6.2 Assimilation of inorganic nitrogen
6.2.1 Nitrogen fixation
Nitrogen is incorporated into cell constituents through transamina- tion reactions using glutamate or glutamine as the amino group donor. Glutamate and glutamine are synthesized from ammonia.
Gaseous nitrogen (N2) is very stable as it possesses a triple bond.
When reduced or oxidized forms of nitrogen (fixed nitrogen) are not available, some prokaryotes reduce the structurally stable N2
to ammonia to use as the nitrogen source, investing a large amount of energy in the form of ATP and reduced electron carriers. Ammonia fixed in this way serves as the nitrogen source for all forms of organ- isms, just as photosynthesis supplies organic materials as the energy source for many organisms. Biological N2fixation is estimated as high as 1.31014g a year, which is more than twice the amount fixed industrially and naturally by lightning (51013g). The fixed nitrogen returns to gaseous nitrogen through nitrification (Section10.2) and denitrification (Section9.1), which, together with biological nitrogen fixation, constitute the nitrogen cycle (Figure6.2).
6.2.1.1 N2-fixing organisms
A wide variety of prokaryotes have the ability to fix N2(Table6.2). These include certain photosynthetic prokaryotes, anaerobic and aerobic bacteria, and archaea. Some fix N2in a symbiotic relationship with plants, while others can do so in the free-living state. Bacteria belonging
Figure 6:1 Catabolism and anabolism.
Monomers are synthesized from carbon skeletons and inorganic substances such as ammonia, sulfate and phosphate that are obtained from the growth medium or cellular environment. The monomers are polymerized into proteins, nucleic acids, polysaccharides,
phospholipids and other macromolecules. Catabolism supplies not only carbon skeletons, but also ATP and NADPH required for anabolic processes. The size of the squares in the diagram represents the relative content in theEscherichia colicell summarized in Table6.1. The numbers of individual monomers are shown in the squares.
to the genusRhizobiumare well known as symbiotic nitrogen fixers with legumes. Alder trees host nitrogen-fixingFrankia alni, an actinomycete- like organism.Anabaena azollaeis an example of a symbiotic nitrogen- fixing cyanobacterium associated with a fern,Azolla.
6.2.1.2 Biochemistry of N2fixation
Nitrogen reduction to ammonia can be expressed as:
N2+3H2+2H+ 2NH4+ (ΔG0′=–39.3kJ/mol NH4+)
This is an exergonic reaction, but requires a high activation energy due to the stable triple bond in N2. For this reason, the chemical N2 fixation process employs a high temperature (300–6008C) and high pressure (200–800 atm). Nitrogen-fixing microbes produce the
Table 6.2. Examples of nitrogen-fixing prokaryotes Bacteria
Cyanobacteria Anabaena azollae Gloeocapsaspp.
Mastigocladus laminosus Photosynthetic bacteria Chromatium vinosum
Rhodopseudomonas viridis Rhodospirillum rubrum Heliobacterium chlorum Strict anaerobes Acetobacterium woodii
Clostridium pasteurianum Desulfovibrio vulgaris Desulfotomaculum ruminis Aerobes and facultative
anaerobes Azotobacter paspali Azotobacter vinelandii Azospirillum lipoferum Bacillus polymyxa Beijerinkia indica Derxia gummosa Frankia alni
Halobacterium halobium Klebsiella pneumoniae Methylococcus capsulatus Methylosinus trichosporium Mycobacterium flavum Pseudomonas azotogensis Rhizobium japonicum Thiobacillus ferrooxidans Archaea
Methanogens Methanosarcina barkeri Methanococcus maripaludis
Methanobacterium thermoautotrophicum
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enzyme nitrogenase, which reduces nitrogen under normal physio- logical conditions.
N I T R O G E N A S E
N2 fixation is catalyzed by nitrogenase. Nitrogenase is a complex protein consisting of azoferredoxin and molybdoferredoxin in a 2:1 ratio. Both of the enzymes are [Fe-S] proteins, and molybdenum is contained in molybdoferredoxin. Azoferredoxin is a homodimer of a protein containing a [4Fe-4S] cluster, and molybdoferredoxin consists of 2 molecules of 2 proteins containing 2 molybdenum and 28 iron and sulfur atoms (Table 6.3). Molybdoferredoxin reduces nitrogen with the reducing equivalents provided by azoferredoxin. Based on their functions, molybdoferredoxin is termed dinitrogenase, and azoferredoxin is termed dinitrogenase reductase. Since the redox potential of azoferredoxin is low (0.43 V), ferredoxin or flavodoxin is believed to be the electron donor for its reduction. In this process, dissociated azoferredoxin from molybdoferredoxin is reduced, accepting the electrons from low redox potential ferredoxin or fla- vodoxin followed by ATP binding. Molybdoferredoxin binds N2
before forming a nitrogenase complex with the ATP-reduced azofer- redoxin complex. At this point, electrons are transferred from azo- ferredoxin to molybdoferredoxin with ATP hydrolysis. Since azoferredoxin is a one electron carrier, the reduction of a nitrogen molecule requires six oxidation–reduction cycles with the hydrolysis of at least 16 ATP molecules (Figure6.3).
