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Journal of Bacteriology, September 1999, p. 5530-5533, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A Hyperactive NAD(P)H:Rubredoxin Oxidoreductase
from the Hyperthermophilic Archaeon Pyrococcus
furiosus
Kesen
Ma and
Michael W. W.
Adams*
Department of Biochemistry and Molecular
Biology and Center for Metalloenzyme Studies, University of Georgia,
Athens, Georgia 30602
Received 17 March 1999/Accepted 17 June 1999
 |
ABSTRACT |
NAD(P)H:rubredoxin oxidoreductase (NROR) has been purified from the
hyperthermophilic archaeon Pyrococcus furiosus. The enzyme is exceedingly active in catalyzing the NADPH-dependent reduction of
rubredoxin, a small (5.3-kDa) iron-containing redox protein that had
previously been purified from this organism. The apparent Vmax at 80°C is 20,000 µmol/min/mg, which
corresponds to a
kcat/Km value of
300,000 mM
1 s1. The apparent
Km values measured at 80°C and pH 8.0 for
rubredoxin, NADPH, and NADH were 50, 5, and 34 µM, respectively. The
enzyme did not reduce P. furiosus ferredoxin. NROR is a
monomer with a molecular mass of 45 kDa and contains one flavin adenine
dinucleotide molecule per mole but lacks metals and inorganic sulfide.
The possible physiological role of this hyperactive enzyme is discussed.
| |
TEXT |
Pyrococcus furiosus is a
member of the hyperthermophilic archaea, microorganisms that thrive at
extreme temperatures and inhabit shallow and deep-sea volcanic
environments (23, 24). It is an obligate anaerobe and grows
optimally at 100°C by the fermentation of carbohydrates and peptides
to organic acids, CO2 and H2. If elemental
sulfur (S0) is present in the medium, it is reduced to
H2S. The primary electron acceptor for these oxidative
fermentative pathways is the small (7.5-kDa), iron-sulfur-containing
redox protein ferredoxin (1). The oxidation of reduced
ferredoxin is thought to be coupled to H2 production and
S0 reduction via ferredoxin:NADP oxidoreductase and
sulfhydrogenase (12, 15). Sulfhydrogenase is a bifunctional
enzyme which catalyzes the oxidation of NADPH and the reduction of
protons and S0 to H2 and H2S,
respectively (15).
A second small, iron-containing redox protein termed rubredoxin (~5.3
kDa) has also been purified from P. furiosus (3). Rubredoxin from several species of anaerobic bacteria has been characterized (11), but its physiological function is not
clear. Neither sulfhydrogenase or ferredoxin:NADP oxidoreductase
efficiently reduces rubredoxin with NADH or NADPH as the electron donor
(12, 15). An intriguing question, therefore, is whether
P. furiosus contains an NAD(P)H-dependent enzyme which
specifically uses rubredoxin as an electron acceptor. Herein the
purification and properties of such an enzyme, which is termed
NAD(P)H:rubredoxin oxidoreductase (NROR), are described.
Purification of NROR.
NROR activity was determined
anaerobically by the NADPH-dependent reduction of P. furiosus rubredoxin at 494 nm (molar absorptivity of 9,220 M1 cm1 [3]) at 80°C. The
reaction mixture (2.0 ml) contained 100 mM EPPS
[N-(2-hydroxyethyl)-piperazine-N'-(3-propanesulfonic
acid)] buffer (pH 8.0), NADPH (0.3 mM), and rubredoxin (10 µM). As
determined by this assay, the specific activity of cell extracts from
six different batches of P. furiosus cells was 2.2 ± 0.8 µmol of rubredoxin reduced/min/mg. The enzyme appeared to be
located in the cytoplasm, since after ultracentrifugation
(110,000 × g for 2 h) of the cell extract, more
than 90% of the activity was in the supernatant fraction. This also
contained 83% of the cellular glutamate dehydrogenase activity, a
known cytoplasmic enzyme (14, 21). From a small-scale purification of NROR by using the NADPH-dependent reduction of rubredoxin to measure its activity, it was found that the pure enzyme
also catalyzed the NADH-dependent reduction of benzyl viologen (BV). In
this case the assay mixture (2.0 ml) contained 50 mM CAPS
[3-(cyclohexylamino)-1-propanesulfonic acid] buffer (pH 10.2), NADH
(0.3 mM), and BV (1 mM). A molar absorptivity of 7,800 M1
cm1 was used for reduced BV. This NADH:BV oxidoreductase
assay was used for the large-scale purification of NROR, where 1 U of
activity equals 1 µmol of NADH oxidized/min/mg.
