Nitrogenase is a highly O2-sensitive enzyme. The mechanism of O2 sensitivity is not known, but both components become irreversibly inactivated on exposure to atmospheric levels of O2. This may be an accidental relic of the evolution of nitrogenase at a time when the earth’s atmosphere still contained very little O2, or it may be a fundamental feature of the dinitrogen reduction reaction. In any case, protection of nitrogenase from O2damage is a very important aspect of N2-fixation, and a surprising variety of mechanisms have been adopted by N2-fixing organisms.
The first solution is to avoid the problem altogether, as occurs in obligate anaerobes such asClostridium pasteurianum. Similarly, many facultative anaerobes, such asK. pneumoniae, have a system of genetic control that ensures that nitrogenase is only synthesized when the ambient O2tension is very low (Gussinet al., 1986), the optimum oxygen concentration for N2-fixation inK. pneumoniaebeing 0.03mM in comparison with the concentration of 225mM found in air-saturated water (Hill, 1992).
All other organisms face a paradox. They have an absolute requirement for O2to generate the ATP and reducing equivalents required by nitrogenase, and yet a high pO2 will inactivate the enzyme. The optimum conditions for nitrogenase activity in an aerobic organism therefore occur when the supply of O2exactly matches respi- ratory demand: if the O2supply is insufficient, then respiration, and consequently many other metabolic reactions, including nitrogenase activity, will be reduced; on the other hand, if the oxygen tension rises above the rate of respiratory consumption, nitrogenase activity will be damaged. The simplest way for obligate aerobes to cope with this dilemma is to fix N2only in microaerobic conditions – that is, when the pO2is well below atmospheric concentrations.
Microaerobiosis can be achieved in a number of ways, almost all of which require exploitation or creation of a physical barrier that restricts O2 diffusion in combination with respiratory consumption of O2. Some organisms, such as Azospirillum brasilense, use a ‘behavioural’ strategy by migrating to regions of intrinsically low pO2. This movement results in the characteristic pellicle formation observed when they are grown in culture (Chapter 2). Other organisms show a different behaviour, aggregating to form colonial structures in the centre of which the pO2is lowered. Such behaviour is seen in a number of unicellular cyanobacteria, and in the characteristically huge colonies ofDerxia gummosa(Chapter 2).
Another strategy to obtain microaerobic conditions is to restrict nitrogenase to specialized cells that have a wall with very low permeability to O2– for example, the vesicles formed byFrankia(Fig. 3.2) and the heterocysts of filamentous cyano- bacteria. Cyanobacteria have a particularly tricky problem in that they actually produce O2during oxygenic photosynthesis, and they solve this by either spatial or temporal separation of photosynthesis and N2-fixation.
Spatial separation occurs among members of sections IV and V of the fila- mentous cyanobacteria (Chapter 2). Cells at regular intervals along the filament (commonly about every ten cells) differentiate into heterocysts. These are cells in which photosynthesis ceases – photosystem II (PSII) becomes inactive – so enabling
high levels of nitrogenase activity without the problem of evolution of O2 within the same cell. They are usually larger than their adjacent vegetative cells in the cyanobacterial filament, and are enclosed within a double-layered envelope that includes novel polysaccharide and glycolipid components which render them almost impermeable to O2. Heterocysts are terminally differentiated cells in that they can neither divide nor de-differentiate – in this respect they are perhaps analogous to rhizobial bacteroids.
There are also N2-fixing species among most of the non-heterocystous fila- mentous and among the unicellular genera of the cyanobacteria. In these species, there is temporal separation of photosynthesis and N2-fixation. InGloeocapsathis was shown to be related to light–dark cycles, photosynthesis occuring in the light and nitrogenase activity in the dark (Mullineauxet al., 1981). In another unicellular cyanobacterium,Synechococcussp., synchronized cell cultures were used to show that photosynthesis and nitrogenase activity were in fact separated in two phases of the cell cycle. Maximal O2evolution, and hence photosynthesis, occurred just before cell division and it declined thereafter to reach a minimum value halfway through the cell cycle. Patterns of nitrogenase activity were exactly the inverse of this, reaching a maximum when photosynthesis was at a minimum and vice versa. This temporal separation was maintained for at least one cell division cycle in continuous light (Mitsui et al., 1986). It was later shown that, for Synechococcus, nitrogenase is synthesizedde novoevery cycle of activity (Huanget al., 1988).
