The structure of the F₄₂₀-dependent sulfite-detoxifying enzyme from Methanococcales reveals a prototypical sulfite-reductase with assimilatory traits

Abstract

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.

Transcriptomics sheds light on N2-fixation strategies employed by a thermophilic member of theMethanococcales

“…Series of incubations with different NH4Cl concentrations, ranging from 0.1 to 16.8 mM, showed that M. thermolithotrophicus required a minimum of 10 mM NH4Cl for best growth (Fig. 1A) in our optimized medium (see Materials and Methods for composition) (39,40).…”

“…Series of incubations with different NH 4 Cl concentrations, ranging from 0.1 to 16.8 mM, showed that M. thermolithotrophicus required a minimum of 10 mM NH 4 Cl for best growth (Fig. 1A) in our optimized medium (see Materials and Methods for composition) (39, 40). Higher NH 4 Cl concentrations of 25-200 mM NH 4 Cl did not result in higher cell yields, and a 500 mM excess of NH 4 Cl led to a decrease (Fig.…”

“…Nitrogen acquisition by M. thermolithotrophicus. We selected M. thermolithotrophicus DSM 2095 due to its remarkable chemolithoautotrophic capabilities and fast growth at 65 °C in mineral medium (38,39). Series of incubations with different NH4Cl concentrations, ranging from 0.1 to 16.8 mM, showed that M. thermolithotrophicus required a minimum of 10 mM NH4Cl for best growth (Fig.…”

“…In addition to batch diazotrophic cultures, M. thermolithotrophicus was successfully grown in a 10 L fermenter continuously supplied with H 2 /CO 2 and N 2 . To maintain the sulfur source in the medium, we replaced Na 2 S used in batch cultures (which would be flushed out as H 2 S) with Na 2 SO 3 or Na 2 SO 4 (see Materials and Methods)(39). Diazotrophic growth was not affected by HSO 3 - , a known inhibitor of methanogenesis (Fig.…”

“…If nitrate would be imported and reduced in the cell, the oxidant nitrite would be generated and could damage the highly oxidation-sensitive methanogenic machinery (61). A gene coding for a F420-sulfite reductase isoform (also belonging to Fsr group 1 but different to the one naturally expressed under sulfite condition described in (39)), which co-occurs with narK and narB , would be a plausible candidate for nitrite detoxification, since Fsr has been recognized to catalyze nitrite reduction (39). M. thermolithotrophicus has been reported to be able to grow on nitrate as the only source of nitrogen (62), which might involve these genes.…”