– –
– Figure 6:2 The nitrogen cycle.
(Sprent, J. I. 1979,The Biology of Nitrogen-fixing Organisms, Figure 1.1.
McGraw-Hill, Maidenhead)
Nitrogenase can reduce various other substances in addition to dinitrogen (Table6.4). The ability to reduce acetylene to ethylene is exploited in a simple nitrogenase assay method. Protons are reduced by the nitrogenase complex to H2during normal N2fixation.
Table 6.3. The nitrogenase complex ofRhizobiumspp.
Characteristics
Azoferredoxin (dinitrogenase reductase)
Molybdoferredoxin (dinitrogenase)
Molecular weight 70 000 230 000
Subunit 2 4a
Iron 8b 28
Molybdenum 0 2
Acid-labile sulfide 8 28
aTwo subunits each of two peptides with molecular weights of 55 000 and 60 000.
bOne [4Fe-4S] for each subunit.
i
Figure 6:3 N2reduction by the nitrogenase complex.
(Sprent, J. I. 1979,The Biology of Nitrogen-fixing Organisms, Figure 2.1. McGraw-Hill, Maidenhead.)
Azoferredoxin (dinitrogenase reductase) is reduced, coupled with the oxidation of ferredoxin or flavodoxin, and binds ATP.
Molybdoferredoxin (dinitrogenase) binds N2and forms a nitrogenase complex with the reduced azoferredoxin-ATP complex.
Electrons required to reduce N2are transferred from azoferredoxin to molybdoferredoxin, and this reaction repeats six times to reduce one molecule of N2.
6 . 2 A S S I M I L A T I O N O F I N O R G A N I C N I T R O G E N 131
Most nitrogen fixers synthesize alternative nitrogenases contain- ing vanadium and iron, or iron only under molybdenum-limited conditions. These have a lower activity than molybdenum-containing nitrogenase.
E L E C T R O N C A R R I E R S
Ferredoxin or flavodoxin supply the electrons required for nitrogen reduction. Photosythetic organisms reduce them by light reactions, and obligate anaerobes reduce ferredoxin by pyruvate:ferredoxin oxidoreductase or hydrogenase. Aerobes reduce them through a reverse electron transport mechanism (Section10.1) using reduced pyridine nucleotides. H2produced from the nitrogenase reaction is used to reduce ferredoxin by the action of the hydrogenase.
6.2.1.3 Bioenergetics of N2fixation
The YATP of Klebsiella pneumoniae was measured to be 4.20.2 g/mol ATP under nitrogen-fixing conditions and 10.91.5 g/mol ATP with ammonia. These figures show that nitrogenase consumes 29 ATP to reduce one dinitrogen. This figure is much higher than the predicted ATP consumption by the nitrogenase complex. This might be because of the energy consumed in the reverse electron transport process to reduce the low redox potential electron carriers.
6.2.1.4 Molecular oxygen and N2fixation
All nitrogenases known to date are inactivated irreversibly by mole- cular oxygen. O2is required to synthesize ATP needed for N2fixation through aerobic respiration, and photosystem II (Section 11.4) pro- duces O2in N2-fixing cyanobacteria. To avoid irreversible inactiva- tion of nitrogenase by O2, N2-fixing organisms therefore employ various protection mechanisms against O2.
Species ofRhizobiumfix N2in a symbiotic association with legumes.
When they infect the plant root, nodules are formed through trans- formation of the plant cells, and the bacterial cells become irregularly formed (bacteroidal) instead of rod-shaped. They synthesize ATP through aerobic respiration using energy source(s) and O2 supplied by the host plants. The O2is supplied bound to leghemoglobin which is
Table 6.4. Substances reduced by the nitrogenase complex
Substrate Product(s)
N2 2NH4þ
N3
N2, NH4þ
N2O N2
HCN CH4, NH4þ
, CH3NH2
CH3CN C2H6, NH4þ
CH2CHCN C3H6, NH4þ
, C3H8
C2H2 C2H4
2Hþ H2
similar in structure and function to myoglobin in animals. Molecular oxygen is not involved in the respiratory process. The fixed nitrogen is transferred to the host plant (Figure6.4). Other symbiotic N2-fixers have a similar relationship with the host plant.
