Metabolism of Nitrogen Fixation | Microbiology

In this article we will discuss about the metabolism of nitrogen fixation.

Nitrogenase enzyme, which is chiefly associated with fixing of nitrogen, is inactivated by oxygen. This inactivation may be reversible or irreversible, the latter clearly being a more serious problem. How it arose is an open question? One suggestion is that oxygen sensitivity results from evolution of nitrogenase in days before oxygen was present in atmosphere.

An alternative proposal is that in order to reduce such a stable substrate as nitrogen gas, nitrogenase is invariably able to pass electrons to stronger oxidizing agents of comparable size, such as oxygen.

Thus nitrogenase-oxygen complexes would be expected and if stable, these would prevent the reduction of nitrogen. Reversible inactivation may be a way of regulating its activity, oxygen may also regulate nitrogenase synthesis. Each and every type of nitrogen fixing organism, therefore, has some means to counter the effects of oxygen.

(i) Anaerobic Microorganisms:

There are two types of anaerobic nitrogen fixers: photosynthetic and non-photosynthetic. In non-photosynthetic nitrogen fixers, ATP for nitrogen fixation is provided by substrate level phosphorylation. Certain species like K. pneumoniae fix nitrogen better when grown micro-aerophilically. This is because it can use glucose. Low oxygen coupled with low combined nitrogen may stimulate nitrogenase synthesis.

In Rhodospirillum rubrum photosynthesis does not involve oxygen evolution but generates ATP. Nitrogenase activity is switched off in dark and switched on again in light. This is due to reversible modification of di-nitrogenase reductase enzyme. Inactive form of di-nitrogenase reduc­tase has an ADP-ribosyl group specifically attached to it.

In the presence of light this group is removed by an enzyme, DRAG (di-nitrogenase reductase activating glycohydrolase), and in dark it is replaced using NAD and Mg-ADP using enzyme DRAT (di-nitrogenase reductase ADP-ribosyl transferase). DRAT is controlled by ammonium and glutamine, hence it ensures that nitrogenase is switched off when combined nitrogen is available.

This phenomenon also occurs in Azotobacter, Rhizobium, K. pneumoniae and Methylococcus capsulatus. Therefore, photosynthetic bacteria do not have problem of internally generated oxygen and they may have to only guard against low levels of oxygen from outside. Reversible inhibition of nitrogenase by oxygen can also be influenced in R. rubrum by DRAG/DRAT system.

(ii) Cyanobacteria:

These microorganisms carry out higher level of photosynthesis i.e. generate oxygen in light. They have also to face the problem of externally supplied oxygen. These organisms solve the problem of oxygen inactivation of nitrogenase by separating photosynthesis and nitrogen fixation in space or time.

Heterocystous cyanobacteria carry out nitrogen fixation in heterocysts and photosynthesis in vegetative cells. These organisms can fix nitrogen in light as well as dark. In dark it requires oxygen and ATP for nitrogen fixation generated by respiratory processes, and in light ATP is generated in heterocysts which contains only part of photosynthetic apparatus. Heterocysts lack both O₂ evolution and CO₂ fixation but retain the ability to generate ATP and reductant.

Heterocysts are connected to vegetative cells by large pores which permit a photosynthate disaccharide to heterocysts and glutamine out of heterocysts. Sugars passing in, help to generate NADPH by pentose phosphate pathway or scavange oxygen via respiratory processes. Heterocyst wall allows sufficient nitrogen in but restrict oxygen diffusion to a level which can be removed by respiration.

Non-heterocystous cyanobacteria carry out separate photosynthesis and nitrogen fixation in time (day/night respectively). Aerobic organisms can fix nitrogen in light and dark, but when grown in alternating light/dark cycles, fix 95% nitrogen during dark.

Thus, it separates photosynthesis from nitrogen fixation. Atmospheric oxygen is used to generate ATP by respiration. In certain cyanobacteria control of nitrogenase is at transcriptional level acting like an endogenous rythm.

(iii) Free Living Aerobic Microorganisms:

Azotobacter is the most studied and best example of free living aerobic nitrogen fixers. It produces another FeS protein which complexes with two nitrogenase proteins to form a three membered oxygen stable and inactive complex. This is a short term response to increased pO₂.

The overall system represents a series of modifications of respiration e.g. cytochrome O increases as pO₂ decreases and coupled with an increase in phosphorylating efficiency. There is, therefore, a branched pathway of respiration for different conditions (Fig. 14.2).

Under low pO₂ conditions, electron flow from NADPH via cytochrome d to oxygen, by passing phosphorylation. Under high pO₂, the pathway from malate and NADPH is followed. Thus at high pO₂, oxygen is taken up rapidly and inactivated.

Fig. 14.2 : Electron transport pathway in Azotobacter.

Under low pO₂, oxygen may limit phosphorylation. Carbon compounds and reducing equivalents then tends to accumulate. This leads that acetyl CoA, instead of entering tricarboxylic acid cycle is used with NADPH for synthesis of poly β hydroxybutyrate, a major reserve product.

Colony morphology is also altered similarly to changes in pO₂ e.g. Azospirillum when grows without combined nitrogen on semisolid agar can move to that part of culture which has a suitable pO₂ for nitrogenase activity.

Association with an oxygen consuming organism is a natural way of finding a niche with required level of oxygen. Azospirillum sp. do this by colonizing with a wide variety of plant roots.

Therefore, cyanobacteria show a number of associations with wide variety of organisms e.g. R. capsulata fixes nitrogen under aerobic conditions with Bacillus megaterium. Chloropseudomonas ethylicum now appears to be a mixture of at least two species, one being aerobic and non-nitrogen fixer and other nonaerobic but nitrogen fixer.

(iv) Symbiotic Microorganisms:

These microorganisms fix nitrogen by formation of nodules in the roots of the symbiotic plant. Nodules have a variable diffusion resistance to oxygen, coupled with leghaemoglobin (a haemoglobin like pigment found in nodules) provide a very good system for tailoring oxygen demand to supply rhizobia; best example of these type of organisms can also develop branched respiratory pathways and multiple forms of cytochromes linked to varying oxygen sensitivity.

Control of oxygen diffusion is additionally associated with intercellular space system which may vary with species and/or environment and through the development of special walls in nitrogenase containing vesicles.

Fig. 14.3 : Regulation N₂ fixation in Kiebslella pneumonlae by nif gene products.