Growth, Products, and Application
4.2 Nitrogenase
Nitrogenase (EC 1.7.99.2) catalyzes the reduction of molecular nitrogen to ammonia:
This enzyme can also catalyze the reduction of a number of other compounds with triple bonds 4 7 9 - 4 8 1 ) and the irreversible energy-dependent reduction of protons to molecular hydrogen 132,175,334,482).
Nitrogenase is also involved in a specific exchange reaction which proceeds in the presence of N2 23,483,484). Since the ability to catalyze H2 evolution is probably the common property of the nitrogenases of all microorganisms, it may be assumed that all N2-fixing species may, in principle, evolve hydrogen, due to the function of this enzyme.
Recently, several reviews on the properties of nitrogenase have been published 23, 2 4 , 1 3 2 , 4 8 1 , 4 8 3 - 4 8 5 , 5 3 0 , 5 3 1 )
Nitrogenases of all microorganisms consist of two components, a molybdenum iron-containing protein (MoFe-protein, component I or molybdoferredoxin) and an iron-containing protein (Fe-protein, component II or azoferredoxin). Sometimes, the Fe-protein is called nitrogenase reductase and the MoFe-protein nitrogenase itself 175, 486,487)
The MoFe-protein is a high molecular weight complex (200-300 x 103) and consists of four subunits (Table 9).
In comparison with the MoFe-protein the molecular weight of the Fe-protein is lower (M ~ 51-73 x 103 (Table 9)). It consists of two subunits with the same molecular weight (27.5-34.6 x 103).
The MoFe-protein of nitrogenases contains 18-38 atoms of non-heme iron, 18-38 atoms of acid-labile sulfur, and 1-2 atoms of molybdenum per molecule 132,
175,442,488) . About half the iron and molybdenum is bound to a lowpotential cofactor:
these metals may be detached from the MoFe-protein by acid treatment 481,489,490). Four atoms of non-heme iron and four atoms of acid-labile sulfur (S2-) are found in a dimer of the Fe-protein (Table 9).
4.2.1 Mechanism of Catalysis and H2 Evolution
All nitrogenase-catalyzed reactions demand the participation of both components of the nitrogenase. A recombination of the isolated components of the nitrogenases from different organisms is possible 408,409).
For nitrogenase to function, energy in the form of ATP and Mg ions are necessary.
According to the available data, 12-30 mol of ATP are required for the reduction of each N2 molecule to NH3 1 3 2 , 4 8 8 ). This enables a transfer of electrons from the Fe-protein to the MoFe-protein 175,479). With the exception of the exchange reaction, in all cases nitrogenase activity also depends on the presence of a low-potential reductant 132,491).
For the activation of R. rubrum Fe-protein 3 2 6 , 4 9 2 , 4 9 4 ) as well as perhaps that of Rh. palustris 207), Rh. capsulata 202), and Az. lipoferum 409) a specific membrane-bound enzyme is necessary under certain conditions. The molecular weight of this enzyme from R. rubrum which is stabilized in the presence of Mn is about 20500 202,284).
The affinity of nitrogenase to N2 is higher than its affinity to the other substrates.
The Michaelis constant for N2 lies in the range 0.03 to 0.1 x 10-3 M whereas for other substrates this constant is only 1.2 x 10-2-3.6 x 10- 4 M 483,485).
Nitrogenase catalyzes the formation of HD from D2 and of H20 from H2 and D2O4 8 3 , 4 8 4 ). The formation of HD is stimulated by N2. It is probably formed by exchange reaction of D2 with the hydrogen of the intermediate products of the reduc- tion (bound to the enzyme) of diimide or hydrazine with N2. It is assumed that such "exchange" reaction (H+/D20) is carried out by nitrogenase (E) in the following way 2 3 , 4 8 4 ).
Nitrogenase-catalyzed H2 evolution was first observed when dithionite, ATP, and Mg2+ were added to enzyme preparations from Ab. vinelandii and R. rubrum482). The most intense evolution of H2 catalyzed by nitrogenase both in vitro and in vivo occurs in the absence of N2 which is a competitive inhibitor of this process485,495). Nevertheless, nitrogenase-catalyzed H2 evolution may proceed in the presence of N2, both in whole cells and in cell-free preparations.