Cyanobacteria obtain the reducing equivalents and ATP for ana- bolism through oxygenic photosynthesis generating molecular oxy- gen. Heterocystous cyanobacteria such asAnabaena andNostoc spp.
transform 5–10% of normal vegetative cells within the filaments into heterocysts which lack oxygenic photosystem II to protect nitrogen- ase from molecular oxygen produced by photosystem II. Heterocysts fix nitrogen using electrons transported from neighbouring normal cells, and in return the heterocysts supply fixed nitrogen (Figure6.5).
Unicellular cyanobacteria operate photosystem I only when nitro- genase activity is high, and photosystem II appears with the accumu- lation of fixed nitrogen within the cell. Under N2-fixing conditions, O2is not generated (Figure6.6).
Aerobic bacteria protect their nitrogenase through different mechanisms. Azotobacter vinelandii keeps the O2 concentration low using a high affinity terminal oxidase, cytochromed, under nitrogen- fixing conditions instead of the normal cytochromeo(Section5.8.2.3).
Some nitrogenases of aerobes such asAzotobacter chroococcumandDerxia gummosa reversibly change their structure in the presence of O2
(Figure6.7).
Figure 6:4 Roles of the host plant andRhizobiumin symbiotic N2fixation.
WhenRhizobiuminfects the legume plant the host root cells form nodules, and the bacterial cells become irregular bacteroids. The host supplies the carbon and energy source, and O2in the leghaemoglobin (Lb) bound form. The bacterium fixes and supplies nitrogen to the host plant.
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6.2.1.5 Regulation of N2fixation
When an organism faces a surplus of fixed nitrogen or is starved of energy sources, nitrogenase activity is not needed. To avoid the waste of energy under these conditions, N2fixation is regulated both at the transcriptional level of the genes and by controlling enzyme activity.
Nitrogenase activity is inhibited by ammonia and under starvation conditions with a low adenylate energy charge (EC). Inhibition by ammonia is reversible and the response is very quick. This regulation is referred to as the ammonia switch. When ammonia accumulates, an arginine residue of azoferredoxin (dinitrogenase reductase) is
Figure 6:5 N2fixation in heterocysts of heterocystous cyanobacteria.
(Gottschalk, G. 1986,Bacterial Metabolism, 2nd edn., Figure 10.3. Springer, New York)
Under N2-fixing conditions, heterocystous cyanobacteria transform 5–10% of the cells in the filament into heterocysts which lack photosystem II to protect the nitrogenase from O2. Heterocysts fix N2using carbon and energy sources obtained from normal vegetative cells, and supply fixed nitrogen to the normal cells.
PSI, photosystem 1.
Figure 6:6 Growth and N2
fixation in unicellular cyanobacteria.
(Sprent, J. I. 1979,The Biology of Nitrogen-fixing Organisms, Figure 2.4, McGraw-Hill, Maidenhead) Unicellular cyanobacteria do not operate photosystem II under N2- fixing conditions to protect nitrogenase from O2. When fixed nitrogen is accumulated they grow normally with photosystems I and II.
*–*, nitrogenase activity;–, O2evolution;*–*, chlorophyll/
phycocyanin ratio (phycocyanin is a constituent of the antenna molecule of photosystem II).
bound with ADP-ribose available from NADþ. The nitrogenase com- plex is inactive with ADP-ribose. Nitrogenase activity is controlled to less than 10% when ATP/ADP is around 1 (EC value of around 0.6).
MOLYBDOFERREDOXIN MOLYBDOFERREDOXIN
AZOFERREDOXIN AZOFERREDOXIN-
ribose-ADP active
NAD+ +NH4
–NH4 ADP-ribose
inactive nicotinamide
+
+
Ammonia inhibits the transcription of genes of the nitrogenase complex and this regulation is elaborate. Genes for the nitrogenase complex (nifregulon) consist of 7 operons with 20 genes inKlebsiella pneumoniae(Figure6.8). In addition to these genes, nitrogen control genes,ntr(Section12.2.2), are also involved in their regulation.
Nitrogen control genes consist of ntrA,B, andC. NtrA is a sigma factor of the RNA polymerase (N,54), NtrC and NtrB are NRIand NRII, respectively (Figure12.22, Section 12.2.2). They regulate the transcrip- tion and activity of enzymes related to ammonia metabolism. When the ammonia concentration is low, four molecules of UMP bind with NRIIwhich in turn phosphorylates NRI. The phosphorylated NRIacti- vates NtrA(N) to transcribenifAandnifL. NifA is an activator for the transcription of othernifgenes, NifL is a repressor protein (Figure6.9).
NtrA, NtrB and NtrC regulate not only the nif regulon but also other enzymes related to ammonia metabolism such as glutamine synthetase, and the transport and utilization of arginine, proline and histidine. The regulation of many operons with different functions by a single regulator is referred to as a global control system or multigene system. This is discussed later (Section12.2).