Nitrous: Cracking the cycle

Greenhouse nitrogen

The greenhouse gas nitrous oxide undergoes partial decomposition depending on environmental conditions. Now, researchers in Germany have determined the structure of an enzyme, N₂O-reductase, that breaks down the gas. The structure reveals the surprising presence of four copper atoms and two sulfur atoms, as opposed to one, at its active centre.

N₂O-reductase reduces nitrous oxide to nitrogen gas and an enzyme highly sensitive to oxygen concentration. Agriculture releases large amounts of nitrous oxide gas into the atmosphere from fertilised fields through bacterial respiratory pathways of nitrification and denitrification. The gas has many other anthropogenic and non-industrial sources, such as from hypersaline ponds in the Antarctic. Overall nitrous oxide has a significant role in the nitrogen cycle. Moreover, given that nitrous oxide is a greenhouse gas three-hundred times more potent than the more infamous carbon dioxide, although not quite as long lived, this is a serious issue for climate change. Moreover, nitrous oxide under bombardment by cosmic rays in the upper atmosphere can also cause ozone destruction.

Anja Pomowski and Oliver Einsle of the Albert-Ludwigs-Universitaet Freiburg, Walter Zumft of of the Karlsruher Institut fur Technologie and Peter Kroneck of the Universitaet Konstanz, in Germany, have thoroughly investigated the functionality and mechanisms of this important enzyme from the microbe Pseudomonas stutzeri, in studies that strictly excluded dioxygen from the equation and so focused on the "purple" as opposed to "pink" form. Previous research had not made the important distinction between the purple and pink forms thus giving rise to several incongruities in the data emerging from various laboratories. The enzyme was first identified in 1982 and has been the focus of considerable research ever since. Writing in the journal Nature, the team attempts to fill the gaps in previous incomplete structural studies of the protein and reconcile data that did not explain the behaviour of the enzyme clearly.

Direct structure

"This structure represents the first direct observation, to our knowledge, of nitrous oxide bound to its reductase," the team says, "and sheds light on the functionality of metalloenzymes that activate inert small-molecule substrates." The researchers point out that the principle of using distinct clusters for substrate activation and for reduction might be found in other biochemical systems such as in the well-known nitrogen-fixing enzyme, nitrogenase. "It was of decisive importance that all steps of our investigation were executed in the absence of air oxygen," adds Zumft, now a retired professor. "In contact with oxygen, parts of the enzyme react and the enzyme changes its structure," he explains.

The team points out that their structure does not fix the process of activation and reduction of nitrous oxide by the enzyme. Indeed, the separation between the molecule of gas and the requisite copper cluster (as supported by EPR and UV-Vis spectroscopy) suggests that a stronger interaction might be needed and that would demand a conformational change. Nevertheless, they add that the positioning of nitrous oxide between the two metal centres does point to a reaction mechanism involving the precise orientation of the gas molecule by the protein and that this is somehow activated by the copper cluster.

The structure will now allow the reaction sequence of the enzyme to be modelled more effectively so that future investigations can garner details and improve our understanding of how environmental conditions affect the process of nitrogen reduction and the role of agriculture in the nitrogen cycle. "The current study provides interesting and complementary insight into the nitrogen cycle," explains Kiese from the KIT Institute of Meteorology and Climate Research. Nitrous oxide and nitrogen production on fields, pastures, and in forests depends on a multitude of often opposing effects.

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.