Metal clusters, MoFe-protein

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. 10. The putative transition-state complex formed between the Fe protein MgADP AlFj and the MoFe protein. For simplicity only one a/3 pair of subunits of the MoFe protein is shown. The polypeptides are indicated by ribbon diagrams and the metal-sulfur clusters and MgADP AlFi" by space-filling models (MOLSCRIPT (196)). The figure indicates the spatial relationship between the metal-sulfur clusters of the two proteins in the complex.

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. 

For further details about the progress in the syntheses of the structural models of the clusters in MoFe protein, please see the other recent reviews,33 together with those dealing with the reactions of metal-sulfur clusters relating to the nitrogen-fixation chemistry.34... 

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. 

The so-called midpoint potential, Em, of protein-bound [Fe-S] clusters controls both the kinetics and thermodynamics of their reactions. Em may depend on the protein chain s polarity in the vicinity of the metal-sulfur cluster and also upon the bulk solvent accessibility at the site. It is known that nucleotide binding to nitrogenase s Fe-protein, for instance, results in a lowering of the redox potential of its [4Fe-4S] cluster by over 100 mV. This is thought to be essential for electron transfer to MoFe-protein for substrate reduction.11 3... 

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. 

Core extrusion studies—removal of the iron-sulfur cluster intact from the enzyme surroundings—have been carried out and the iron-cluster types in proteins identified through the process shown in equation 6.10.18 DMS0/H20 is the protein unfolding solvent for this process. By this method, Fe-protein and MoFe-protein metal-sulfur clusters have been removed from the holoenzyme for separate analysis by many instrumental techniques. 

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). 

Figure 24-3 Structures of the metal-sulfide clusters of the MoFe-protein.

The conversion of dinitrogen to ammonia is one of the important processes of chemistry. Whereas the technical ammonia synthesis requires high temperature and pressure (1), this reaction proceeds at room temperature and ambient pressure in nature, mediated by the enzyme nitrogenase (2). There is evidence that N2 is bound and reduced at the iron-molybdenum cofactor (FeMoco), a unique Fe/Mo/S cluster present in the MoFe protein of nitrogenase. Although detailed structural information on nitrogenase has been available for some time (3), the mechanism of N2 reduction by this enzyme is still unclear at the molecular level. Nevertheless, it is possible to bind and reduce dinitrogen at simple mono- and binuclear transition-metal systems which allow to obtain mechanistic information on elemental steps involved... 

Combined EPR and 57Fe Mossbauer spectroscopic studies of the MoFe protein in various overall oxidation states39 40,42,44 48,49) have provided strong evidence for the presence of six metal clusters two M centers that are the protein-bound form of the FeMo-cofactor (a novel Mo-Fe-S cluster) and four unusual tetranuclear iron clusters referred to as the P clusters. These will be discussed separately below. In addition, Mossbauer spectra of MoFe proteins from Azotobacter vinelandii, Clostridium pas-teurianum, and Klebsiella pneumoniae all show an additional component termed S39 40 42, which accounts for 6% of the total Fe present (2 Fe per molecule) and which has Mossbauer parameters (AEq = 1.35 mm/s d = 0.60 mm/s) different from those expected for likely impurities such as high-spin Fe2+ or Fe3+ ( adventitious iron). At present, it is difficult to decide whether species S is an unusual and persistent impurity or an integral part of the MoFe protein. 

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...

The MoFe protein is an aifli tetramer of Mr 220 kDa, and its a and subunits are encoded by the nifD and nifK gene, respectively (Figure 1(a) and Table 1). It contains, in preparations with the highest activity, 2 molybdenum (Mo), 30 to 34 iron (Fe), and an approximately equivalent number of acid-labile sulfur (S ) atoms (Table 1). This metal content is consistent with the presence of two different types of unique metal clusters in the protein, that is, the [8Fe-7S] cluster (P cluster), which is bridged between each a/3 subunit pair, and the [Mo-7Fe-9S-homocitrate] cluster (FeMo cofactor or FeMoco), which is located within each a subunit (Figure la). ... 

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