The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S 0 ). Growth rates were highest on media containing peptides and S 0 , with or without maltose. Growth did not occur on the peptide medium without S 0 . S 0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S 0 with or without maltose were the same, suggesting that S 0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S 0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S 0 -reducing enzyme in this organism and the mechanism of the S 0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.Hyperthermophiles are microorganisms that grow optimally at 80°C and above (46,47). Virtually all of them are strict anaerobes, and most are heterotrophs. All of the heterotrophs utilize peptides as a carbon source, and most use elemental sulfur (S 0 ) as a terminal electron acceptor leading to H 2 S production. The most studied of the S 0 -reducing, heterotrophic hyperthermophiles are species of Pyrococcus. Most of these organisms only utilize peptide-related substrates as a carbon source and show no significant growth in the absence of S 0 (9,12,19,36). Notable exceptions are Pyrococcus furiosus, P. woesei, and P. glycovorans, which are capable of metabolizing poly-and oligosaccharides, as well as peptides (2, 4, 10). P. furiosus and P. woesei can also grow to high cell densities in the absence of S 0 . The pathways of peptide and carbohydrate metabolism have been well studied in P. furiosus (1, 7). Glycolysis appears to occur via a modified Embden-Meyerhof pathway (Fig. 1) (22,35). This pathway is unusual in that the hexose kinase and phosphofructokinase steps are dependent on ADP rather than ATP, and a novel tungsten-containing enzyme termed glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR) replaces the expected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase. Amino acid catabolism in P. furiosus is thought to involve four distinct 2-keto acid oxidoreductases that convert transaminated amino acids into their corresponding coenzyme A (CoA) derivatives (Fig. 2) (3,15,31,32). These CoA derivatives, together with acetylCoA produced from glycolysis via pyruvate...

A gene (yacK) encoding a putative multicopper oxidase (MCO) was cloned from Escherichia coli, and the expressed enzyme was demonstrated to exhibit phenoloxidase and ferroxidase activities. The purified protein contained six copper atoms per polypeptide chain and displayed optical and electron paramagnetic resonance (EPR) spectra consistent with the presence of type 1, type 2, and type 3 copper centers. The strong optical A 610 (⌭ 610 ‫؍‬ 10,890 M ؊1 cm ؊1) and copper stoichiometry were taken as evidence that, similar to ceruloplasmin, the enzyme likely contains multiple type 1 copper centers. The addition of copper led to immediate and reversible changes in the optical and EPR spectra of the protein, as well as decreased thermal stability of the enzyme. Copper addition also stimulated both the phenoloxidase and ferroxidase activities of the enzyme, but the other metals tested had no effect. In the presence of added copper, the enzyme displayed significant activity against two of the phenolate siderophores utilized by E. coli for iron uptake, 2,3-dihydroxybenzoate and enterobactin, as well as 3-hydroxyanthranilate, an iron siderophore utilized by Saccharomyces cerevisiae. Oxidation of enterobactin produced a colored precipitate suggestive of the polymerization reactions that characterize microbial melanization processes. As oxidation should render the phenolate siderophores incapable of binding iron, yacK MCO activity could influence levels of free iron in the periplasm in response to copper concentration. This mechanism may explain, in part, how yacK MCO moderates the sensitivity of E. coli to copper.

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Hydrogenase from the hyperthermophilic archaeon, Pyrococcus furiosus, catalyzes the reversible activation of H(2) gas and the reduction of elemental sulfur (S degrees ) at 90 degrees C and above. The pure enzyme, modified with polyethylene glycol (PEG), was soluble (> 5 mg/mL) in toluene and benzene with t(1/2) values of more than 6 h at 25 degrees C. At 100 degrees C the PEG-modified enzyme was less stable in aqueous solution (t(1/2) approximately 10 min) than the native (unmodified) enzyme (t(1/2) approximately 1 h), but they exhibited comparable H(2) evolution, H(2) oxidation, and S degrees reduction activities at 80 degrees C. The H(2) evolution activity of the modified enzyme was twice that of the unmodified enzyme at 25 degrees C. The PEG-modified enzyme did not catalyze S degrees reduction (at 80 degrees C) in pure toluene unless H(2)O was added. The mechanism by which hydrogenase produces H(2)S appears to involve H(2)O as the proton source and H(2) as the electron source. The inability of the modified hydrogenase to catalyze S degrees reduction in a homogeneous non-aqueous phase complicates potential applications of this enzyme.