The model hyperthermophilic archaeon, Pyrococcus furiosus, grows optimally at 100 ℃ by fermentation. This generates ATP through substrate-level phosphorylation and reduced ferredoxin (Fd), which is used by a membrane-bound hydrogenase, MBH, to reduce protons to H2 and generate a Na+ gradient. This is used by ATP synthase to generate additional ATP through electron transport phosphorylation. Herein, we used deletion strains of MBH and a related multiple resistance and pH (Mrp) antiporter to demonstrate the different mechanisms by which the Na+ gradient is generated and utilized by the cell. During growth in the presence of elemental sulfur (S0), the expression of genes encoding MBH rapidly decreases and expression of the membrane-bound sulfane sulfur reductase (MBS) increases. MBS oxidizes Fd and reduces sulfane sulfur derived from S0 and generates a Na+ gradient. However, the catalytic mechanism remains unknown. Herein, we determined the cryo-EM structure of MBS and showed that it uses a novel [4Fe-4S] cluster to directly catalyze the reduction of sulfane sulfur. Additionally, the structure provided insights into the evolution of both MBS and MBH and established their close relationship with Complex I of the mitochondrial respiratory chain. We also took advantage of the robust genetic system of P. furiosus in order to heterologously express respiratory systems from other hyperthermophiles. A thiosulfate reductase (Tsr) and arsenate respiratory reductase (Arr) were heterologously expressed from the hyperthermophile Pyrobaculum aerophilum. Both Tsr and Arr were the first characterized archaeal examples of their respective enzyme families, and both were shown to utilize molybdenum instead of tungsten in their active sites, the first examples of active molybdoenzymes being expressed by and purified from P. furiosus. The Arr-expressing strain was also able to utilize arsenate as a terminal electron acceptor, the first example of P. furiosus using non-native substrates as terminal electron acceptors.