The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91؇C by the fermentation of peptides and reduces elemental sulfur (S 0 ) to H 2 S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S 0 in the medium, and no growth was observed in the absence of S 0 . The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S 0 concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S 0 limitation. This was accompanied by increased amounts of H 2 and alcohol (ethanol and butanol) from cultures grown with limiting S 0 . Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M r of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under S 0 limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.Hyperthermophiles are a recently discovered group of microorganisms that have the remarkable property of growing at temperatures of 90ЊC and above (1, 2, 64, 65). They have been isolated from a variety of geothermally heated environments, and almost all are classified as Archaea (formerly Archaeobacteria [75]). The majority are strict anaerobes, and most are obligately dependent upon the reduction of elemental sulfur (S 0 ) to H 2 S for optimal growth. Various organic compounds and molecular H 2 serve as electron donors for the apparent respiration of S 0 . The mechanism of S 0 reduction in one autotrophic hyperthermophile, Pyrodictium brockii, an obligate S 0 -reducing species which grows optimally at 105ЊC, has been investigated. This organism was shown to have a primitive membrane-bound electron transport chain for coupling H 2 oxidation and H 2 S production (52), although the S 0 -reducing entity was not purified.On the other hand, some of the heterotrophic hyperthermophiles are able to grow well in the absence of S 0 . These have fermentative-type metabolisms and produce H 2 during growth, although they also produce H 2 S if S 0 is added to the growth medium. S 0 reduction was thought to be...