Overview of Nitrogen Metabolism:- Nitrogen Is Fixed by Enzymes of the Nitrogenase Complex
Only certain prokaryotes can fix atmospheric nitrogen. These include the cyanobacteria of soils and fresh and salt waters, other kinds of free-living soil bacteria such as Azotobacter species, and the nitrogen-fixing bacteria that live as symbionts in the root nodules of leguminous plants. The first important product of nitrogen fixation is ammonia, which can be used by all organisms either directly or after its conversion to other soluble compounds such as nitrites, nitrates, or amino acids. The reduction of nitrogen to ammonia is an exergonic reaction:
N2+3H2→2NH3 ΔG0=-33.5 kJ/mol
The N≡N triple bond, however, is very stable, with a bond energy of 930 kJ/mol. Nitrogen fixation therefore has an extremely high activation energy, and atmos pheric nitrogen is almost chemically inert under normal conditions. Ammonia is produced industrially by the Haber process (named for its inventor, Fritz Haber), which requires temperatures of 400 to 500 C and nitrogen and hydrogen at pressures of tens of thousands of kilopascals (several hundred atmospheres) to provide the necessary activation energy. Biological nitrogen fixation, however, must occur at biological temperatures and at 0.8 atm of nitrogen, and the high activation barrier is overcome by other means. This is accomplished, at least in part, by the binding and hydrolysis of ATP. The overall reaction can be written
N2+10H+ +8e-+16ATP→2NH4++16ADP+16Pi +H2
Biological nitrogen fixation is carried out by a highly conserved complex of proteins called the nitrogenase complex (Fig. 22–2), the crucial components of which are dinitrogenase reductase and dinitrogenase (Fig. 22–3). Dinitrogenase reductase (Mr 60,000) is a dimer of two identical subunits. It contains a single 4Fe 4S redox center (see Fig. 19–5), bound between the subunits, and can be oxidized and reduced by one electron. It also has two binding sites for ATP/ADP (one site on each subunit). Dinitrogenase (Mr 240,000), a tetramer with two copies of two different subunits, contains both iron and molybdenum; its redox centers have a total of 2 Mo, 32 Fe, and 30 S per tetramer. About half of the iron and sulfur is present as two bridged pairs of 4Fe-4S centers called P clusters; the remainder is present as part of a novel iron-molybdenum cofactor. A form of nitrogenase that contains vanadium rather than molybdenum has been discovered, and some bacterial species can produce both types of nitrogenase systems. The vanadium-containing enzyme may be the primary nitrogen-fixing system under some environmental conditions, but it is not yet as well characterized as the molybdenum-dependent enzyme. Nitrogen fixation is carried out by a highly reduced form of dinitrogenase and requires eight electrons: six for the reduction of N2 and two to produce one molecule of H2 as an obligate part of the reaction mechanism. Dinitrogenase is reduced by the transfer of electrons from dinitrogenase reductase (Fig. 22–2). The dinitrogenase tetramer has two binding sites for the reductase. The required eight electrons are transferred from reductase to dinitrogenase one at a time: a reduced reductase molecule binds to the dinitrogenase and transfers a single electron, then the oxidized reductase dissociates from dinitrogenase, in a repeating cycle. Each turn of the cycle requires the hydrolysis of two ATP molecules by the dimeric reductase. The immediate source of electrons to reduce dinitrogenase reductase varies, with reduced ferredoxin (p. 733; see also Fig. 19–5), reduced flavodoxin, and perhaps other sources playing a role. In at least one species, the ultimate source of electrons to reduce ferredoxin is pyruvate (Fig. 22–2). The role of ATP in this process is somewhat un usual. As you will recall, ATP can contribute not only chemical energy, through the hydrolysis of one or more of its phosphoanhydride bonds, but also binding en ergy (pp. 196, 301), through noncovalent interactions that lower the activation energy. In the reaction carried out by dinitrogenase reductase, both ATP binding and ATP hydrolysis bring about protein conformational changes that help overcome the high activation energy of nitrogen fixation. The binding of two ATP molecules to the reductase shifts the reduction potential (E0) of this protein from -300 to -420 mV, an enhancement of its reducing power that is required to transfer electrons to dinitrogenase. The ATP molecules are then hydrolyzed just before the actual transfer of one electron to dinitrogenase.
