Hydrogenotrophic methanogens are strictly anaerobic microorganisms that thrive at the thermodynamic limit of Life by oxidizing H₂ and reducing CO₂ into methane (CH₄). They are one of the main contributors to global CH₄ emissions, the second most impactful greenhouse gas, but also a valuable biofuel to heat our houses. Therefore, understanding these ancient archaea (more than 3.4 billion years old) and their metabolism is of particular interest in times of climate change. Methanogens live worldwide in environmental niches without oxygen, such as the deeper layers of marine sediments, hydrothermal vents or as part of the gut microbiome, even in human ones. Those environments are rich in sulphides (S^2-), which methanogens assimilate as a source of sulphur for their growth and replication. However, sulphite (SO₃^2-), an oxidized form of sulphur, is a poison for them as it inhibits the enzyme generating CH₄ and therefore collapses their central energy metabolism.¹

The F₄₂₀-dependent sulphite reductase (Fsr)

Despite its toxicity, certain marine methanogens from the order Methanococcales have been shown to not only tolerate SO₃^2- but even grow on it as a sole source of sulphur.^2,3 In other words, they turn poison into food. In their natural environments, they are occasionally exposed to SO₃^2- when oxygenated seawater enters the sediment or vent plumes. Partial oxidation of S^2- results in the formation of SO₃^2-, which the methanogens need to detoxify immediately.⁴ This is possible due to the F₄₂₀-dependent sulphite reductase Group I or, in short, Fsr – a single enzyme that harbours two catalytic units. One reason why Fsr is very efficient at detoxifying SO₃^2- is because it uses the reduced F₄₂₀ cofactor as a source for electrons.

F₄₂₀ is the molecule that gives a bright blue fluorescence to methanogens under fluorescent microscopy (see figure) because of its natural abundance.⁵ Once F₄₂₀ is oxidized, it can quickly be recycled by the reducing power of H₂ oxidation.⁶ Furthermore, the overall reaction is highly exergonic, which promotes a fast rate of SO₃^2- reduction:

HSO₃^- + 3F₄₂₀H₂ → HS^- + 3H₂O + 3F₄₂₀    ∆G⁰′ = -135 kJ mol^-1

The crystal structures of Fsr from Methanothermococcus thermolithotrophicus and Methanocaldococcus jannaschii elucidated the detailed reaction mechanism of the enzyme. One-half of Fsr, the F₄₂₀H₂-oxidase, transfers the electrons from the F₄₂₀H₂ to its flavin (FAD). From here, the electrons are passed via a six-[4Fe–4S] cluster relay to its other half, a sulphite reductase harbouring a siroheme. The SO₃^2- anion is attracted to the active site of the sulphite reductase by a positively charged cavity, followed by a tight interaction between the SO₃^2- and the siroheme, which carries out the six-electron reduction reaction to generate S^2-. The chain of [4Fe-4S] clusters is essential to facilitate the electron flow, and it allows a tight electronic coupling between F₄₂₀H₂ oxidation and SO₃^2- reduction, as attested by electron paramagnetic resonance experiments.

A primitive sulphite reductase

Since Fsr has been isolated from (hyper)thermophilic Methanococcales, it has been shown to have a high-temperature tolerance (>85 °C)² and it enables the methanogens to survive even high SO₃^2- concentrations (up to 40 mM for Methanothermococcus thermolithotrophicus). Besides being of interest to chemists because of its efficiency and robustness, Fsr's sulphite reductase domain might provide new evolutionary insights. Sulphite reductases are considered to be ancient enzymes that have shaped our Earth for at least 3.47 billion years.⁷ While dissimilatory sulphite reductases (dSirs, here, referred to as DsrAB) are used by microorganisms to conserve energy, assimilatory sulphite reductases (aSirs) are needed to generate S^2- for biomass production (i.e., for amino acids like cysteine, methionine, or cofactors, etc.). Based on structural and phylogenetic studies, it has been proposed that aSirs and dSirs originated from a common ancestor. This progenitor was most likely much simpler than the modern sulphite reductases, which arose through the duplication of the sulphite reductase domain. The crystal structures of Fsr revealed that it contains an interesting mix of features from both assimilatory and dissimilatory sulphite reductases. Its overall architecture resembles a dSir, but the molecular mechanism behind SO₃^2- reduction to S^2- is identical to assimilatory ones. Furthermore, Fsr does not contain certain extensions, as reported for aSirs and dSirs, and is the most compact and “primitive” sulphite reductase that has been structurally characterised so far. Because of its “primitive organisation”, Fsr would provide a plausible picture of an ancient sulphite reductase prototype. However, its evolution is still debatable, as the origin of Fsr remains uncertain.

Besides Fsr Group I, the closely related Fsr Group II, which is present in some genomes of anaerobic methane oxidisers (ANMEs) and Methanosarcinales, seems to serve a different function. Fsr Group II, recombinantly produced in a methanogen, did not reduce SO₃^2- but performed nitrite reduction, albeit at a low rate.⁸

Cultivating hydrogenotrophic methanogens can be challenging as they require a strictly anaerobic atmosphere, a supply of H₂ and CO₂, and S^2- as a sulphur source. However, they are excellent gas bio-converters used in biotechnology. Using SO₃^2- in bioreactors will circumvent the need to use hydrogen sulphide as a sulphur substrate, which is corrosive, explosive and highly toxic for humans. This opens opportunities for safer biotechnological applications and simplifies the studies of these important microorganisms. An optimal solution would be to find a methanogen that reduces sulphate, which is cheap, abundant, and a completely safe sulphur source. One methanogen has already been reported to grow on SO₄^2- – M. thermolithotrophicus.⁹ Most likely, Fsr orchestrates the last reaction of this sulphate reduction pathway because one of its intermediates would be sulphite. Furthermore, we showed that Fsr from M. thermolithotrophicus reduces nitrite at the same rate as sulphite. Nitrite is another poison that could damage the CH₄-generating machinery. However, the physiological contribution of Fsr in this process needs further investigation.

References:

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Top image provided by the author.