Highly washed membrane preparations from cells of the hyperthermophilic archaeon Pyrococcus furiosus contain high hydrogenase activity (9.4 mol of H 2 evolved/mg at 80°C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with n-dodecyl--D-maltoside and purified by multistep chromatography in the presence of Triton X-100. The purified preparation contained two major proteins (␣ and ) in an approximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained ϳ1 Ni and 4 Fe atoms/mol. The reduced enzyme gave rise to an electron paramagnetic resonance signal typical of the so-called Ni-C center of mesophilic NiFe-hydrogenases. Neither highly washed membranes nor the purified enzyme used NAD(P)(H) or P. furiosus ferredoxin as an electron carrier, nor did either catalyze the reduction of elemental sulfur with H 2 as the electron donor. Using N-terminal amino acid sequence information, the genes proposed to encode the ␣ and  subunits were located in the genome database within a putative 14-gene operon (termed mbh). The deduced sequences of the two subunits (Mbh 11 and 12) were distinctly different from those of the four subunits that comprise each of the two cytoplasmic NiFe-hydrogenases of P. furiosus and show that the ␣ subunit contains the NiFe-catalytic site. Six of the open reading frames (ORFs) in the operon, including those encoding the ␣ and  subunits, show high sequence similarity (>30% identity) with proteins associated with the membrane-bound NiFe-hydrogenase complexes from Methanosarcina barkeri, Escherichia coli, and Rhodospirillum rubrum. The remaining eight ORFs encode small (<19-kDa) hypothetical proteins. These data suggest that P. furiosus, which was thought to be solely a fermentative organism, may contain a previously unrecognized respiratory system in which H 2 metabolism is coupled to energy conservation.Hydrogenases catalyze the reversible reduction of protons to hydrogen gas. They are found in a wide variety of microorganisms and enable them to use H 2 as a source of reductant under either aerobic or anaerobic conditions. Alternatively, fermentative-type organisms utilize hydrogenase to dispose of reductant without the need of terminal electron acceptors other than protons (1, 3). Hydrogenases can be divided into two major types, depending on the metals they contain (5). The so-called iron-only hydrogenases have high specific activities and usually function to evolve H 2 . Their catalytic site is comprised of a novel 6Fe cluster (26,29). The active site of nickel-and ironcontaining hydrogenases (NiFe-hydrogenases), on the other hand, consists of a binuclear NiFe center (12, 36). The NiFehydrogenases are less active than their Fe-only counterparts, and their physiological role is usually to oxidize H 2 . In aerobic H 2 -oxidizing bacteria, NiFe-hydrogenases can function both as cytoplasmic, NAD-reducing enzymes and as part of conventional membrane-bound (MB) respiratory chains where O 2 is the terminal electron acceptor (4, 9). In contrast, in anaerobic ...