The transition element molybdenum is essential for most organisms and occurs in more than 60 enzymes catalyzing diverse oxidation–reduction reactions (7,8). Although the element is capable of existing in oxidation states from 0 to VI, only the higher oxidation states of IV, V, and VI are important in biological systems. The functions of molybdenum in plants and other organisms are related to the valence changes that it undergoes as a metallic component of enzymes (9). With the exception of bacterial nitrogenase, molybdenum-containing enzymes in almost all organisms share a similar molybdopterin compound at their catalytic sites (7,8). This pterin is a molybdenum cofactor (Moco) that is responsible for the correct anchoring and positioning of the molybdenum center within the enzyme so that molybdenum can interact with other components of the electron-transport chain in which the enzyme participates (7). Molybdenum itself is thought to be biologically inactive until complexed with the cofactor, Moco.

Several molybdoenzymes including nitrogenase, nitrate reductase, xanthine dehydrogenase, aldehyde oxidase, and possibly sulfite oxidase are of significance to plants. Because of its involvement in the processes of N2 fixation, nitrate reduction, and the transport of nitrogen compounds in plants, molybdenum plays a crucial role in nitrogen metabolism of plants (10).


Nitrogenase
The observation of Bortels (1) that molybdenum was necessary for the growth of Azotobacter was the first indication that molybdenum played a role in biological processes. It is now well established that molybdenum is required for biological N₂ fixation, an activity that is facilitated by the molybdenum- containing enzyme nitrogenase. Several types of asymbiotic bacteria, such as Azotobacter, Rhodospirillum, and Klebsiella, are able to fix atmospheric N₂, but of particular importance to agriculture is the symbiotic relationship between Rhizobium and leguminous crops (10). Nitrogenases from different organisms are similar in nature, and they catalyze the reduction of molecular nitrogen (N₂) to ammonia (NH₃) in the following reaction (11):


N2 + 8H⁺ + 8e^- + 16ATP ? 2NH₃ + H2 + 16ADP + 16Pi


One of the great wonders in nature is how the process of N2 fixation takes place biologically at normal temperatures and atmospheric pressure (12), when in the Haber–Bosch process, the same reaction performed chemically requires temperatures of 300 to 500°C and pressures of >300 atm (13). According to Mishra et al. (11), nearly all nitrogenases contain the same two proteins, both of which are inactivated irreversibly in the presence of oxygen: an Mo–Fe protein (MW 200,000) and an Fe protein (MW 50,000 to 65,000). The Mo–Fe protein contains two atoms of molybdenum and has oxidation–reduction centers of two distinct types: two iron–molybdenum cofactors called FeMoco and four Fe-S (4Fe-4S) centers. The Fe–Mo cofactor (FeMoco) of nitrogenase constitutes the active site of the molybdenum-containing nitrogenase protein in N₂-fixing organisms (14). The effect of biological N2 fixation on the global nitrogen cycle is substantial, with terrestrial nitrogen inputs in the range of 139 to 170 × 106 tons of nitrogen per year (15). Despite the importance of molybdenum to N₂-fixing organisms and the nitrogen cycle, the essential nature of molybdenum for plants is not based on its role in N2 fixation. The primary breach of the Arnon and Stout criteria of essentiality (6) is that many plants lack the ability to fix atmospheric N2 and therefore do not require molybdenum for the activity of nitrogenase. In addition, the process of N2 fixation is not essential for the growth of legumes if sufficient levels of nitrogen fertilizers are supplied (11,16).



Nitrate Reductase
The essential nature of molybdenum as a plant nutrient is based solely on its role in the NO₃^- reduction process via nitrate reductase. This enzyme occurs in most plant species as well as in fungi and bacteria (12), and is the principal molybdenum protein of vegetative plant tissues (17). However, the requirement of molybdenum for nitrogenase activity in root nodules is greater than the requirement of molybdenum for the activity of nitrate reductase in the vegetative tissues (18). Because nitrate is the major form of soil nitrogen absorbed by plant roots (19), the role of molybdenum as a functional component of nitrate reductase is of greater importance in plant nutrition than its role in N2 fixation. Like other molybdenum enzymes in plants, nitrate reductase is a homodimeric protein. Each identical subunit can function independently in nitrate reduction (9), and each consists of three functional domains: the N-terminal domain associated with a molybdenum cofactor (Moco), the central heme domain (cytochrome b557), and the C-terminal FAD domain (7,20). This enzyme occurs in the cytoplasm and catalyzes the reduction of nitrate to nitrite (NO₂^-) in plants (19):


NO₃^- + 2H⁺ + 2e₂^- ? NO₂^- + 2H₂O


Nitrate and molybdenum are both required for the induction of nitrate reductase in plants, and the enzyme is either absent (21), or its activity is reduced (22), if either nutrient is deficient. In deficient plants, the induction of nitrate reductase activity by nitrate is a slow process, whereas the induction of enzyme activity by molybdenum is much faster (10). It has been demonstrated that the molybdenum requirement of plants is higher if they are supplied nitrate rather than ammonium (NH⁴⁺) nutrition (23)—an effect that can be almost completely accounted for by the molybdenum in nitrate reductase (12).


Xanthine Dehydrogenase
In addition to the enzymes nitrogenase and nitrate reductase, molybdenum is also a functional component of xanthine dehydrogenase, which is involved in ureide synthesis and purine catabolism in plants (8). This enzyme is a homodimeric protein of identical subunits, each of which contains one molecule of FAD, four Fe-S groups, and a molybdenum complex that cycles between its Mo(VI) and Mo(IV) oxidation states (9,13). Xanthine dehydrogenase catalyzes the catabolism of purines to uric acid (7):


purines → xanthine → uric acid


In some legumes, the transport of symbiotically fixed N2 from root to shoot occurs in the form of ureides, allantoin, and allantoic acid, which are synthesized from uric acid (10). Although xanthine dehydrogenase is apparently not essential for plants (10), it can play a key role in nitrogen metabolism for certain legumes for which ureides are the most prevalent nitrogen compounds formed in root nodules (9). The poor growth of molybdenum-deficient legumes can be attributed in part to poor upward transport of nitrogen because of disturbed xanthine catabolism (10).



Aldehyde Oxidase
Aldehyde oxidases in animals have been well characterized, but only recently has this molybdoenzyme been purified from plant tissue and described (24). In plants, aldehyde oxidase is considered to be located in the cytoplasm where it catalyzes the final step in the biosynthesis of the phytohormones indoleacetic acid (IAA) and abscisic acid (ABA) (8). These hormones control diverse processes and plant responses such as stomatal aperture, germination, seed development, apical dominance, and the regulation of phototropic and gravitropic behavior (25,26). Molybdenum may therefore play an important role in plant development and adaptation to environmental stresses through its effect on the activity of aldehyde oxidase, although other minor pathways exist for the formation of IAA and ABA in plants (7).



Sulfite Oxidase
Molybdenum may play a role in sulfur metabolism in plants. In biological systems the oxidation of sulfite (SO₃^2-) to sulfate (SO₄^2-) is mediated by the molybdoenzyme, sulfite oxidase (10). Although this enzyme has been well studied in animals (27), the existence of sulfite oxidase in plants is not well established. Marschner (9) explains that the oxidation of sulfite can be brought about by other enzymes such as peroxidases and cytochrome oxidase, as well as a number of metals and superoxide radicals. It is therefore not clear whether a specific sulfite oxidase is involved in the oxidation of sulfite in higher plants (28) and, consequently, also whether molybdenum is essential in higher plants for sulfite oxidation.