Purification and characterization of a membrane-bound hydrogenase from thehyperthermophilic archaeon Pyrococcus furiosus

Highly mashed membrane preparations from cells of the hyperthermophilic arc haeon Pyrococcus furiosus contain high hydrogenase activity (9.4 mu mol of H-2 evolved/mg at 80 degrees C) using reduced methyl viologen as the electr on donor. The enzyme was solubilized with n-dodecyl-beta-D-maltoside and pu rified by multistep chromatography in the presence of Triton X-100. The pur ified preparation contained two major proteins (alpha and beta) in an appro ximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained si milar to 1 Ni and 4 Fe atoms/mol. The reduced enzyme gave rise to an electr on paramagnetic resonance signal typical of the so-called Ni-C center of me sophilic NiFe-hydrogenases. Neither highly washed membranes nor the purifie d enzyme used NAD(P)(H) or P. furiosus ferredoxin as an electron carrier, n or did either catalyze the reduction of elemental sulfur with H-2 as the el ectron donor. Using N-terminal amino acid sequence information, the genes p roposed to encode the alpha and beta subunits mere located in the genome da tabase within a putative 14-gene operon (termed mbh). The deduced sequences of the two subunits (Mbh 11 and 12) were distinctly different from those o f the four subunits that comprise each of the two cytoplasmic NiFe-hydrogen ases of P. furiosus and show that the alpha subunit contains the NiFe-catal ytic site. Six of the open reading frames (ORFs) in the operon, including t hose encoding the alpha and beta subunits, show high sequence similarity (> 30% identity) with proteins associated with the membrane-bound NiFe-hydroge nase complexes from Methanosarcina barkeri, Escherichia coli, and Rhodospir illum rubrum. The remaining eight ORFs encode small (<19-kDa) hypothetical proteins. These data suggest that P. furiosus, which was thought to be sole ly a fermentative organism, may contain a previously unrecognized respirato ry system in which H-2 metabolism is coupled to energy conservation.