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Abstract
The model hyperthermophilic archaeon, Pyrococcus furiosus, grows optimally at 100 C by fermenting carbohydrates and peptides. In the absence of elemental sulfur (S0), the reduced ferredoxin (Fd) generated from the glycolysis pathway transfers electrons to a membrane-bound hydrogenase (MBH) for H2 production and Na+ gradient generation, which is used by ATP synthase for energy production. Two soluble hydrogenases termed SHI and SHII are thought to recycle the H2 for nicotinamide cofactor generation. SHI has been extensively studied and is widely used in several applications, such as in vitro H2 production. To scale up these applications, large amounts of purified SHI are required. Herein, we engineered P. furiosus to overproduce SHI by increasing the expression of the genes encoding the maturation accessory proteins and devised a 500-g wet wt. cell-scale purification procedure. In the presence of S0, the expression of the genes encoding MBH, SHI and SHII decreases dramatically and MBH is replaced by a membrane-bound oxidoreductase of unknown function. Herein we show that this enzyme is a sulfane reductase (MBS). Like MBH, MBS is proposed to oxidize Fd and generate a Na+ gradient but it reduces sulfane sulfur derived from S0. A sulfane-containing substrate was discovered for MBS and the enzyme was purified in membrane-associated and soluble forms and these were biochemically characterized. P. furiosus was also utilized as a platform for the heterologous expression of enzymes to study respiratory systems from other hyperthermophiles. An arsenate respiratory reductase (Arr) was heterologously-expressed from the archaeon Pyrobaculum aerophilum in P. furiosus. This is the first archaeal Arr to be purified and biochemically characterized and a novel type of arsenite resistance was observed in the Arr-expressing P. furiosus strain. In addition, this strain was able to use arsenate as the terminal electron acceptor to support growth. This represents the first example of P. furiosus using a non-native substrate as a terminal electron acceptor other than S0 and protons.