FIGURE 22–2 Nitrogen fixation by the nitrogenase complex. Electrons are transferred from pyruvate to dinitrogenase via ferredoxin (or flavodoxin) and dinitrogenase reductase. Dinitrogenase reductase re duces dinitrogenase one electron at a time, with at least six electrons required to fix one molecule of N2. An additional two electrons are used to reduce 2 H+ to H2 in a process that obligatorily accompanies nitrogen fixation in anaerobes, making a total of eight electrons re quired per N2 molecule. The subunit structures and metal cofactors of the dinitrogenase reductase and dinitrogenase proteins are described in the text and in Figure 22–3.
FIGURE 22–3 Enzymes and cofactors of the nitrogenase complex. (PDB ID 1N2C) (a) In this ribbon diagram, the dinitrogenase subunits are shown in gray and pink, the dinitrogenase reductase subunits in blue and green. The bound ADP is red. Note the 4Fe-4S complex (Fe atoms orange, S atoms yellow) and the iron-molybdenum cofactor (Mo black, homocitrate light gray). The P clusters (bridged pairs of 4Fe-4S complexes) are also shown. (b) The dinitrogenase complex cofactors without the protein (colors as in (a)). (c) The iron-molybdenum co factor contains 1 Mo (black), 7 Fe (orange), 9 S (yellow), and one molecule of homocitrate (gray).
Another important characteristic of the nitrogenase complex is an extreme lability in the presence of oxy gen. The reductase is inactivated in air, with a half-life of 30 seconds; dinitrogenase has a half-life of 10 minutes in air. Free-living bacteria that fix nitrogen cope with this problem in a variety of ways. Some live only anaerobically or repress nitrogenase synthesis when oxygen is present. Some aerobic species, such as Azotobacter vinelandii, partially uncouple electron trans fer from ATP synthesis so that oxygen is burned off as rapidly as it enters the cell (see Box 19–1). When fixing nitrogen, cultures of these bacteria actually increase in temperature as a result of their efforts to rid themselves of oxygen. The symbiotic relationship between leguminous plants and the nitrogen-fixing bacteria in their root nodules (Fig. 22–4) takes care of both the energy requirements and the oxygen lability of the nitrogenase complex. The energy required for nitrogen fixation was probably the evolutionary driving force for this plant-bacteria association. The bacteria in root nod ules have access to a large reservoir of energy in the form of abundant carbohydrate and citric acid cycle intermediates made available by the plant. This may allow the bacteria to fix hundreds of times more nitrogen than their free-living cousins can fix under conditions generally encountered in soils. To solve the oxygen-toxicity problem, the bacteria in root nodules are bathed in a solution of the oxygen-binding heme protein leghemoglobin, produced by the plant (although the heme may be contributed by the bacteria). Leghemoglobin binds all available oxygen so that it cannot interfere with nitrogen fixation, and efficiently delivers the oxygen to the bacterial electron-transfer system. The benefit to the plant, of course, is a ready supply of reduced nitrogen. The efficiency of the symbiosis between plants and bacteria is evident in the enrichment of soil nitrogen brought about by leguminous plants. This enrichment is the basis of crop rotation methods, in which plantings of nonleguminous plants (such as maize) that extract fixed nitrogen from the soil are alternated every few years with plantings of legumes such as alfalfa, peas, or clover. Nitrogen fixation is the subject of intense study, because of its immense practical importance. Industrial production of ammonia for use in fertilizers requires a large and expensive input of energy, and this has spurred a drive to develop recombinant or transgenic organisms that can fix nitrogen. Recombinant DNA techniques (Chapter 9) are being used to transfer the DNA that encodes the enzymes of nitrogen fixation into non-nitrogen-fixing bacteria and plants. Success in these efforts will depend on overcoming the problem of oxygen toxicity in any cell that produces nitrogenase.
FIGURE 22–4 Nitrogen-fixing nodules. (a) Root nodules of bird’s-foot trefoil, a legume. The flower of this common plant is shown in the in set. (b) Artificially colorized electron micrograph of a thin section through a pea root nodule. Symbiotic nitrogen-fixing bacteria, or bacteroids (red), live inside the nodule cells, surrounded by the peribacteroid membrane (blue). Bacteroids produce the nitrogenase complex that converts atmospheric nitrogen (N2) to ammonium (NH4); without the bacteroids, the plant is unable to utilize N2. The infected root cells provide some factors essential for nitrogen fixation, including leghemoglobin; this heme protein has a very high binding affinity for oxygen, which strongly inhibits nitrogenase. (The cell nucleus is shown in yellow/green. Not visible in this micrograph are other organelles of the infected root cell that are normally found in plant cells.)
