THE METABOLISM OF INORGANIC NITROGEN AND ITS COMPOUNDS IN MICRO‐ORGANISMS

Summary The most important advance made recently in studies with biological nitrogen fixation has been the preparation of active cell extracts of micro‐organisms that will fix nitrogen gas. This has been achieved in a variety of bacteria and blue‐green algae. Thus, at last it is now possible to study the nitrogen‐fixing system outside the cell. Results with these extracts support earlier work on whole cells showing that ammonia is the ‘key intermediate’ in nitrogen fixation, but evidence for earlier intermediates is less definite. Using the stable isotope 15 N, and more recently the radioactive species 13 N, it is clear that should intermediates be formed between nitrogen and ammonia they must be tightly bound to the enzyme complex, since ammonia is the first free labelled product. Results of 13 N experiments suggest that products of fixation are formed before ammonia, and it is likely that the nitrogen‐activating enzyme is itself labelled. In some extracts the hydrogen‐donating system has been separated from the nitrogen‐fixing component. Neither alone fixes nitrogen but together they activate the gas, resulting in ammonia formation. In many anaerobic micro‐organisms fixation is linked to the metabolism of pyruvate, i.e. the phosphoroclastic reaction yielding hydrogen, carbon dioxide and acetyl phosphate, but the precise mechanism is not known. A new non‐haem iron protein, ferredoxin, first isolated from Clostridium pasteurianum , is an electron carrier in pyruvate metabolism. It is the most electronegative natural carrier isolated thus far (estimates between –0–35 and – 0–42 V.) and it is primarily concerned in hydrogen metabolism of micro‐organisms. Another quite distinct species of non‐haem iron, which may be more directly associated with the nitrogen‐fixing component, has been identified by electron paramagnetic resonance spectroscopy. This type of iron has a wide distribution in animals, plants and microorganisms. It is likely that these newly identified non‐haem iron species are the forerunners of others not yet identified in biological materials. They are likely to be as important as the well‐known haem iron components, i.e. the cytochromes, in cell metabolism. Molybdenum was found, by electron paramagnetic spectroscopy (EPR), to undergo valency changes in nitrogen‐fixing particles from Azotobacter reduced first with hydrogen and reoxidized with nitrogen. Since these effects were not observed in particles from cells grown with ammonium salts, where the hydrogen‐donating system is still intact but the nitrogen‐fixing component is absent, it was concluded that molybdenum may link the two systems in the active particles. Evidence that these EPR changes for non‐haem iron and molybdenum are also linked to the respiratory chain to oxygen was obtained by using the EPR technique. Minute amounts of cobalt or vitamin B 12 are required for nitrogen fixation in nodulated plants and also in free‐living nitrogen fixers, e.g. Azotobacter vinelandii. The coenzyme, 3.5‐benzimadazolylcobamide, has been identified in Azotobacter and in root nodules. Since only minute amounts of the B 12 coenzyme are required, it is more likely that it functions in the synthesis of enzymes concerned in nitrogen fixation rather than directly as a cofactor. Cell‐free extracts have not yet been prepared from the symbiotic bacteria in root nodules but it has been proposed that fixation may occur in the membranes surrounding the bacteroids in the nodules. Bacteroids in the nodules are bathed in haemoglobin; since the pigment has a high affinity for oxygen it may prevent free oxygen from coming into direct contact with the nitrogen fixing system and thus competing with nitrogen for the reducing power generated by the bacterioids. This concept is similar to the alternative electron transfer scheme to oxygen or nitrogen proposed for Azotobacter. By oxidizing inorganic nitrogen compounds, nitrifying bacteria derive the energy required for the fixation of carbon dioxide and other cell processes associated with growth. These organisms do not utilize sugars, organic acids or amino acids as a source of carbon. Difficulties encountered in culturing these bacteria have been largely overcome and thus sufficient cells for biochemical studies can now be grown in batch or semi‐continuous cultures. Recent success in preparing cell‐free extracts of Nitrosomonas and Nitrobacter has led to an elucidation of the enzymes concerned in the oxidation processes. Components of the respiratory chain are involved in the dehydrogenation of hydroxylamine in Nitrosomonas and of nitrite in Nitrobacter. Cytochrome oxidase facilitates reoxidation of the reduced chain by oxygen in both organisms. It has been proposed that the intermediate between hydroxylamine and nitrite is nitrohydroxylamine but there is insufficient evidence to be certain. Rapid oxidation of ammonia has not been achieved in cell extracts, because the ammonia is probably metabolized in the intact cell membranes which are readily disrupted and deactivated during cell breakage. Phosphorylation occurs during these oxidations and the energy is conserved in adenosine triphosphate. Although the path of carbon dioxide fixation has not been followed in detail in nitrifying bacteria, preliminary information shows that 3‐phosphoglyceric acid is labelled very early after exposure of the cells to 14 CO 2 . Other products formed are similar to those found when carbon dioxide is utilized by green plants. The utilization of nitrate nitrogen can be conveniently discussed under assimilation when nitrate is reduced to ammonia and incorporated into cell nitrogen or dissimilation when nitrate, usually under anaerobic conditions, is used as an alternative hydrogen acceptor to oxygen. Denitrification may be regarded as a special type of dissimilation in which nitrogen gas, nitrous and nitric oxides are produced, thus eliminating nitrite which can prove toxic to bacteria when allowed to accumulate. Since there are striking similarities between enzymes and intermediates in both the assimilation and denitrification processes, it is likely the latter is an adaptation of the former rather than a de novo formation of an entirely new pathway. The two types of nitrate reductases are of interest in relation to their function. Thus the slow assimilatory enzyme is a molybdoprotein only, but the faster turn‐over required of the dissimilatory or respiratory system as an alternative hydrogen acceptor to oxygen necessitates cytochrome components to make it a more efficient electron‐transfer system. It is of interest that all nitrate reductase enzymes examined contain molybdenum. Results thus far support the view that the inorganic route from nitrate to ammonia is an important reductive pathway. This does not rule out an alternative or even a simultaneous reduction sequence involving organo‐nitro compounds but present‐day evidence for the latter is not convincing.