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Microbe Performs Rocket Science, Vital for Life on Earth

David Coppedge
Image: Cassini Saturn orbiter via JPL/NASA.

Anammox. It’s a good term to learn. Wikipedia stresses its importance:

Anammox, an abbreviation for ANaerobic AMMonium OXidation, is a globally important microbial process of the nitrogen cycle. The bacteria mediating this process were identified in 1999, and at the time were a great surprise for the scientific community. It takes place in many natural environments… [Emphasis added.]

A team of European scientists found something very interesting about the bacteria. Publishing in Nature, the researchers tell how they ascertained the structure of a molecular machine that performs chemical wizardry using rocket science.

Anaerobic ammonium oxidation (anammox) has a major role in the Earth’s nitrogen cycle and is used in energy-efficient wastewater treatment. This bacterial processcombines nitrite and ammonium to form dinitrogen (N2) gas, and has been estimated to synthesize up to 50% of the dinitrogen gas emitted into our atmosphere from the oceans. Strikingly, the anammox process relies on the highly unusual, extremely reactive intermediate hydrazine, a compound also used as a rocket fuel because of its high reducing power. So far, the enzymatic mechanism by which hydrazine is synthesized is unknown. Here we report the 2.7 Å resolution crystal structure, as well as biophysical and spectroscopic studies, of a hydrazine synthase multiprotein complex isolated from the anammox organism Kuenenia stuttgartiensis. The structure shows an elongated dimer of heterotrimers, each of which has two unique c-type haem-containing active sites, as well as an interaction point for a redox partner. Furthermore, a system of tunnels connectsthese active sites. The crystal structure implies a two-step mechanism for hydrazine synthesis: a three-electron reduction of nitric oxide to hydroxylamine at the active site of the γ-subunit and its subsequent condensation with ammonia, yielding hydrazine in the active centre of the α-subunit. Our results provide the first, to our knowledge, detailed structural insight into the mechanism of biological hydrazine synthesis, which is of major significance for our understanding of the conversion of nitrogenous compounds in nature.

The Job of Anammox Bacteria

Dinitrogen gas (N2) is a tough nut to crack. The atoms pair up with a triple bond, very difficult for humans to break without a lot of heat and pressure. Fortunately, this makes it very inert for the atmosphere, but life needs to get at it to make amino acids, muscles, organs, and more. Nitrogenase enzymes in some microbes, such as soil bacteria, are able break apart the atoms at ambient temperatures (a secret agricultural chemists would love to learn). They then “fix” nitrogen into compounds such as ammonia (NH3) that can be utilized by plants and the animals that eat them. To have a nitrogen cycle, though, something has to return the N2 gas to the atmosphere. That’s the job of anammox bacteria.

Most nitrogen on earth occurs as gaseous N2 (nitrogen oxidation number 0). To make nitrogen available for biochemical reactions, the inert N2 has to be converted to ammonia (oxidation number -III), which can then be assimilated to produce organic nitrogen compounds, or be oxidized to nitrite (oxidation number +III) or nitrate (+V). The reduction of nitrite in turn results in the regeneration of N2, thus closing the biological nitrogen cycle.

Let’s take a look at the enzyme that does this, the “hydrazine synthase multiprotein complex.” Rocket fuel; imagine! No wonder the scientific community was surprised. The formula for hydrazine is N2H4. It’s commonly used to power thrusters on spacecraft, such as the Cassini Saturn orbiter and the New Horizons probe to Pluto. Obviously, the anammox bacteria must handle this highly reactive compound with great care. Here’s their overview of the reaction sequence. Notice how the bacterium gets some added benefit from its chemistry lab:

Our current understanding of the anammox reaction (equation (1)) is based on genomic, physiological and biochemical studies on the anammox bacterium K. stuttgartiensis. First, nitrite is reduced to nitric oxide (NO, equation (2)), which is then condensed with ammonium-derived ammonia (NH3) to yield hydrazine (N2H4, equation (3)). Hydrazine itself is a highly unusual metabolic intermediate, as it is extremely reactive and therefore toxic, and has a very low redox potential (E0’ = -750 mV). In the final step in the anammox process, it is oxidized to N2, yielding four electrons (equation (4)) that replenish those needed for nitrite reduction and hydrazine synthesis and are used to establish a proton-motive force across the membrane of the anammox organelle, the anammoxosome, driving ATP synthesis.

