A New Type of Sulfite Reductase, a Novel Coenzyme F420-dependent Enzyme, from the MethanarchaeonMethanocaldococcus jannaschii
Eric F. Johnson, Biswarup Mukhopadhyay
2005
Journal of Biological Chemistry
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
more »
... y of 0.57 mol sulfite reduced min ؊1 mg ؊1 protein. The cellular level of Fsr protein is comparable to that of methyl-coenzyme M reductase, an enzyme essential for methanogenesis and a possible target for sulfite. Purified Fsr reduces sulfite to sulfide using reduced F 420 (H 2 F 420 ) as the electron source (K m : sulfite, 12 M; H 2 F 420 , 21 M). Therefore, Fsr provides M. jannaschii an anabolic ability and protection from sulfite toxicity. The N-terminal half of the 70-kDa Fsr polypeptide represents a H 2 F 420 dehydrogenase and the C-terminal half a dissimilatory-type siroheme sulfite reductase, and Fsr catalyzes the corresponding partial reactions. Previously described sulfite reductases use nicotinamides and cytochromes as electron carriers. Therefore, this is the first report of a coenzyme F 420 -dependent sulfite reductase. Fsr homologs were found only in Methanopyrus kandleri and Methanothermobacter thermautotrophicus, two strictly hydrogenotrophic thermophilic methanogens. fsr is the likely ancestor of H 2 F 420 dehydrogenases, which serve as electron input units for membranebased energy transduction systems of certain late evolving archaea, and dissimilatory sulfite reductases of bacteria and archaea. fsr could also have arisen from lateral gene transfer and gene fusion events. 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 METHODS Growth 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 Hewlett-Packard 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 to Bradford (15). Gel filtration chromatography was also conducted as described previously (16) but with the following modifications. The mobile phase was made anaerobic by purging it with helium and was maintained such under a helium blanket. To the anaerobic column with a constant flow of mobile phase, an anaerobic Fsr sample was applied via autoinjection from a sealed vial. These precautions ensured a separation under anaerobic condition. Enzyme Assays-F 420 -dependent sulfite reductase (Fsr) activity was assayed spectrophotometrically under strictly anaerobic conditions. It 2 F 420 :phenazine oxidoreductase; FqoF and FpoF, H 2 F 420 dehydrogenase subunit of Fqo and Fpo, respectively; MV ϩ , methylviologen; MV 0 , reduced methylviologen; MES, 2-(N-morpholino)ethanesulfonic acid; ORF, open reading frame. FIGURE 4. UV-visible spectrum of M. jannaschii Fsr. The full spectrum (inset) and an expanded version (350 -750 nm) are shown. A 300-l anaerobic solution containing 38 g of homogenous enzyme (as isolated), 25 mM potassium phosphate buffer, pH 7, and 440 mM NaCl was analyzed.
doi:10.1074/jbc.m503492200
pmid:16048999
fatcat:gdhjsl4o6fhgpnrkuz22zbikjm