Deep-sea microbe reveals how nitrogen fixation survives near-boiling heat

Deep beneath the ocean surface, in volcanic vent systems where fluids can push past the boiling point of water, lives a microorganism that performs one of the most chemically demanding feats in biology. Researchers at the Max Planck Institute for Marine Microbiology in Bremen, together with structural biologists at the Institut de Biologie Structurale in Grenoble, have now purified and visualised the enzyme that makes this possible: a nitrogenase of extraordinary heat stability, isolated from the deep-sea archaeon Methanocaldococcus infernus. The work, published in Nature Communications, offers the most detailed look yet at an enzyme that may resemble the ancient ancestor of all nitrogenases, and it captures a reaction state never before seen in a molybdenum-containing member of the family.

Nitrogen fixation is the conversion of atmospheric nitrogen gas, which makes up roughly 78 percent of the air we breathe, into ammonia that living cells can incorporate into amino acids, nucleotides and other essential molecules. The obstacle is the nitrogen-nitrogen triple bond, one of the strongest chemical bonds known in nature, which locks the two atoms together with an energy that neither plants nor animals can unlock. Only certain microorganisms, equipped with the enzyme nitrogenase, can break it under the mild conditions of ambient pressure and temperature that life tolerates. Industrial chemistry, by contrast, needs the Haber-Bosch process, with its high temperatures, high pressures and substantial energy consumption, to accomplish the same transformation at scale.

The nitrogenase enzyme carries at its heart the most complicated metallocofactor known in biology, a cluster of metal and sulfur atoms that orchestrates the reduction of N2 to ammonia. Nitrogenases come in three main varieties, distinguished by the metal at the centre of the cofactor: molybdenum, vanadium or iron alone. The molybdenum form is the best studied and generally the most efficient, yet how the different metal centres enable the breaking of the triple bond, and how the three enzyme families relate to one another evolutionarily, remain open questions in biochemistry.

Methanocaldococcus infernus offered an unusual opportunity to probe those questions. The archaeon thrives in marine volcanic areas, and the team led by Tristan Wagner managed to cultivate it in the laboratory under conditions that forced it to fix nitrogen at temperatures above 90 degrees Celsius. How, Wagner wondered, could an enzyme tasked with splitting the N2 triple bond remain functional in heat that denatures most proteins within seconds? The answer, it turns out, lies in a molecular architecture of remarkable robustness.

When the researchers isolated the nitrogenase directly from the microbe, they found that the protein does not begin to fall apart until around 90 degrees Celsius, and a fraction of it even survives at 98 degrees, just shy of water’s boiling point. Nevena Maslać, the study’s first author, compared the fate of ordinary proteins in such conditions to egg white cooked in hot water, noting that this enzyme is instead built for exactly those extremes. Intriguingly, the enzyme is not active at room temperature; it produces ammonia only when hot. That thermal rigidity proved to be a scientific gift, because it stabilised reaction states that normally collapse before they can be studied.

Obtaining a detailed picture of the enzyme demanded a tour de force spanning microbial physiology, native enzyme purification, biochemistry and structural biology, all conducted under strictly oxygen-free conditions. Nitrogenase metallocofactors are exquisitely sensitive to oxygen, which irreversibly damages them, so every step of the work had to exclude air. The team crystallised the enzyme and examined the crystals at the Institut de Biologie Structurale in Grenoble, using the on-site synchrotron, a circular particle accelerator that generates intense X-rays capable of resolving atomic-scale structure.

The resulting images delivered a near-atomic-resolution view of the simplest nitrogenase known to date, and one with a strikingly hybrid character. The archaeal enzyme combines structural features of all three nitrogenase families, the molybdenum, vanadium and iron-only forms. This mosaic architecture supports the idea that ancestral nitrogenases, the ancient systems from which the modern families evolved, may have resembled this archaeal enzyme more closely than their bacterial counterparts. In other words, studying M. infernus’s nitrogenase may be the closest researchers can get to examining a molecular fossil of the original nitrogen-fixing machinery.

Confirming that the enzyme truly carried a molybdenum cofactor pushed the synchrotron instrumentation to its absolute limits, according to Wagner. The measurement succeeded, detecting the characteristic molybdenum signal, but it also delivered an unexpected bonus. The team observed a so-called turnover state of the enzyme, a configuration believed to represent an intermediate step in the nitrogen reduction reaction, that had previously been captured only in vanadium and iron-only nitrogenases. Seeing this state in a molybdenum-containing enzyme for the first time stunned the researchers, and it carries a significant implication: nitrogenases of all three metal types may universally follow the same mechanistic pattern when they dismantle the N2 triple bond.

The significance of understanding nitrogen fixation extends well beyond deep-sea microbiology. Nitrogen-fixing microorganisms such as M. infernus do more than make ammonia; they are also major players in Earth’s carbon cycle, generating roughly half of the methane present in the atmosphere. Wagner and colleagues point to a future in which such heat-loving organisms, or the enzymes they carry, could serve as biological platforms for converting gases into useful products, including methane and ammonia, powered by green hydrogen as an energy source. Enzymes that operate at near-boiling temperatures could offer advantages of reaction speed and stability that their mesophilic cousins cannot match.

Agriculture stands to gain as well. Wagner speculates about a future in which crops might one day obtain nitrogen directly from atmospheric N2, freeing farming from its heavy dependence on industrial fertilisers. Today’s fertiliser production through the Haber-Bosch process consumes substantial energy and is associated with greenhouse gas emissions, while the overuse of fertilisers drives eutrophication of waterways and other environmental damage. For now, however, the study delivers something more fundamental: an updated molecular view of one of biology’s most remarkable chemical reactions, seen through an enzyme that keeps working at temperatures where almost everything else in the cell falls apart.

Subject of Research: Structure and mechanism of a hyperthermophilic archaeal nitrogenase involved in biological nitrogen fixation

Article Title: Molecular insight into biological nitrogen fixation close to the boiling point

Article References: Molecular insight into biological nitrogen fixation close to the boiling point. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: nitrogenase, nitrogen fixation, Methanocaldococcus infernus, deep-sea hydrothermal vents, metallocofactor, molybdenum, hyperthermophile, structural biology, synchrotron, ammonia, Haber-Bosch, Nature Communications