An enzyme with exceptional heat resistance converts atmospheric nitrogen into ammonia, an important fertiliser and biofuel
Illustration of the core of the heat-stable nitrogenase from a deep-sea microorganism, showing the metallocofactor which breaks nitrogen gas into ammonia.
© Tristan Wagner / Max Planck Institute for Marine Microbiology. Illustration by Benjamin Large, Sc·EYE·nce
To the point
- A remarkable nitrogenase: Scientists at the Max Planck Institute for Marine Microbiology and the Institut de Biologie Structurale in Grenoble have purified a nitrogen-fixing enzyme from a deep-sea microorganism that shows extreme heat-stability and is possibly of ancient origin.
- A new state: The molybdenum-containing enzyme has been captured in a “turnover” state, highlighting a universal mechanistic principle among nitrogenases.
- A promising outlook: A better understanding of the nitrogen-fixation reaction holds great potential to tackle the climate crisis and environmental problems.
Breaking one of nature’s toughest chemical bonds
Microorganisms can do remarkable things – for example nitrogen fixation, the conversion of nitrogen gas (N₂) into a form that organisms can use. Although nitrogen gas makes up around 78 per cent of Earth’s atmosphere, plants and animals can not directly use it. The two nitrogen atoms are held together by superglue: a chemical triple bond. But some microorganisms have a superpower: They can fix the nitrogen by breaking these bonds and converting N₂ into ammonia, which can then be used to build biological molecules. One such superhero is the deep-sea archaeon Methanocaldococcus infernus. This microorganism lives in marine volcanic areas, where temperatures of vent fluids can exceed the boiling point of water.
That sparked curiosity of the scientists from Tristan Wagner’s laboratory at the Max Planck Institute for Marine Microbiology in Bremen. They “tamed” this microorganism, forcing it to fix N2 even at temperatures above 90 °C. “How do they do it, in such heat? And how can the enzyme splitting the N2 triple bond work under these conditions?” Wagner asked himself.
The enzyme that provides the superpower of fixing N2 is called nitrogenase, an enzyme which contains the most complicated metallocofactor known in biology. Metallocofactors are metallic helper molecules, bound to an enzyme and essential for its function. The best-studied and most performant nitrogenases have a metallocofactor containing molybdenum, but they also exist in vanadium- and iron-only forms. How these different forms are related to one another and exactly how their metal centres enable them to break the N₂ triple bond remains an active area of research.
A remarkable nitrogenase built for extreme conditions
“The nitrogenase found in Methanocaldococcus infernus is remarkable because it seems to share traits of the molybdenum, vanadium and iron forms. This type of nitrogenase could be similar to a common nitrogenase-ancestor, the ancient system all of them evolved from. Thus, it could deliver common principles in the nitrogenase reaction,” says Wagner.
The researchers were able to isolate the nitrogenase directly from the microbe. This enzyme proved exceptionally stable at high temperatures, as the protein starts to fall apart only at 90 °C, and some of it even survived at 98 °C.
“This proves that this enzyme is designed to function under conditions in which most proteins would rapidly decay, like egg white cooked in hot water”, says first author Nevena Maslać from the Max Planck Institute for Marine Microbiology. “It is not active at room temperature. Rather, we show that it only produces ammonia at high temperatures. Its extreme stability allowed us to study states of the nitrogenase that are usually difficult to capture.”
State-of-the-art methods for a molecular dissection
First author Nevena Maslać measuring the nitrogenase activity in a glovebox to avoid oxygen damage to the sensitive metallocofactor.
© Tristan Wagner / Max Planck Institute for Marine Microbiology
Getting a detailed picture of this nitrogenase did not require the researchers to travel to the deep sea, but it did require a tour de force combining microbial physiology, native enzyme purification, biochemistry and structural biology-all under strictly oxygen-free conditions to avoid irreversible damage to its metallocofactors.
For this, the researchers obtained the enzyme in a crystal form and studied it at the Institut de Biologie Structurale in Grenoble, France. Here, they used the on-site synchrotron-a circular particle accelerator producing powerful X-rays. First, the scientists unveiled the molecular structure of the nitrogenase. They achieved a near-atomic-resolution view of the simplest nitrogenase known to date. It combines structural features of all three known nitrogenase families-molybdenum-, vanadium- and iron-only nitrogenases. This supports the idea that ancestral nitrogenases may have been more similar to this archaeal enzyme than to their bacterial counterparts. Next, the researchers wanted to ensure that the nitrogenase contained the molybdenum metallocofactor. “Our search for the molybdenum was technically extremely challenging and required the experts at the synchrotron to push their instrument to its absolute limits”, says Wagner.
In the end, the measurement not only delivered the typical molybdenum signal but held another surprise: “We were stunned to look at a so far unobserved state in a molybdenum-containing nitrogenase!” Until now, the so-called “turnover” state, potentially showing an intermediate step in the reaction, has been captured solely in the vanadium and iron-only forms. Observing this state also in a molybdenum enzyme suggests that nitrogenase universally follows the same pattern to break down N2.
From deep-sea chemistry to future biotechnology
A better understanding of nitrogen fixation is important for much more than just the deep sea. Nitrogen-fixing microorganisms such as M. infernus not only make nitrogen available in the form of ammonia, but they are also major players in Earth’s carbon cycle by generating half of the methane we find in the atmosphere. In the future, such organisms could potentially be explored as biological platforms for converting gases into useful products, including methane and ammonia, using green hydrogen as an energy source.
“And what if crops could one day obtain nitrogen directly from atmospheric N₂?” Wagner speculates. That could make agriculture less dependent on industrial fertilisers. Today, fertiliser production through the Haber-Bosch process requires substantial energy and is associated with greenhouse gas emissions, while excessive fertiliser use contributes to eutrophication and other environmental problems. “For now, the study provides something more fundamental: an updated molecular view of one of biology’s most remarkable chemical reactions.”