The ratio of the rates of the reduction of H+ and N2 varies considerably for nitrogenases of different N2-fixing organisms 75,479). But even for pN2 values which are sufficiently high for nitrogen fixation, about 25 % of energy may be spent on H2
evolution by nitrogenase 132,479) . Nitrogenase-catalyzed H2 evolution by Anabaena sp. 7120 heterocysts decreases at 68% of N2 in the atmosphere. However, in the presence of 18% acetylene this evolution is entirely suppressed. From this it follows that acetylene and N2 inhibit H2 evolution and affect nitrogenase in different ways 479).
The rates of both H2 evolution and N2 reduction, catalyzed by nitrogenase, depend on the ratios of the MoFe-protein to the Fe-protein 23,411) and of ATP to ADP496).
It was demonstrated for X. autotrophicus nitrogenase preparations that an increase in the MoFe-protein content causes a rise of H2 evolution in the N2 atmosphere 411). H2 evolution is probably related to the normal mechanism of nitrogenase function in the course of which six electrons are spent for the reduction of N2 and at least two for the production of H2 23,442,479,484) Accordingly, the process of nitrogenase catalysis may be described by the following equation 76)
The binding sites of N2 and H2 on a nitrogenase are apparently different. Carbon monoxide and carbamyl phosphate do not affect H2 evolution while they inhibit N2
demonstrated by means of an artificial system containing vanadium as a catalyst497). This mechanism is in line with the view that nitrogenase can catalyze the transfer of four rather than of two electrons 481,497). The feasibility of such process was reduction and all other nitrogenase-catalyzed reactions483).
It is assumed481, that H2 production is catalyzed by a nitrogenase form with a distorted structure of its binuclear center. Such a form of nitrogenase is able to evolve H2 and to catalyze the slow exchange of deuterium, as well as to reduce compounds requiring at least one or two electrons. But it is incapable of N2 reduction.
4.2.2 Regulation of Biosynthesis and Activity
For the synthesis of nitrogenase to take place, it is necessary to provide the growing bacteria with iron and molybdenum since these elements are included into the enzyme molecule 132,200,201) . In the case of some purple bacteria the presence of manganese is also important. Manganese is essential for the action of the nitrogenase- activating enzyme 202).
Of considerable importance for nitrogenase biosynthesis by various bacteria is the oxygen content. Although Azotobacter and several other microorganisms are able to grow under aerobic conditions due to nitrogen assimilation, for nitrogenase synthesis lower pO2 values are favourable 2 3 , 2 4 , 4 8 4 , 4 8 8 , 5 3 2 ). A number of N2-fixing organisms that require oxygen for their growth synthesize nitrogenases only under microaerobic or anaerobic conditions. Examples are Az. lipoferum 498) and certain cyanobacteria 23, 3 4 , 1 7 4 , 2 1 9 , 2 3 1 , 4 9 9 ) . Nitrogenase synthesis by K. pneumoniae, which is a facultative anaerobe, demands strictly anaerobic conditions 23,484).
Considerable concentrations of NH4+ exert a repressable effect on nitrogenase synthesis by various bacteria 2 4 , 5 3 , 1 3 2 , 1 7 4 , 1 8 7 , 4 8 8 ).
According to a number of data on K. pneumoniae, Ab. vinelandii, the purple bacteria, and some other microorganisms, repression and derepression of nitrogenase synthesis depends on the state of glutamine synthetase, this enzyme plays an important role in the metabolism of ammonium, especially when its concentration is low. The following data furnish evidence of the role of glutamine synthetase in the regulation of nitrogen- ase synthesis. Firstly, it was shown that the K. pneumoniae mutants and the mutants of certain purple bacteria with derepressed nitrogenase are ineffective in glutamine synthetase. Secondly, in the presence of methionine sulfone, methionine sulfoximine and other compounds which are bound to glutamine synthetase and thus prevent it
from its function, a number of bacteria are able to synthesize nitrogenase in the presence of NH4+ 24,132,232,484,488,500) .
It is supposed that glutamine synthetase acts as a positive effector of nitrogenase synthesis when it is present in the active deadenylated form which is the case when there is a deficiency of ammonium. However, regulation of the activity of glutamine synthetase itself (through adenylation-deadenylation) does not take place in all In- fixing organisms. For example, it was not discovered in the Cyanobacteria 24). It cannot be ruled out that regulation of nitrogenase synthesis is carried out not by glutamine synthetase but by glutamine and other products synthesized from ammonium as the result of its functioning24,132,500-502).
Along with the regulation of synthesis, the activity of nitrogenase may change, depending on the conditions of the N2-fixing bacteria.
