Biochemistry - The Chemical Reactions of Living Cells Volume 3 - D. Metzler 1980

Metabolism of Nitrogen-Containing Compounds
N2 Fixation and Other Transformations of Inorganic Nitrogen Compounds
The Nitrogenase Enzyme System

Cell-free Nitrogenase preparations have been isolated from A wide variety of organisms. All these Enzymes are rapidly inactivated in the presence of oxygen, a factor that greatly hindered early research. Nitrogen Fixation apparently takes place in the anaerobic regions of Cells. It has even been suggested that leghemoglobin protects the nitrogen-fixing enzymes of ROOT nodules from oxygen toxicity. Leghemoglobin may also facilitate O₂ delivery via Facilitated Diffusion to the aerobic Mitochondria of bacteroids under sustained low oxygen tension [8].

The nitrogenase system catalyzes the six-electron reduction of N₂ to ammonia:

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It is also capable of reducing many Other Compounds. For example, the reduction of acetylene to Ethylene [Eq. (14-2)] is a two-electron process:

Another two-electron reduction converts the azide anion into N₂ and NH⁺₄ [Eq. (14-3)]. Cyanide ions yield methane and ammonia [Eq. (14-4)]. The nitrogenase system can also reduce alkyl nitriles and N₂O. In addition, nitrogenases invariably catalyze the reduction of protons to H₂ [Eq. (14-5)].

Early experiments revealed that the fixation of N₂ in cell-free extracts requires sodium Pyruvate. The accumulation of large amounts of CO₂ and H₂ was also observed. Pyruvate was found to be cleaved by pyruvate-formate lyase (Fig. 8-19), supplying the cells with two essential products: ATP and reduced ferredoxin. Pyruvate could be replaced by a mixture of ATP, Mg²⁺, and reduced Fd. Furthermore, reduced ferredoxin could be substituted by the non-biological reductant dithionite (S₂O^2-₄). Because ADP exerts an inhibitory effect on the nitrogenase system, the most effective way to generate ATP was to use an ATP-regenerating system consisting of a mixture of creatine phosphate (Supplement 10-E), creatine kinase, and a small amount of ADP.

In each case, the investigated nitrogenase system could be readily resolved into two components. One of these, azoferredoxin (azoFd, also known as component II), is an extremely oxygen-sensitive Fe-S protein. Azoferredoxin consists of two identical peptide chains, each with a Molecular Weight of 30,000. Each dimer contains four iron atoms, 4S^2-, and 12 titratable thiol groups. The other component is molybdofeferredoxin (MoFd, also referred to as component I), which contains iron, molybdenum, and labile sulfide. This protein features Two Types of peptide chains with molecular weights of ~51,000 and ~60,000, forming a mixed (a₂ß₂) tetramer. Each mixed tetramer contains two molybdenum atoms, ~24 iron atoms, ~24 sulfide ions, and ~30 titratable thiol groups, likely comprising three Fe₄S₄ clusters per molybdenum atom (Chapter 10, Section B). The association of these Proteins yields the nitrogenase complex, which contains two azoFd dimer molecules and one MoFd molecule (Fig. 14-1).

FIG. 14-1. Proposed Structure OF THE nitrogenase molecule.

Upon reduction of azoFd, an EPR signal with g = 1.94 is observed, which is typical of Fe-S proteins (Chapter 10, Section B). This signal is strongly influenced by the interaction of Mg²⁺ and ATP; however, ATP has no effect on the complex set of EPR signals observed upon the reduction of MoFd. These and other observations led to the concept that azoFd serves as the electron carrier responsible for the reduction of the molybdenum contained in MoFd. The resulting Mo(IV) subsequently reduces N₂ via a two-electron process accompanied by The formation of Mo(VI) [Eq. (14-6)]:

According to this scheme, the complete reduction of N₂ to two molecules of ammonia requires three sequential two-electron steps. An alternative process is the reduction of two protons to H₂, as indicated by the dashed arrows in Eq. (14-6). ATP apparently drives the electron flow in a manner analogous to the ATP-driven "reverse electron flow" in the Respiratory Chain (Chapter 10, Sections D and 7), which is also shown schematically in Eq. (14-6). It has been demonstrated [9] that ATP binds tightly to azoFd and shifts E⁰' (pH 7.5) from —0.29 to —0.40 V. Surprisingly, most researchers have found that 4 to 5 molecules of ATP are consumed per two electrons transferred. The reduction of N₂ by reduced ferredoxin [Eq. (14-7)] is thermodynamically spontaneous:

However, N₂ is chemically exceptionally inert. Therefore, to overcome the high activation barrier, ATP Hydrolysis may need to be coupled to the operation of the nitrogenase system in more than one way [10]. It is also possible that the in vivo ATP requirement is lower than that in isolated systems.

