by a molecular twofold rotation axis, and each subunit consists of three domains, designated a, an, and alll and p, p, and jSin, respectively. In the dithionite-reduced state, the P cluster of the NifB MoFe protein, hke that of its wild-type counterpart, is assigned to the P state and is located between domains a I and pi. However, the FeMo cofactor of the wild-type MoFe protein, which occupies a cavity formed between domains... 

With the availability of the crystal structures for both nitrogenase proteins, some general features of the complex between the two proteins may be addressed. Two residues of Fe-protein that have been identified as interacting with the MoFe-protein, Arg 100 (109) and Glu 112 (110),... 

The MoFe proteins from Clostridiumpasteurianum and from Azotobacter vinelandii have been crystallized. For the former protein, crystals of space group P2i are obtained, with two molecules per unit cell of dimensions 70 X 151 X 122 A. There is good evidence for a molecular two-fold axis, which presumably relates equivalent sites in the two a/3 dimers that make up the protein molecule.Preliminary refinement reveals that the two FeMoco units per protein are about 70 A apart and the four P clusters are grouped in two pairs. 

The X-ray crystal structures of MoFe protein, alone or in complex with the Fe protein, have been reported for A. vinelandii,C. pasteunanum, and K. pneumoniae, and all of these structures are highly conserved on the basis of both primary sequence and three-dimensional structure. The MoFe protein of A. vinelandii is an azPz heterotetramer consisting of a pair of ap dimers related by a molecular twofold rotation axis (Figure The homologous a and P subunits include three domains, designated al, all, and alll and pi, pH, and jSIII, respectively, all of which exhibit parallel /3-sheet/a-helical type of polypeptide folds. Domains of both subunits contribute ligands to the P cluster. These ligands are located in a common core of a four-stranded, parallel /3-sheet flanked by a-helices and additional /3-strands. The P clusters are located between... 

As mentioned above, a FeMo cofactor-deficient MoFe protein was obtained from a nifB deletion strain of A. vinelandii, and its X-ray crystal structure has been resolved at 2.3 A.Like the wild-type A. vinelandii MoFe protein (see Section 2.2), this MoFe protein (designated NifB MoFe protein) is an tetramer consisting of a pair of a/S... 

The FeMoco has been observed in three redox states. When the enzyme is isolated in the presence of dithionite, the FeMoco is in the M ( native ) form. Most c stallographic studies have taken place on enzyme in which the FeMoco is in the M state. M° can be generated by one-electron oxidation of M with dyes, and can be reactivated by reduction. The M° /M redox potential is dependent on the organism from which the nitrogenase is derived, lying in the range OmV to — 180mV. " The X-ray crystal structure of MoFe protein with the FeMoco in the M° state shows no major differences from M is the turnover state of the enzyme,... 

Iron-vanadium nitrogenase was isolated in 1986. Its nitrogen-reducing activity is 1/3-1/2 of that for molybdenum nitrogenase, and the VFe protein is spectroscopically different from the analogous MoFe protein. There is no X-ray crystal structure of any component of iron-vanadium nitrogenase. 

Finally, the FeMoeo is transferred to nifDK (apo-MoFe protein) to form the holo-MoFe protein with the help of ATP and several proteins nifH, 7, and GroEL. The X-ray crystal structure of nifDK shows a positively charged channel through which FeMoeo could enter. " The mf system for synthesis of the FeVco is thought to be similar to the nif system, with added assistance in the last step by vnfG, a third subunit of VFe nitrogenase that is necessary for activity. 

A famous example is the structure of Nitrogenase MoFe-Protein, a protein that contains a Fe7MoS9 cluster. The inside of this cluster is about 4 A wide with six iron atoms closest to the centre, and older crystal stmctures had been determined at resolutions of about 2 A. Termination of the Fourier summation at that resolution creates an artefactual miiumum in the electron density of about —0.2 electrons about 2 A away from each iron atom. These spurious minima from all heavy atoms in the... 

The Mo enzyme consists of two components (1) the Fe protein (molecular weight 57,000 daltons), which contains iron and sulfur (4 atoms of each per protein) and (2) the MoFe protein (220,000 daltons, Uz subunits), which contains both metals (1 atom Mo, 32 atoms Fe). Each also contains S ions (ca. one per iron), which act as bridging ligands for the metals. The protein contains special Fe—S clusters called P clusters that have EPR resonances like those of no other Fe—S cluster. A soluble protein-fiee molybdenum and iron-containing cluster can be separated from the enzyme. This iron-molybdenum cofactor, or FeMo-co, was known to have approximately 1 Mo, 7-8 Fe, 4-6 S ", and one molecule of homocitrate ion. As for the P cluster, there was no agreement on the structure of FeMo-co for many years. In purified form FeMo-co does crystallize, and it can restore N2 reducing activity to samples of mutant N2ase that are inactive because they lack FeMo-co. On the other hand, no crystal structure of FeMo-co proved possible, and no synthetic model complex was found that could activate the mutant enzyme. 

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