Nitrogenase MoFe protein function

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. 

A comprehensive description of the mechanism of molybdenum nitrogenase has been provided by the Lowe-Thorneley scheme 102) (Figs. 8 and 9). In this scheme the Fe protein (with MgATP) functions as a single electron donor to the MoFe protein in the Fe protein cycle (Fig. 8), which is broken down into four discrete steps, each of which may be a composite of several reactions ... 

It is becoming clear that the MgATP hydrolysis is not required to induce protein-protein electron transfer, but its role in nitrogenase function is still undefined. The most likely hypothesis at the moment is that its hydrolysis, on the Fe protein, induces important changes in the MoFe protein, presumably by altering the conformation of the enzyme complex. Nevertheless, the nature of the changes in the MoFe protein remain obscure. 

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. 

Fe4(p3-S)3(p2-S)3, a 10-atom cluster present in the nitrogenase M center. Fragment condensation was used by the authors of reference 37 to synthesize [MoFe6S6(CO)i6 2 from the fragments Fe2S2(CO)612 and [Mo(CO)4I3] . None of the clusters mentioned so far mimic either the total structure or any function of those present in MoFe-protein. 

Mayer, S.M., Lawson, D.M., Gormal, C.A., Roe, S.M. and Smith B.E. (1999) New insights into structure-function relationships in nitrogenase A 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein, J. Mol Biol., 292, 871-891. 

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). 

As noted earlier, nitrogenase is made up of two proteins, the iron protein, and the molybdenum-iron protein, and will be linked to an electron-transport chain. The iron protein accepts electrons from this chain (a ferredoxin or flavodoxin in vivo, or dithionite in vitro) and transfers them to the molybdenum-iron protein. The MoFe protein is then able to reduce a number of substrates in addition to dinitrogen. No replacement electron donor will function instead of the iron 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 most comprehensive model for the function of molybdenum nitrogenase in the reduction of N2 is that of Lowe and Thomeley, which was developed almost two decades ago. This model describes two aspects of nitrogenase catalysis, the Fe protein cycle and the MoFe protein cycle. 

Figure 9 The MoFe protein cycle of the molybdeniun nitrogenase." The cycle describes a plausible sequence of events during the reduction of N2 to 2NH3 and H2. M represents one functional half of the MoFe protein, which is composed of one afi subunit pair plus one FeMo cofactor and one P cluster. Subscripts 0 to 7 indicate the number of electrons transferred to M from the Fe protein via the Fe protein cycle. It is plausibly assumed that each transferred electron is neutralized by the addition of one proton. Each dotted arrow corresponds to one completed Fe protein cycle (shown in Figure 7)...

In addition to the mechanistic role in the nitrogenase enzymatic function, Fe-protein also participates at several stages in the biosynthesis of the nitrogenase proteins. Fe-protein is essential for the production of active MoFe-protein and is involved in both the synthesis of FeMo-cofactor and its insertion into cofactor-deficient MoFe-protein (40-42). Fe-protein may also function as an activator for the expression of alternative nitrogenases (43). In turn, formation of active Fe-protein requires the nifM gene product (44, 45), which perhaps functions either in cluster insertion or in promoting the correct subunit-subunit and subunit-cofactor interactions in the Fe-protein dimer (i.e., a chaperone-type role). The significant sequence conservation observed in the Fe-protein family may reflect the structural constraints associated with these diverse aspects of Fe-protein function. 

For the process in vivo flavodoxin is the external electron donor. For isolated nitrogenase, dithionite 8204 is traditionally used as electron donor. If dithionite is the external donor, the oxidized iron protein with 2MgADP (after the electron is transferred) then dissociates from the MoFe protein. This dissociation, which initially seemed necessary for enzyme function, is, however, apparently a result of the salt effect of dithionite, the concentration of which for effective electron transfer must be sufficiently large. 

The component proteins of Mo nitrogenase are MoFe protein, an 2)32 tetramer encoded by the nifDK genes, and an Fe protein, a y2 dimer encoded by nifH. The formation of an active enzyme requires, in addition to these structural genes, the functions of several other nif genes. The nif M in some unknown way activates the Fe protein polypeptide and is essential for its function. A number of nif genes are involved in FeMoco biosynthesis (nifHBENVQ) and are therefore essential for the synthesis of an active MoFe protein. 

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