In some Bacteria, such as the strict anaerobe Azotobacter, NADPH serves as the electron donor for N₂ reduction. AzoFd appears to preferentially accept electrons from a carrier chain that includes at least a conventional bacterial ferredoxin (Fd) and a specialized one-electron acceptor, azotoflavin [11]. This flavoprotein, which is somewhat larger than the flavodoxins (Chapter 8, Section I, 5), apparently plays a specific role in N₂ fixation. The putative electron transfer system is outlined in the equation

Supplement 14-A

Molybdenum

It has long been recognized that molybdenum is an essential element for plant growth, although no conclusive evidence for its mandatory presence in animal diets has yet been obtained. Nevertheless, it has been found in at least three animal enzymes and an additional four bacterial and plant enzymes^a,b. Aldehyde oxidase, Liver xanthine oxidase (Vol. 2, p. 265), and related xanthine dehydrogenases from certain bacteria contain molybdenum, which is essential for catalytic activity. Liver sulfite oxidase (Chapter 14, Section G), bacterial and plant nitrate reductase (Chapter 10, Section E.2), bacterial formate dehydrogenase (Chapter 9, Section B, 3), and nitrogenase (this chapter) complete the list of known enzymes whose activity depends on the presence of molybdenum.

Molybdenum is a second-row transition metal and one of the few heavy elements known to be essential for life. In its most stable oxidized state, Mo(VI), molybdenum possesses a filled 4s shell and 4d orbitals available for coordination bonding with anionic ligands. Coordination numbers of 4 or 6 are preferred, but at least eight ligands can bind to molybdenum. Most complexes are formed with the oxycation MoO²⁺₂. When two Water molecules are coordinated to this ion, their protons become sufficiently acidic to dissociate completely, leaving the complex as the molybdate ion, MoO^2-₄. Other oxidation states range from Mo(III) to Mo(V). In these less oxidized states, the tendency of protons in coordinated ligands to dissociate is diminished; for example, Mo(H₂O)³⁺₆ does not release protons even in strongly basic environments. Molybdenum readily forms dimeric and polymeric ions linked by oxygen bridges.

All molybdenum-containing enzymes have molecular weights of 100,000 or higher and frequently contain two molybdenum atoms. However, there is no evidence that the molybdenum atoms themselves form a dimer; rather, the dimer is formed by the protein subunits to which the metal is bound. E. coli nitrate reductase is known to contain a large subunit with a molecular weight of 150,000, a smaller peptide of molecular weight 55,000, one Mo atom, 12 non-heme iron atoms, and 12 acid-labile sulfides^c. It also forms dimers and tetramers. Evidence suggests that molybdenum in all these enzymes is present as part of a low-molecular-weight cofactor^d.

The precise mechanism of molybdenum's participation in catalysis remains unknown. The Mo(III) and Mo(V) states are paramagnetic, but readily detectable EPR signals correspond solely to the Mo(V) state. This signal is easily recognized by its characteristic six-line hyperfine structure. Such a signal has been recorded for xanthine oxidase, nitrate reductase, and sulfite oxidase upon interaction with their substrates. However, nitrogenase yields only EPR signals characteristic of iron. Furthermore, there is no evidence suggesting that N₂ reacts directly with molybdenum atoms in nitrogenase. Nevertheless, it is tempting to speculate that the presence of molybdenum in nitrogenase is related to the ability of Mo(VI) to accept three electrons, yielding Mo(III). Two Mo(III) atoms, by subsequently releasing three electrons each, could supply the six electrons required for reaction (14-10).

Tungsten can compete with molybdenum in the Organism. For instance, rats fed a diet containing 100 ppm of tungsten synthesize a tungsten-containing sulfite oxidase that is no longer functionally competent^e. However, under these conditions, even larger amounts of the metal-free apoprotein accumulate. These rats also produce an inactive, metal-free xanthine oxidase^f. Apparently, tungsten somehow interferes with the incorporation of molybdenum into enzyme molecules. Most of the molybdenum in nitrogen-fixing Azotobacter bacteria is sequestered in a specialized protein dedicated to molybdenum storage^g.

^a Bowden, F. L., in Techniques and Topics in Bioinorganic Chemistry (C. A. McAuliffe, ed.), pp. 207–267, Macmillan, New York, 1975.

⁶ A Symposium on Molybdenum, J. Less-Common Met., 36, 405—533 1974.

^B Lund K., DeMoss J. A., JBC, 251, 2207—2216 (1976).

^r Nason A., Lee K., Y., Pan S.-S., Erickson R. H., J. Less-Common Met., 36, 449—459 (1974).

^d Johnson J. J., Cohen H. J., Rajagopalan K. V., JBC, 249, 5046—5055 (1974).

^e Johnson J. J., Wand W. R., Cohen H. J., Rajagopalan, JBC, 249, 5056— 5061 (1974).

^zh Brill W. J., Am. Chem. Soc., Cent. Meet., New York, 1976 Abstracts INOR 138 (1976).