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Metal cubes, protein landscapes and the global nitrogen cycle

Nitrogen occurs in all living organisms. Bacteria play a key role in the global N cycle as they possess the enzymes that convert atmospheric nitrogen gases into compounds that can be used by other organisms. How do these enzymes work? This is an interesting question for scientists and industry alike because modern methods used for the production of nitrogen for use in plant fertilisers and other applications are very efficient. Prof. Dr. Oliver Einsle and his team at the University of Freiburg have found a way to investigate the reactive centres of bacterial enzymes. All nitrogen-converting enzymes contain metal ions, and it is these metal ions that mediate the underlying chemical reactions. What happens inside the bacterial proteins? And how can detailed insights be obtained?

Nitrogen is a constituent element of amino acids and thus of proteins. DNA also contains nitrogen atoms. Nitrogen is required by the human body to function properly. However, higher organisms such as humans are only able to take up nitrogen in reduced form, namely as ammonium ion, and are therefore unable to use nitrogen in the form of nitrogen gas (N2) or in oxidised form (NO2, N2O or NO) in the Earth's atmosphere. Plants transform atmospheric nitrogen into a form suitable for use by other organisms. They form symbiotic relationships with nitrogen-fixing bacteria that convert atmospheric nitrogen into ammonium ions. The plants then incorporate the ammonium ions into amino acids which humans take up with their food. When we die, bacteria metabolise nitrogenous compounds, thereby turning oxides back into nitrogen gases that are subsequently discharged into the atmosphere. This process is known as denitrification.

Superior to industry

The unique metal cluster in the centre of the nitrogenase enzyme. Iron (grey) and sulphur atoms (yellow) are arranged in two concentric spherical shells surrounding a light atom (carbon, nitrogen or oxygen). Parts of the protein molecule that anchor the cluster in the enzyme are shown above and below the metal centre. © Prof. Dr. Oliver Einsle

Nitrogenase is one of those enzymes that play a key role in the fixation of nitrogen. This complex protein is found in many bacteria that live in symbiotic relationships with plants. It can convert N2 into ammonium ions, thereby helping feed atmospheric nitrogen into food chains. The centre of nitrogenase enzymes contains two cubes made up of iron and sulphur atoms. One of these so-called iron-sulphur clusters, which also contains molybdenum, mediates the actual enzymatic reaction. "Very unusual chemical reactions occur in the enzyme's centre, which consists of only a few inorganic atoms," said Prof. Dr. Oliver Einsle from the Institute of Organic Chemistry and Biochemistry at the University of Freiburg. "This enzyme is able to do naturally what the industrial Haber-Bosch process can only achieve at high pressure and temperatures. Moreover, the Haber-Bosch process generates much less ammonia than the nitrogenase enzyme," said Einsle.

The Haber-Bosch process is used to turn N2 into ammonium ions which can then be used to produce nitrate fertiliser. However, production of around one t ammonia requires a great deal of energy, corresponding to the quantity of energy required to produce around 1.7 t CO2. Moreover, only about 50 per cent of the fertiliser produced reaches the plants; the other 50 per cent are degraded by soil bacteria. Scientists who are investigating metalloproteins such as nitrogenase therefore always ensure they are aware of effective industrial use of metalloproteins. "It would be nice if we were able to give plants the ability to convert atmospheric nitrogen into the form required," said Einsle implying that a detailed understanding of bacterial enzymes is needed. The scientists are interested to find out where the educt (i.e. N2 gas) is stored in the reactive centre of the enzyme. Which intermediary steps are used for the transfer of electrons from iron atoms to nitrogen? One possible approach is to analyse the enzyme's structure. Once the researchers know the conformation of a specific protein, they will be able to make initial assumptions on what happens during the reaction.

Seafarers and terra incognita

Einsle and his team possess the know-how and the technical equipment to crystallise proteins. In order to generate a model of a three-dimensional protein structure, the scientists determine the spatial distribution of electrons in crystals. However, this is far from being a standard procedure, quite the contrary. It can take up to several months for the scientists to isolate the proteins from the sample tissue and purify them. And they are then faced with an extremely sophisticated step: “In principle, the cultivation of protein crystals is an alchemical process,” said Einsle. “Trial and error are the keywords. This is how we find the pressure, temperature and concentration conditions that proteins require to form a crystal. Sometimes, it is necessary to carry out thousands of experiments before the proteins crystallise and there is never any guarantee of success.”

