Denitrification and producers of nitrous oxide ( a global warming menace)

 

Denitrification is a form of respiration. Only microorganisms break down nitrate to nitrous oxide or nitrogen, in order to make energy. This process is part of the nitrogen cycle, which is the recycling of nitrogen containing compounds through the biosphere. Nitrate (NO3-) is often a component of fertilizers and with the greater use of fertilizers in our world, the importance of the processes by which bacteria break them down becomes important. It matters whether organisms break down nitrate to nitrous oxide (N2O) or nitrogen (N2), because nitrogen is pretty unreactive in the atmosphere but, nitrous oxide has over 200 times the global warming potential of carbon dioxide.denitrification_2

Figure 2. Denitrification (breaking down nitrate to nitrogen)

Each of the reactions shown above require an enzyme ( a protein which catalyzes a chemical reaction) to perform the reaction. I work primarily with the enzymes which catalyze the reaction of the step which produces nitrous oxide, i.e when nitric oxide (NO) is reduced to nitrous oxide. This enzyme is called the nitric oxide reductase (NOR). All NORs in microbes are related to one another because they have structural similarities and similar amino acids to make them up.

How important are the similarities and differences in amino acids of proteins? 

The arrangement of amino acids within proteins can change the structure of the protein and its function. For eg, polypeptides labelled ACAACHGALH and ACAGCHGGLH (each of the alphabets is code for a different amino acid), only differ by a single amino acid and if scientifically modeled, we can tell that the structure and function of the two polypeptides may be affected. We can use sequence alignments which tell us where these differences are, and other techniques which tell us how different the amino acid compositions of proteins are, to identify how similar some related proteins are, and how different.

Using techniques like the above, we identified NORs which perform the same function as cNOR and qNOR (two nitrous oxide producing enzymes which were found before) but were different enough to have some very different components in the enzyme. They were different enough that they were previously never identified as NORs.

However, we looked at sequences in bacterial genomes that were different enough and then purified the protein corresponding to the sequence from the organism. Then. we used biochemical techniques to verify that these proteins were doing the chemical reaction that we thought they were, i.e making nitrous oxide from nitric oxide. We did this by incubating the protein with nitric oxide (NO) and using gas chromatography to see what the end product was. We were also able to see that the bacteria was breaking down nitrate to nitrous oxide and eventually nitrogen by observing the products made by the bacterium using gas chromatography.

The ability to guess what functions proteins perform from the similarity and difference of amino acid sequences of proteins is extremely important for us to be able to predict what functions unknown and previously unstudied bacteria perform. This is because our current technology allows us to easily figure out the sequences of proteins (encoded in the genes) bacteria can make. Knowing what proteins can do on that basis of sequence therefore gives us a big advantage in predicting environments and selecting bacteria useful for us to study.