We’ve discussed ATP synthase before. It’s that rotary engine in all life that runs on proton motive force. Here, we see that some of the protons needed for ATP synthesis come from the hydrazine reaction machine. Cool!

What Does the Anammox Enzyme Look Like?

They say it has tunnels between the active sites. The “hydrazine synthase” module is “biochemically unique.” Don’t look for a common ancestor, in other words. It’s part of a “tightly coupled multicomponent system” they determined when they lysed a cell and watched its reactivity plummet. Sounds like what biochemist Michael Behe would call an irreducibly complex system.

The paper’s diagrams of hydrazine synthase (HZS) show multiple protein domains joined in a “crescent-shaped dimer of heterotrimers” labeled alpha, beta, and gamma, constituted in pairs. The machine also contains multiple haem units (like those in hemoglobin, but unique) and “one zinc ion, as well as several calcium ions.” It’s a good thing those atoms are available in Earth’s crust.

How Does It Work?

Part of the machine looks like a six-bladed propeller. Another part has seven blades. Everything is coordinated to carefully transfer electrons around. This means that charge distributions are highly controlled for redox (reduction-oxidation) reactions (i.e., those that receive or donate electrons). The choice of adverbs suggests that their eyes were lighting up at their first view of this amazing machine. Note how emotion seasons the jargon:

Intriguingly, our crystal structure revealed a tunnel connecting the heme αI and –γI sites (Fig. 3a). This tunnel branches off towards the surface of the protein approximately halfway between the haem sites, making them accessible to substrates from the solvent. Indeed, binding studies show that heme αI is accessible to xenon (Extended Data Fig. 4c). Interestingly, in-between the α- and γ-subunits, the tunnel is approached by a 15-amino-acid-long loop of the β-subunit (β245-260), placing the conserved βGlu253, which binds a magnesium ion, into the tunnel.

We would need to make another animation to show the machine in action, but here’s a brief description of how it works. The two active sites, connected by a tunnel, appear to work in sequence. HZS gets electrons from cytochrome c, a well-known enzyme. The electrons enter the machine through one of the haem units, where a specifically placed gamma unit adds protons. A “cluster of buried polar residues” transfers protons to the active center of the gamma subunit. A molecule named hydroxylamine (H3NO) diffuses into the active site, assisted by the beta subunit. It binds to another haem, which carefully positions it so that it is “bound in a tight, very hydrophobic pocket, so that there is little electrostatic shielding of the partial positive charge on the nitrogen.” Ammonia then comes in to do a “nucleophilic attack” on the nitrogen of the molecule, yielding hydrazine. The hydrazine is then in position to escape via the tunnel branch leading to the surface. Once they determined this sequence, a light went on:

Interestingly, the proposed scheme is analogous to the Raschig process used in industrial hydrazine synthesis. There, ammonia is oxidized to chloramine (NH2Cl, nitrogen oxidation number -I, like in hydroxylamine), which then undergoes comproportionation with another molecule of ammonia to yield hydrazine.

(But that, we all know, is done by intelligent design.)

So here’s something you can meditate on when you take in another breath. The nitrogen gas that comes into your lungs is a byproduct of an exquisitely designed, precision nanomachine that knows a lot about organic redox chemistry and safe handling of rocket fuel. This little machine, which also knows how to recycle and reuse all its parts in a sustainable “green” way, keeps the nitrogen in balance for the whole planet. Intriguing. Interesting. As Mr. Spock might say, fascinating.

This article was originally published in 2015.