Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus : Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase

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Abstract
The hyperthermophilic archaeon Pyrococcus furiosus uses carbohydrates as a carbon source and produces acetate, CO2, and H2 as end products. When S0 is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S0 reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (Mr, 100,000) and is encoded by PF1186. This designation was previously assigned to the gene encoding an enzyme that reduces coenzyme A disulfide, which is a side reaction of NSR. Whole-genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to sevenfold within 10 min of S0 addition. This primary response to S0 also involves the up-regulation (>16-fold) of a 13-gene cluster encoding a membrane-bound oxidoreductase (MBX). The cluster encoding MBX is proposed to replace the homologous 14-gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S0 addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S0. A secondary response to S0 is observed 30 min after S0 addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins termed SipA and SipB. This novel S0-reducing system involving NSR and MBX has been found so far only in the heterotrophic Thermococcales and is in contrast to the cytochrome- and quinone-based S0-reducing system in autotrophic archaea and bacteria.