Journal of Bacteriology, June 2000, p. 3423-3428, Vol. 182, No. 12
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Department of Biochemistry and Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602
Received 29 November 1999/Accepted 21 March 2000
Highly washed membrane preparations from cells of the
hyperthermophilic archaeon Pyrococcus furiosus contain high
hydrogenase activity (9.4 µmol of H2 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 ~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 H2 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 H2
metabolism is coupled to energy conservation.
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