The coenzyme F420-dependent sulfite reductase (Fsr group I) protects hydrogenotrophic methanogens, one of the main contributors in worldwide methane emission, from toxic sulfite. Fsr is a single peptide composed of a F420H2-oxidase and a novel class of sulfite reductase. Both catalytic domains have been proposed to be the ancestors of modern F420-oxido/reductases and dissimilatory/assimilatory sulfite reductases. Here, we describe the X-ray crystal structures of Fsr natively isolated from Methanocaldococcus jannaschii (MjFsr) and Methanothermococcus thermolithotrophicus (MtFsr), respectively refined to 2.30 Å and 1.55 Å resolution. In both organisms, Fsr oligomerizes as a 280-kDa homotetramer, where each siroheme‒[4Fe‒4S] is catalytically active, in contrast to dissimilatory homologues. The siroheme‒[4Fe‒4S], embedded in the sulfite reductase domain, is electronically connected to the flavin in the F420H2-oxidase domain by five [4Fe‒4S]-clusters. EPR spectroscopy determined the redox potentials of these [4Fe‒4S]2+/1+ clusters (-435 to -275 mV), through which electrons flow from FAD to the siroheme‒[4Fe‒4S]2+/1+ (siroheme, -114 mV; [4Fe‒4S] -445 mV). The electron relay is mainly organized by two inserted ferredoxin modules, which stabilize the higher degree of oligomerization. While the F420H2-oxidase part is similar to the β-subunit of F420-reducing hydrogenases, the sulfite reductase domain is structurally analogous to dissimilatory sulfite reductases, whereas its siroheme‒[4Fe‒4S] cofactor is bound in the same way as in assimilatory ones. Accordingly, the reaction of MtFsr is unidirectional, reducing sulfite or nitrite with F420H2. Our results provide the first structural insights into this unique fusion, a snapshot of a primitive sulfite reductase that turns a poison into an elementary block of Life.
Domestication of CO 2-fixation became a worldwide priority enhanced by the will to convert this greenhouse gas into fuels and valuable chemicals. Because of its high stability, CO 2-activation/fixation represents a true challenge for chemists. Autotrophic microbial communities, however, perform these reactions under standard temperature and pressure. Recent discoveries shine light on autotrophic acetogenic bacteria and hydrogenotrophic methanogens, as these anaerobes use a particularly efficient CO 2capture system to fulfill their carbon and energy needs. While other autotrophs assimilate CO 2 via carboxylation followed by a reduction, acetogens and methanogens do the opposite. They first generate formate and CO by CO 2-reduction, which are subsequently fixed to funnel the carbon toward their central metabolism. Yet their CO 2-reduction pathways, with acetate or methane as end-products, constrain them to thrive at the "thermodynamic limits of Life". Despite this energy restriction acetogens and methanogens are growing at unexpected fast rates. To overcome the thermodynamic barrier of CO 2-reduction they apply different ingenious chemical tricks such as the use of flavin-based electron-bifurcation or coupled reactions. This mini-review summarizes the current knowledge gathered on the CO 2-fixation strategies among acetogens. While extensive biochemical characterization of the acetogenic formate-generating machineries has been done, there is no structural data available. Based on their shared mechanistic similarities, we apply the structural information obtained from hydrogenotrophic methanogens to highlight common features, as well as the specific differences of their CO 2-fixation systems. We discuss the consequences of their CO 2reduction strategies on the evolution of Life, their wide distribution and their impact in biotechnological applications.
Methanogenic archaea are main actors in the carbon cycle but are sensitive to reactive sulfite. Some methanogens use a sulfite detoxification system that combines an F420H2-oxidase with a sulfite reductase, both of which are proposed precursors of modern enzymes. Here, we present snapshots of this coupled system, named coenzyme F420-dependent sulfite reductase (Group I Fsr), obtained from two marine methanogens. Fsr organizes as a homotetramer, harboring an intertwined six-[4Fe–4S] cluster relay characterized by spectroscopy. The wire, spanning 5.4 nm, electronically connects the flavin to the siroheme center. Despite a structural architecture similar to dissimilatory sulfite reductases, Fsr shows a siroheme coordination and a reaction mechanism identical to assimilatory sulfite reductases. Accordingly, the reaction of Fsr is unidirectional, reducing sulfite or nitrite with F420H2. Our results provide structural insights into this unique fusion, in which a primitive sulfite reductase turns a poison into an elementary block of life.
Methanothermococcus thermolithotrophicus is the only known methanogen that grows on sulfate as its sole sulfur source, uniquely uniting methanogenesis and sulfate reduction. Here we use physiological, biochemical and structural analyses to provide a snapshot of the complete sulfate reduction pathway of this methanogenic archaeon. We find that later steps in this pathway are catalysed by atypical enzymes. PAPS (3′-phosphoadenosine 5′-phosphosulfate) released by APS kinase is converted into sulfite and 3′-phosphoadenosine 5′-phosphate (PAP) by a PAPS reductase that is similar to the APS reductases of dissimilatory sulfate reduction. A non-canonical PAP phosphatase then hydrolyses PAP. Finally, the F420-dependent sulfite reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that the sulfate reduction pathway is present in several methanogens, the sulfate assimilation pathway in M. thermolithotrophicus is distinct. We propose that this pathway was ‘mix-and-matched’ through the acquisition of assimilatory and dissimilatory enzymes from other microorganisms and then repurposed to fill a unique metabolic role.
By growing on sulfate as the sole source of sulfur, Methanothermococcus thermolithotrophicus breaks a dogma: the ancient metabolic pathways methanogenesis and sulfate-reduction should not co-occur in one organism due to toxic intermediates and energetic barriers. Using a complementary approach of physiological, biochemical, and structural studies, we provide a snapshot of the complete sulfate-reduction pathway of the methanogenic archaeon. While the first two reactions proceed via an ATP-sulfurylase and APS-kinase, common to other organisms, the further steps are catalysed by non-canonical enzymes. 3'-phosphoadenosine-5'-phosphosulfate (PAPS) released by the APS-kinase is converted into sulfite and 3'-phosphoadenosine-5'-phosphate (PAP) by a new class of PAPS-reductase that shares high similarity with the APS-reductases involved in dissimilatory sulfate-reduction. The generated PAP is efficiently hydrolysed by a PAP-phosphatase that was likely derived from an RNA exonuclease. Finally, the F420-dependent sulfite-reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that genes of the sulfate-reduction pathway are present in various methanogens, M. thermolithotrophicus uses a distinct way to assimilate sulfate. We propose that its entire sulfate-assimilation pathway was derived from a mix-and-match strategy in which the methanogen acquired assimilatory and dissimilatory enzymes from other microorganisms and shaped them to fit its physiological needs.
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