ADP shows an inhibitory influence on nitrogenase. Thus, an increase of the ADP/ATP ratio in the cells may lead to a sharp decrease in nitrogenase activity 484, 488)
Oxygen is one of the most significant factors affecting not only the synthesis but also the activity of the nitrogenases of various microorganisms. Furthermore, oxygen may cause inactivation of nitrogenase which is generally irreversible. Therefore, microorganisms are able to perform N2 fixation and to evolve H2, due to nitrogenase activity only under anaerobic conditions. Comparatively few bacteria may perform these processes under microaerobic and even less under aerobic conditions53,132,231, 4 8 4 , 5 0 3 , 5 3 3 ) . Nevertheless, from the latter case it does not follow that nitrogenases of these microbes are more stable to oxygen than that of anaerobes. Nitrogenase activity in the presence of O2 is explained by the ability of these organisms to protect the enzyme against the inactivating effect of O2 23,24,484,488,516,534) .
In the case of Azotobacter and probably some other N2-fixing organisms, a high respiration rate is of considerable importance 23,534). Due to this high rate there is not sufficient time available for O2 to inactivate nitrogenase (respiratory protection).
Besides, Azotobacter and several other N2-fixing organisms possess slime capsules which may also be important for the protection of nitrogenase against oxygen.
Location of nitrogenase in cells may be significant for its protection against oxygen (conformational protection).
Nitrogenase of the filamentous cyanobacteria may function in vivo under aerobic conditions. As mentioned above, it is located in heterocysts with a very thick envelope. Moreover, in contrast to vegetative cells, heterocysts do not evolve O2, but are actively involved in the consumption of the latter. So in this case, a further way of protecting nitrogenase against O2 is effective, namely the special separation of N2 fixation from photosynthesis which is accompanied by O2 evolution.
In the nodules of leguminous plants leghemoglobin apparently takes part in O2
protection of the nitrogenase of bacteroides and lowers the concentration of oxygen.
It is also suggested that H2 production by N2-fixing organisms might be a way of protecting nitrogenase against high oxygen concentrations 23,83,131).
However, the mechanism of nitrogenase inactivation by O2 remains unclear.
Probably, it is related to certain reaction products of oxygen, e.g. to O2*, O2 and Apart from O2, the inhibition of the nitrogenase activity of the purple bacteria and cyanobacterium An. cylindrica is caused by ammonium477,535). However, the
effect of ammonium is reversible, and when this compound is assimilated by the cells, nitrogenase becomes active again187,191,194,207-209,464,504-506,535). There are also data on the inhibition of H2 evolution by ammonium in Rhizobium leguminosarum bacteroids536). The inhibiting effect of NH4+ on the nitrogenase of R. rubrum, Rh. palustris and Rh. capsulata depends on the growth conditions of the latter.
Ammonium does not effect nitrogenase of the cells grown under nitrogen-limiting conditions 21 •535). It was supposed that glutamine synthetase takes part not only in the regulation of nitrogenase synthesis but also in the regulation of nitrogenase activity as well as in some bacteria 207,2°8,507).
According to recent results, inhibition of nitrogenase activity of R. rubrum in the presence of NH4+ is determined by the conversion of this enzyme to an inactive re- gulated form. Simultaneously, the conversion of glutamine synthetase to the inactive adenylated form is observed 494). However, the nitrogenase activity is possibly not regulated by the direct participation of glutamine synthetase but affected by glutamine or other products formed as a result of this activity of the enzyme500,501,505,508). Recent data prove that glutamine synthetase is not directly involved in the regulation of Rh. palustris and Rh. sphaeroides nitrogenase 522).
Along with ammonium, the activity of nitrogenase of An. cylindrica and of several purple bacteria is inhibited in the presence of glutamine, asparagine, urea, NO3-, and NO2- 1 8 7 , 2 0 7 , 2 0 8 , 4 6 4 , 5 0 4 . 5 0 6 , 5 0 9 ) However, these compounds do not affect all In- fixing organisms 484).
Meanwhile, it is known that nitrogenase activity, assayed via the reduction of N2
or C2H2, is inhibited in different bacteria by carbamyl phosphate and by CO.
However, H2 evolution, is not influenced by these substances484).
It should be pointed out that the capability of microorganisms for N2 fixation and for nitrogenase- or hydrogenase-mediated H2 evolution may depend on the activity of other cellular components involved in these processes. Oxygen, for instance, may not only cause the inactivation of nitrogenase and hydrogenase but, in certain microorganisms also oxidize the electron carriers interacting with the en- zymes. As a result, H2 formation by the cells ceases 448).