Affiliation:
1. Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602
Abstract
ABSTRACT
Highly washed membrane preparations from cells of the hyperthermophilic archaeon
Pyrococcus furiosus
contain high hydrogenase activity (9.4 μmol of H
2
evolved/mg at 80°C) using reduced methyl viologen as the electron donor. The enzyme was solubilized with
n
-dodecyl-β-
d
-maltoside and purified by multistep chromatography in the presence of Triton X-100. The purified preparation contained two major proteins (α and β) in an approximate 1:1 ratio with a minimum molecular mass near 65 kDa and contained ∼1 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 α and β 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 α subunit contains the NiFe-catalytic site. Six of the open reading frames (ORFs) in the operon, including those encoding the α and β 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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
105 articles.
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