N2-fixation within a symbiotic association is another very successful means to address the O2problem. The O2physiology of legume nodules has been particularly well studied and comprises two main components. One is a strong and variable physical barrier to O2diffusion in the nodule cortex (Hunt and Layzell, 1993; Witty and Minchin, 1996). The existence of such a variable barrier was deduced from the Fig. 3.2. Vesicles formed byFrankiagrowing in pure culture provide protection for nitrogenase against oxygen damage. (Photograph: A.D.L. Akkermans.)
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observation that external O2concentrations can be increased up to almost 100%
without a significant decline in nitrogenase activity or a significant increase in nodule respiration. Direct evidence for the barrier was subsequently obtained from direct measurements of O2concentrations in the nodule using O2-selective microelectrodes (Wittyet al., 1987). This barrier is located just outside the infected zone of a nodule, possibly in a ‘boundary layer’ where thin-walled cells are very tightly packed without intervening gas spaces (Fig. 3.3). The variable diffusion resistance of this barrier appears to operate, at least partly, through a rapid osmotic response to environmental stimuli which causes cells to collapse or expel water into intercellular spaces. The sec- ond main component of O2protection in the legume nodule is the presence within the infected cells of leghaemoglobin, an O2-binding molecule that serves to provide an adequate supply of O2 for respiration, while keeping it sequestered away from nitrogenase. Other mechanisms are almost certainly involved in the overall reversible response to changes in external O2concentrations within the infected region of the nodule, including enhanced mitochondrial respiration and glycoprotein occlusions (Minchin, 1997). There is also indirect evidence for conformational protection of nitrogenase (see below) in bacteroids of soybean nodules (Denisonet al., 1992).
Fig. 3.3. The change in oxygen concentration across a legume root nodule. (After Wittyet al., 1986.)
A few organisms are able to fix N2at levels of O2close to atmospheric partial pressures.Azotobacteris the best studied and has three protection systems (Kennedy and Toukdarian, 1987). One is known as ‘respiratory protection’, which means that cultures ofAzotobactercan adapt their rate of respiration in step with the external pO2. This is achieved by use of a branched respiratory chain, with different branches generating different amounts of ATP. When the pO2is high, but the requirement for ATP is not correspondingly increased,Azotobactercan divert its respiratory electron flow along a path that leads to minimal generation of ATP, thus burning O2rapidly without overburdening itself with ATP. Conversely, if the pO2falls,Azotobactercan increase its respiratory efficiency – i.e. generate more ATP per electron transferred – simply by using a different respiratory pathway (Hill, 1992).
Azotobacter has a further temporary means of preventing O2 from reaching nitrogenase, sometimes referred to as ‘conformational protection’. It is mediated by a low molecular weight iron–sulphur protein that binds to the entire nitrogenase complex and thereby protects it from O2 damage. In the process, nitrogenase is temporarily inactivated (Scheringset al., 1983). It is thought that this mechanism is used byAzotobacterto protect its nitrogenase following a sudden change in external pO2, while the organism has time to adapt its respiratory system.
The third mechanism for protection of nitrogenase has been termed ‘auto- protection’ as it is directly mediated by the Fe protein acting to reduce O2to H2O2, which is subsequently converted to H2O by superoxide dismutase. This occurs when the molar ratio of reduced (electron-bearing) Fe protein to O2is four or greater (Hill, 1992). Yet another form of metabolic protection lies in the activity of uptake hydrogen- ases which recapture H2evolved by nitrogenase and, in the process, use O2(see above).