The blue grid shows an experimental electron density map, the result of an X-ray diffraction experiment. A structural model consisting of individual models is subsequently manually incorporated and fitted into this three-dimensional map. © Prof. Dr. Oliver Einsle

Once the researchers have succeeded in producing a crystal, they irradiate the crystals, which are only a few micrometres in size, with X-rays in a so-called X-ray diffractometer. The electrons surrounding the atoms of the proteins deflect the energy-rich rays in a characteristic way. The researchers are then able to measure the diffraction patterns and a computer programme turns them into a complex, three-dimensional grid. It often takes weeks of hard work to interpret the chaos. Are there any characteristic structures that can be matched with known amino acids? Are superordinate structures visible? Gradually, the researchers are coming up with a protein model containing thousands of atoms. This is how Einsle and his team decipher the three-dimensional structure of the nitrogenase enzyme with the two iron-sulphur clusters.

"Crystallography is proper research-based science," said Einsle. "We go out and see something nobody has seen before. Sometimes I feel like a seafarer looking at a terra incognita." At present, the biochemists are working to gain an understanding of what happens on the chemical level in the reactive centre of the nitrogenase enzyme. Which iron atoms mediate these reactions? What is the orientation of the nitrogen molecule in the protein's interior? Which amino acids in the protein are crucial for this? In order to find answers to these questions, the scientists need to combine the results from a broad range of experiments, including from the site-directed mutagenesis of amino acids or biochemical experiments that measure the enzyme's N2 turnover. They are currently investigating how the metal centre is established and incorporated into the protein by manipulating several genes.

An integral part of life sciences research

Spatial structure of the enzyme dinitrogen monoxide reductase (N2O reductase). The protein consists of two amino acid chains (green and blue) that are schematically shown as ribbons. The complete model shows the exact position of more than 10,000 individual atoms. The reactive metal centres of N2O reductase contain copper ions, which are shown in the picture as spheres. © Prof. Dr. Oliver Einsle

Einsle and his team have used crystallography and made good progress with another enzyme, i.e. crimson dinitrogen monoxide reductase (N2O reductase). This enzyme, whose reactive centre contains a cube of copper ions and hence is lilac in colour, also catalyses a step in the global nitrogen cycle. N2O reductase catalyses the final reaction of the denitrification reaction, where N2 is produced from N2O and released into the atmosphere or reduced once again to ammonium ions. Einsle and his team have clarified the spatial structure of the enzyme. In addition, they have found out how the N2O molecules are arranged at the copper cube in the centre of the metalloprotein. The researchers now have to clarify the exact mechanism of the reaction and find out whether the process can be simulated in silico.

Bacteria use the enzymes of the nitrogen metabolism to produce energy. The enzymes are located on the bacterial membranes. The reactions lead to the release of protons (i.e. positively charged hydrogen ions), which are pumped across the membranes, a process during which energy is released. The biochemists in Einsle's group are not only interested in investigating the enzymes of the global nitrogen cycle, but are generally interested in the function of membrane proteins. How do the membrane proteins transport protons? How does this process lead to the generation of energy? Einsle and his team have been in Freiburg for two years now where they benefit from working with teams from the faculties of chemistry, medicine, biology and pharmacy. These groups provide Einsle's group with sample material as well as new ideas and questions. In return, Einsle and his team elucidate the structure of the proteins in which their cooperation partners are interested. "People working in many different disciplines contact us to find out what their proteins look like," said Einsle going on to add "such projects have positive outcomes for both sides". X-ray crystallography has become an integral part of modern life sciences research, just as microscopy did many years ago.

Further information
Prof. Dr. Oliver Einsle
Department of Biochemistry
Institute of Organic Chemistry and Biochemistry
University of Freiburg
Albertstrasse 21
79104 Freiburg
Tel.: +49 (761) 203 6058
Fax: +49 (761) 203 6161
E-mail: einsle(at)bio.chemie.uni-freiburg.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/metal-cubes-protein-landscapes-and-the-global-nitrogen-cycle