Nitrogenases FeMoco structure

In the related V clusters a similar core structure is seen but with shorter Fe4S3 bond distances. The ten atom Fe4( 43-S)3( 4-S)3 core is contained in the FeMoco structure, Figure 3, with the same bond connectivity and similar spatial arrangement and, as such, probably provides the best starting point for development of higher nuclearity clusters related to the nitrogenase cofactors. 

A preparation of the third nitrogenase from A. vinelandii, isolated from a molybdenum-tolerant strain but lacking the structural genes for the molybdenum and vanadium nitrogenases, was discovered to contain FeMoco 194). The 8 subunit encoded by anfG was identified in this preparation, which contained 24 Fe atoms and 1 Mo atom per mol. EPR spectroscopy and extraction of the cofactor identified it as FeMoco. The hybrid enzyme could reduce N2 to ammonia and reduced acetylene to ethylene and ethane. The rate of formation of ethane was nonlinear and the ethane ethylene ratio was strongly dependent on the ratio of nitrogenase components. 

The elucidation of the crystal structures of two high-spin EPR proteins has shown that the proposals for novel Fe-S clusters are not without substance. Two, rather than one novel Fe-S cluster, were shown to be present in nitrogenase, the key enzyme in the biotic fixation of molecular nitrogen 4, 5). Thus the FeMoco-cofactor comprises two metal clusters of composition [4Fe-3S] and [lMo-3Fe-3S] bridged by three inorganic sulfur atoms, and this is some 14 A distant from the P-cluster, which is essentially two [4Fe-4S] cubane moieties sharing a corner. The elucidation of the crystal structure of the Fepr protein (6) provides the second example of a high-spin EPR protein that contains yet another unprecedented Fe-S cluster. 

Figure 2.10 Schematic structures of (a) sulfite reductase of Escherichia coli in which a 4Fe-4S cluster is linked via a cysteine to the iron in a sirohaem (b) P cluster of nitrogenase (c) FeMoCo cluster of nitrogenase (d) the binuclear site in Desulforibrio gigas hydrogenase.

Concurrently with the X-ray crystallographic studies, extended X-ray absorption fine structure (EXAFS) studies confirmed many of the bond distances proposed for nitrogenase s FeMoco cluster. The EXAFS data of reference 25 indicate short Fe-Fe distances of 2.61, 2.58, and 2.54 A for M+, M (resting state), and M forms, respectively. The authors believe that the short M center bond lengths indicate Fe-Fe bonds in this cluster. In another study using dithionite-reduced MoFe-protein Fe-S, Fe-Fe, Fe-Mo distances of 2.32, 2.64, and 2.73 A, respectively, were found in the 1 to 3 A region and Fe-Fe, Fe-S and Fe-Fe distances of 3.8, 4.3, and 4.7 A, respectively, were found in the 3 to 5 A region.30... 

Despite the availability of the molecular structures of the different active sites of the FeMo-nitrogenases, the mechanism of nitrogen fixation remains obscure. The interest of our discussion, however, is centred on the various modes proposed to describe how molecular nitrogen might coordinate the FeMoco. Some of the reported schemes are inspired by the type of coordination found in model compounds. 

Nitrogenases from various nitrogen fixing organisms seem to contain the same Fe/Mo/S structural unit that occurs as an extractable cofactor (FeMoco) (2). Extracts of the Fe-Mo component protein from inactive mutant strains of different microorganisms that do not contain the Fe/Mo/S center are activated upon addition of the FeMoco. 

Intense interest in the basic coordination chemistry of Fe/M/S clusters (M=Mo,W) has emerged in parallel with the advances in understanding the nature of the Fe/Mo/S center in the nitrogenases. Studies directed toward the synthesis of at least a structural analog for the Fe/Mo/S center have been guided by the broadly defined analytical and spectroscopic data on the Fe-Mo protein and the FeMoco. 

Extended X-ray absorption fine structure (EXAFS) studies on the Fe/Mo/S aggregate in nitrogenase have made available structural data that are essential in the design of synthetic analog clusters. Analyses of the Mo K-edge EXAFS of both the Fe-Mo protein and the FeMoco (9) have shown as major features 3-4 sulfur atoms in the first coordination sphere at 2.35 A and 2-3 iron atoms further out from the Mo atom at 2.7 A. The Fe EXAFS of the FeMoco (10,11) shows the average iron environment to consist of 3.4 1.6 S(C1) atoms at 2.25(2) A, 2.3 +0.9 Fe atoms at 2.66(3) A, 0.4 0.1 Mo atoms at 2.76(3) A and 1.2 1.0 0(N) atoms at 1.81(7) A. In the most recent Fe EXAFS study of the FeMoco (11) a second shell of Fe atoms was observed at a distance of 3.75 A. 

The solution of the x-ray crystallographic structures of the nitrogenase proteins has provided a great deal of information but has not solved the fundamental problem of how the enzyme activates and reduces N2. It is now possible to formulate much more informed hypotheses on the enzyme s mode of action, but still major questions remain, e.g., Where on FeMoco does N2 bind How is it activated for reduction and how is this activation coupled to MgATP hydrolysis Are electron and hydron transfers coordinated by the enzyme What are the intermediate steps in N2 reduction Are these steps the same for all three nitrogenases ... 

The structure of FeMoCo (and the dinitrogen-binding protein) is unknown. The environment of the metals is constituted primarily of sulfur atoms, but the interpretation of the X-ray absorption fine structure (EXAFS) data on both the cofactor and protein (117-119) are contentious. It is not known whether the system in any specific part is aqueous or anhydrous, but oxygen destroys the activity. The electron-transfer pathway in the functioning nitrogenase is believed to be as shown in Scheme 22. 

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... 

Regarding the structure and function of nitrogenases in producing ammonia from N2, Sellmann has studied several model systems wherein heterolytic activation of H2 occurs on sulfur ligands (87). A core geometry based on a hybrid of the FeMoco active site structure with a dinuclear diazene complex, [Fe( NHS4 )]2(p-N2H2), is a proposed model (Scheme 4). 

The principle advances in the area has been made using x-ray structural analysis. Crystallographic data have been first produced for the nitrogenase complex of FeP (A2) and FeMoP (Al) from Azotobacter vinelandii (Kim and Rees, 1992) and for the corresponding complex of Cp2 and Cpl from Clostridium pasterianum (Bolen et al., 1993), ). A 1.6 A resolution X-ray crystallographic structure of Klebsiella pneumoniae proteins has been recently reported (Mayer et al., 1999) It was shown that FeMoco sites in Al, Cpl, and Kpl are 70 A apart and FeMoco and P clusters are separated by about 19 A. X-ray structures of the nitrogenase complex and the active site clusters are presented in (Figs. 3.2-3.4). 

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