Mo nitrogenase

Fig. 4. Requirements, substrates, and products of Mo-nitrogenase catalysis, where I is the MoFe protein II the Fe protein and Pi is inorganic phosphate. The generating system is composed of creatine phosphate and creatine phosphokinase to recycle the inhibitory MgADP produced during catalysis to...

In contrast to the situation with the alternative nitrogenases, but with the notable exception of the C. pasteurianum proteins, the component proteins from aU. Mo-based nitrogenases interact as heterologous crosses to form catalyticaHy active enzymes (52). Carbon monoxide, CO, is a potent inhibitor of aU. nitrogenase-cataly2ed substrate reductions, with the exception of reduction (126). Molecular hydrogen has a unique involvement with Mo-nitrogenase... 

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

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

Molybdenum nitrogenase has been the subject of intensive study for more than 30 years, but much less work has been done on the vanadium and iron-only nitrogenases. Consequently, we first review the properties of Mo nitrogenase, and then in later sections outline what is known of the other two enzymes. 

Multinuclear M centre Nitrogenases (Mo, V, Fe) Mo-nitrogenase Dinitrogen to ammonia... 

Figure 1 Structure of the MoFe7SgN cluster of FeMo-co in Mo nitrogenase...

D = -0.7 cm", E/D = 0.26 for the S = 3/2 center in dithionite-reduced AvV (B. J. Hales, unpublished results). MCD magnetization data for dithionite-reduced Avl and Avl (8) indicate that the EPR-detectable S = 3/2 paramagnets are also responsible for the temperature-dependent MCD transitions. Therefore, by analogy with the MoFe protein it seems probable that the S = 3/2 paramagnetic chromophore in dithionite-reduced Avl is a V-Fe-S cluster. However, the EPR and MCD studies indicate that this cluster has magnetic and electronic properties distinct from that of the Mo-Fe-S clusters in conventional Mo nitrogenases. 

A large number of studies devoted to metal-sulfur centers are motivated by the occurrence of such arrangements at the active site of various metalloenzymes [1-13]. Mononuclear complexes with Mo=0 func-tion(s) and possessing sulfur ligands in their coordination sphere have been extensively investigated since they can be seen as models of the active site of enzymes such as nitrate- and DM SO reductases or sulfite- and xanthine oxidases [1-4]. On the other hand, a large variety of mono-, di-, and polynuclear Mo—S centers have been synthesized in order to produce functional models of the Mo-nitrogenase since the exact nature (mono-, di- or polynuclear) of the metal center, where N2 interacts within the iron-molybdenum cofactor (FeMo—co) of the enzyme is still unknown [4-8]. 

In the earlier examples, the binuclear complexes with a M02S3 core clearly cannot be seen as modeling the metal sites in FeMo—co since it is known to contain a single molybdenum center [74]. However, what is suggested by these studies is that several steps (if not all) of the reduction of dinitrogen to ammonia by Mo-nitrogenase could take place at a... 

More complex iron-sulfur centers in the MoFe-protein of Mo-nitrogenase are discussed next. 

The Fe proteins of all three nitrogenases are very similar [1], The Fe protein of Mo nitrogenase is a dimer (Mr —65,000) of equivalent subunits with a single Fe4S4 cluster bound between the subunits by bonds between the cluster Fe atoms and the S atoms of two cysteine residues from each subunit [2], The x-ray crystallographic structure at 0.29 nm resolution of the Fe protein from Azotobacter vinelandii (Av2) has been described (Figure 1) [6], This shows that the Fe4S4... 

The only kinetic study on a V nitrogenase [74] reports that the rates of binding of the reduced Fe protein with the VFe protein and the subsequent electron-transfer rates are very similar to those for Mo nitrogenase, as the components of the V nitrogenase form a slightly weaker electron-transfer complex. 

Careful analysis of the kinetic data for the reduction of CN by Mo nitrogenase from A. vinelandii shows that at high concentrations of CN a constant rate of reduction is observed, that rate depends only on the pH of the solution [128], This observation is inconsistent with the earlier proposal that CN and HCN bind independently, and the revised model is one in which the CN binds to a single site (Figure 21). 

The ability of K. pneumoniae Mo nitrogenase to convert ethyne to ethene has been described [77] in terms of the now familiar scheme (Figure 13). C2H2 can... 

Siemann, S., Schneider, K., Drottboom, M., and Muller, A. (2002) The Fe-only nitrogenase and the Mo nitrogenase from Rhodobacter capsulatus. A comparative study on the redox properties of the metal clusters present in the dinitrogenase components, European Journal of Biochemistry 269, 1650-1661. 

EXAFS studies indicate very little change in the coordination of the Mo atom in Mo-nitrogenase during enzyme turnover an indication that N2 probably does not bind to molybdenum but instead to iron. However, this issue has not been completely resolved. 

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...

Figure 14 EPR powder spectrum of the MoFe protein of Mo-nitrogenase taken at 4K. The spectral g-factors of 4.6, 3.7 and 2.0 represent the lower TOj = 1/2 transition of an 5 = 3/2 system with E/D = 0.05...

Mo Nitrogenase, nitrate reductase, xanthine oxidase, formate dehydrogenase, sulfite oxidase, DMSO reductase... 

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. 

---