The Enzyme Nitrogenase

Industrially, ammonia has been produced from dinitrogen and dihydrogen by the Haber-Bosch process, which operates at very high temperatures and pressures, and utilizes a promoted iron catalyst. Millions of tons of ammonia are generated annually for incorporation into agricultural fertilizers and other important commercial products. The overall reaction is exergonic, as indicated in equation 6.1  

However, the first step of the process, forming diazene (N2H2), has a large positive free energy change, as shown below in equation 6.4. 

As nitrogenase s structure and function have become known at the molecular level through the work of many groups over the past 20 years, chemists have 

H+ shuttled From umiro acid side chains 

P-cluster of MoFe-proteiu. 10 Au below protein surface on 2-fold axis of 0-(f subunits 

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Free-living bacteria are, however, used as the source of the enzyme nitrogenase, responsible for N2 fixation (1,4,26,80), for research purposes because these ate easier to culture. The enzyme is virtually identical to that from the agriculturally important thizobia. These free-living N2-fixets can be simply classified into aerobes, anaerobes, facultative anaerobes, photosynthetic bacteria, and cyanobacteria. 

Fig. 1. Schematic illustration of the enzyme nitrogenase being composed of the molybdenum-iron (MoFe) protein, an oc2p2 tetramer with two unique iron-sulfur clusters (P-cluster) and two iron-molybdenum cofactors (FeMoco), and the iron protein with one [4Fe-4S]-cluster and two ATP binding sites.

In Nature, nitrogen fixation is mediated by the enzyme nitrogenase according to Eq. (1) (6)... 

X-ray crystallographic structures of the enzyme nitrogenase first became available in 1992 with refinements of the structures continuing to the present time. As of this... 

Table 3.4 lists values for A Eq and for some important oxidation and spin states found in bioinorganic molecules. Data are taken from reference 24 and from Table 1 of reference 25 for hemoglobin, myoglobin, and the picket-fence porphyrin model compound, FeTpivPP(l-Melm).25 The myoglobin and hemoglobin model compounds are discussed in Section 4.8.2. Reference 26 provides the Table 3.4 data on iron sulfur clusters found in many bioinorganic species.26 The unusual iron-sulfur and iron-molybdenum-sulfur clusters found in the enzyme nitrogenase are discussed more fully below and in Chapter 6. 

Figure 6.1 Cartoon illustrating the structure of the enzyme nitrogenase.

Molybdate, although present in small amounts in soil, is an essential nutrient for nitrogen fixation, specifically in the enzyme nitrogenase. It does not move readily through soil and is therefore considered to be of limited mobility. 

In this text, iron-sulfur clusters are discussed because they appear in proteins and enzymes (1) cytochrome b(6)f, Rieske [2Fe-2S] cluster (Section 7.5 and Figure 7.26) (2) cytochrome bci, Rieske [2Fe-2S] cluster (Section 7.6 and Figure 7.30) and (3) aconitase, [4Fe-4S] cluster (Section 7.9.2.1, and Figure 7.50). The iron-sulfur protein (ISP) component of the cytochrome b(6)f and cytochrome bci complexes, now called the Rieske ISP, was first discovered and isolated by John S. Rieske and co-workers in 1964 (in the cytochrome bci complex). More information about the RISP is found in Section 7.5.1. Section 7.9.2 briefly discusses other proteins with iron-sulfur clusters—rubredoxins, ferrodoxins, and the enzyme nitrogenase. The nitrogenase enzyme was the subject of Chapter 6 in the hrst edition of this text— see especially the first edition s Section 6.3 for a discussion of iron-sulfur clusters. In this second edition, information on iron-sulfur clusters in nitrogenase is found in Section 3.6.4. See Table 3.2 and the descriptive examples discussed in Section 3.6.4. 

Four distinct types of Fe-S center have now been found in proteins, ranging from mono- to tetranuclear in addition, a novel Mo-Fe-S cluster is present in the enzyme nitrogenase. Synthetic analogs of most of these have been prepared and used to provide insight into the intrinsic properties of the metal-sulfur centers in the absence of protein-imposed constraints. The strategies used to prepare both Fe-S and Mo-Fe-S clusters are described they range from spontaneous self-assembly to the designed synthesis of clusters with specific structural features. 

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 2.6 Diffraction pattern from a crystal of the MoFe (molybdenum-iron) protein of the enzyme nitrogenase from Clostridium pasteurianum. Notice that the reflections lie in a regular pattern, but their intensities (darkness of spots) are highly variable. [The hole in the middle of the pattern results from a small metal disk (beam stop) used to prevent the direct X-ray beam, most of which passes straight through the crystal, from destroying the center of the film.] Photo courtesy of Professor Jeffery Bolin.

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