A New Type of Sulfite Reductase, a Novel Coenzyme F420-dependent Enzyme, from the Methanarchaeon Methanocaldococcus jannaschii

Abstract

Methanocaldococcus jannaschii is a hypertheromphilic, strictly hydrogenotrophic, methanogenic archaeon of ancient lineage isolated from a deep-sea hydrothermal vent. It requires sulfide for growth. Sulfite is inhibitory to the methanogens. Yet, we observed that M. jannaschii grows and produces methane with sulfite as the sole sulfur source. We found that in this organism sulfite induces a novel, highly active, coenzyme F 420 -dependent sulfite reductase (Fsr) with a cell extract specific activity of 0. Methanogenesis by the methanogenic archaea is inhibited by sulfite (1). This oxyanion is a strong nucleophile and is known to be toxic to cells of all types due to its reactivity toward proteins and sulfhydryl groups (2). Methanogens perhaps have an additional reason for sulfite sensitivity. In vitro sulfite reacts with and inactivates purified methylcoenzyme M reductase (3, 4), an essential enzyme for methanogenesis (5). Yet two methanogens, Methanothermococcus thermolithotrophicus and Methanothermobacter thermautotrophicus, have been reported to tolerate and even use sulfite as a sole sulfur source (6, 7). Also, as shown in this report, Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic methanogenic archaeon isolated from a deep-sea hydrothermal vent (8), grows with sulfite. However, the genomes of M. thermautotrophicus and M. jannaschii do not carry a clear homolog of a sulfite reductase (9, 10); the genome sequence of M. thermolithotrophicus is yet to be determined. With the goal of identifying the sulfite detoxification and assimilation mechanisms of these organisms, we have studied sulfite metabolism of M. jannaschii. As shown below, this work has led to the discovery of a new type of sulfite reductase. MATERIALS AND METHODSGrowth of M. jannaschii-The organism was grown on H 2 plus CO 2 (80:20, v/v; 3 ϫ 10 5 Pa) in a mineral salts medium in sealed 500-ml serum bottles, as described previously (8, 11), with either sodium sulfite (1 mM) or sulfide (1 mM) as the sole sulfur source and medium reductant. The cells were harvested by centrifugation at 9600 ϫ g and 4°C anaerobically under an N 2 plus CO 2 atmosphere (80:20, v/v).Methane Measurement-Methane was assayed by use of a HewlettPackard model HP5890 gas chromatograph (Agilent Technologies, Inc., Palo Alto, CA) fitted with a flame ionization detector and a 0.5-mm ϫ 30-m HP-PLOT (aluminum oxide, 15 m) column. The column, detector, and injector were maintained at 100, 150, and 150°C, respectively. The carrier gas (N 2 ) flow rate was 1 ml/min. A methane standard (Matheson Tri-Gas, Montgomeryville, PA) was used for calibration.Protein Analysis-SDS-PAGE was performed according to Laemmli (12), and the same method but employing buffers without SDS and omitting the sample denaturation and reduction step was used for nondenaturing gel electrophoresis. The identity of a polypeptide in a gel band was determined by in-gel trypsin digestion, MALDI-TOF 2 mass spectrometry, and data base searches as described previously (13,14). Protein was assayed according...

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

“…Although both F 420 and NAD ϩ (P) catalyze hydride transfer and facilitate chemically similar reactions, none of the previously described F 420 -dependent enzymes show meaningful similarities to functionally analogous or other type NAD ϩ (P)-dependent enzymes (27,(43)(44)(45). In contrast, at the primary structural level DFTR exhibits 33% identities and 50% similarities to E. coli NTR, and they share several conserved sequence features (Fig.…”

“…NADH, NADPH, dithionite, coenzyme M, and ferredoxin at final concentrations of 0.1, 0.1, 1, and 6 mM and 5 M, respectively, were examined as potential electron donors. The assays were performed in round glass cuvettes sealed with cutoff butyl rubber stoppers with N 2 (1.3 ϫ 10 5 Pa) in the headspace (27), except with ferredoxin as electron carrier H 2 (1.3 ϫ 10 5 Pa) was used. Ferredoxin was from C. pasteurianum, and it was reduced in situ with bidirectional hydrogenase from the same organism, and the respective final concentrations in the assay were 5 M and 50 nM, respectively (76).…”

“…The initial test for the Trx reductase activity in Mj-TrxR was via the commonly used insulin disulfide reduction assay (26), and it was performed under anaerobic conditions (20,27). It utilized the following electron flow: electron donor 3 MjTrxR 3 Mj-Trx 3 insulin.…”

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…The nitrogen atom in position 5 is required for an unpaired electron to efficiently delocalize through the isoalloxazine ring; indeed, radicals of pyrazine groups (of flavins) are much lower energy than those of pyridine groups (of 5-deazaflavins) (7,43). Reflecting this reactivity, F 420 -dependent enzymes mediate diverse hydride transfer reactions that transform CϭC and C §C bonds (28,29,47,48), alcohol and imine groups (49,50), and certain inorganic compounds (51,52). Furthermore, due to the substitution, 5-deazaflavins do not readily undergo single-electron reactions.…”

Characterization of an NADH oxidase of the flavin-dependent disulfide reductase family from Methanocaldococcus jannaschii

“…Geological data (Corliss et al, 1979;Jannasch & Mottl, 1985;Jones et al, 1983) suggest that M. jannaschii could experience minor oxygen exposure within the submarine hydrothermal vents (Johnson & Mukhopadhyay, 2005) or during transport through cold oxygenic water (Huber et al, 1990;Stetter et al, 1993). Also, during low-temperature storage, hyperthermophilic anaerobes are relatively resistant to O 2 exposure (Huber et al, 1990;Jannasch et al, 1992 (1.9 mM) and activation energy (46 kJ mol 21 ) for rMjNox.…”