Respiratory pathways and mechanisms of energy conservation in the hyperthermophilic archaeon, pyrococcus furiosus
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The main objective of this research was the identification, characteriza tion and elucidation of pathways of enzymes involved in hydrogen (H2) and sulfur metabolism in the hyperthermophilic archaeon Pyrococcus furiosus. P. furiosus is an anaerobic hyperthermophilic archaeon that has an optimal growth temperature of 100°C. It is an obligate heterotroph that uses maltose and peptides as carbon and energy sources to produce acetate, organic acids, CO2 and H2 as metabolic end products. H2 production, catalyzed by cytoplasmic hydrogenases, was thought to be an electron sink for fermentative pathways. However, we have isolated a membrane-bound hydrogenase (MBH) that also functions to evolve H2. The enzyme, which contains redox active nickel and iron, is part of a multi-subunit complex that interacts with the cytosolic electron carrier protein ferredoxin, but not with NAD(P)H. Furthermore, the MBH complex has been shown to not only reduce protons to H2, but also couple the reaction to proton translocation resulting in the formation of a membrane potential. Thus established membrane potential is used for energy conservation by a membrane-bound ATPase. The MBH complex, however, is downregulated in the presence of peptides and sulfur. While growth on sugars is unaffected by the presence of sulfur, it is required for growth on peptides and is reduced to H2S with a concomitant decrease in H2 production. A major difference between the metabolism of sugars and peptides is the identity of the primary electron carrier, ferredoxin during glycolysis and NAD(P)H during peptidolysis. We have also isolated a membrane-bound NADH dehydrogenase (NDH) complex, which oxidizes NAD(P)H and the resulting electrons are proposed to be used for the respiratory reduction of sulfur to H2S. The enzyme is expressed when cells are grown with peptides and sulfur and is downregulated during growth on sugars. Thus P. furiosus, which was previously thought to be a fermentative organism, is shown to contain two membranebound energy conserving complexes, which are elegantly regulated to suit the electron carriers used in the metabolism of sugars and peptides in the presence of sulfur.