P. furiosus (DSM 3638) was routinely grown at 90°C in a
600-liter fermentor with maltose as the carbon source as described previously (4). NROR was purified at 23°C under strictly
anaerobic conditions (4). Frozen cells (400 g [wet
weight]) were thawed in 1.5 liters of buffer A (50 mM Tris-HCl [pH
8.0] containing 10% [vol/vol] glycerol, 2 mM dithiothreitol, and 2 mM sodium dithionite) containing DNase I (10 µg/ml) and were lysed by
incubation at 35°C for 2 h. A cell extract was obtained by
centrifugation at 50,000 × g for 80 min. The
supernatant (1.3 liters) was loaded onto a column (8 by 21 cm) of
DEAE-Sepharose Fast Flow (Pharmacia LKB, Piscataway, N.J.) equilibrated
with buffer A. The column was eluted with a linear gradient (9.0 liters) from 0 to 0.5 M NaCl in buffer A, and 90-ml fractions were
collected. Elution of NROR activity started as 0.2 M NaCl was applied
to the column. Those fractions containing NROR activity were combined
(810 ml), concentrated by ultrafiltration (type PM-30 membrane; Amicon, Beverly, Mass.), and washed with buffer B. Buffer B was the same as
buffer A except that sodium dithionite was omitted. The concentrated sample (150 ml) was applied to a column (5 by 12 cm) of blue Sepharose (Pharmacia LKB) equilibrated with buffer B. The column was eluted with
a linear gradient (1.4 liters) of 0 to 2.0 M NaCl in buffer B, and
50-ml fractions were collected. Elution of NROR activity started as 1.6 M NaCl was applied. Those fractions containing NROR activity were
combined (600 ml), concentrated to 30 ml by ultrafiltration (PM-30
membrane), and applied to a column (6 by 60 cm) of Superdex 200 (Pharmacia LKB) equilibrated with buffer B containing 50 mM KCl.
Fractions of 25 ml were collected. Those containing NROR activity were
combined (50 ml) and applied to a column (2.6 by 10 cm) of Q-Sepharose
(high performance; Pharmacia LKB) equilibrated with buffer B. The
column was eluted with a gradient (0.5 liter) of 0 to 1.0 M KCl in
buffer B, and 20-ml fractions were collected. Elution of NROR activity
started as 0.3 M KCl was applied. Those fractions judged to be
homogeneous on the basis of sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis (10) were combined (80 ml), concentrated
by ultrafiltration (PM-30 membrane), and stored as pellets in liquid N2.
The results of a typical purification are given in Table
1. Three peaks of NADH-dependent BV
reduction activity were separated
after chromatography on the blue
Sepharose column (data not shown),
but only data for the peak
corresponding to NROR, which accounted
for ~40% of the total BV
reduction activity, are shown in Table
1. One of the other two peaks
from this column contained ferredoxin:NADP
oxidoreductase, which also
catalyzes the NADH-dependent reduction
of BV (
12), but the
nature of the enzyme responsible for the
third peak of NADH:BV
oxidoreductase activity is not known. The
specific activities of pure
NROR were 490 µmol of NADH oxidized/min/mg
in the NADH:BV
oxidoreductase assay (Table
1) and 2,750 µmol
of NADPH
oxidized/min/mg in the NADPH:rubredoxin assay. In the
latter assay, the
enzyme was purified 1,830-fold, with a recovery
of activity (NROR) of
about 47% relative to that of the cell extract
(1.5 U/mg).
Biophysical properties of NROR.
SDS-polyacrylamide gel
electrophoresis (10) of purified NROR showed only one
protein band, and this had a mass of 45,000 Da (Fig. 1).
By using a calibrated column (1.6 by 60 cm) of Superdex 200 (Pharmacia LKB) equilibrated with 50 mM Tris-HCl
buffer (pH 8.0) containing KCl (200 mM), NROR was eluted with a mass of
51,000 ± 5,000 Da. Therefore, the protein appears to be
monomeric. The N-terminal amino acid sequence,
MKVVIVGNGPGGFELAKQLSQTYEV, was determined after electroblotting the
enzyme from an SDS gel onto a polyvinylidene difluoride protein
sequencing membrane (see, for example, reference
13). Database searches indicated that this sequence
in NROR bound flavin since it showed similarity to the sequences of
such domains present in NADH dehydrogenase of Escherichia
coli (25) and NADH oxidase (nox-1 and nox-2) of
Archaeoglobus fulgidus (9). The presence of a
flavin chromaphore was also suggested by the UV-visible absorption
spectra of the oxidized (as purified) NROR. This exhibited peaks near
375 and 450 nm which decreased in intensity upon the addition of sodium dithionite or NADPH (data not shown). As determined by using a molar
absorptivity of 11,300 M1 cm1 at 450 nm
(2), the holoenzyme contains one flavin moiety per mole,
while plasma emission spectroscopy (13) showed the presence of 1.8 g-atoms of P per mol of enzyme. These data suggest that NROR
contains one flavin adenine dinucleotide (FAD) molecule per mole. This
was confirmed by thin-layer chromatography analysis of the
acid-extracted cofactor which migrated at the same position as that of
FAD (data not shown). Mass spectroscopy analyses, carried out by the
Mass Spectrometry Facility at the University of Georgia, revealed that
the flavin cofactor had a mass of 786 Da, which corresponds to that of
FAD. No metals could be detected NROR (>0.05 atoms/mol) as measured by
plasma emission spectroscopy, while chemical analysis (20)
showed that it contained two cysteinyl residues. The enzyme was
thermostable, with times required for a 50% loss in catalytic activity
(with 0.4 mg/ml in 50 mM EPPS, pH 8.0) at 80 and 95°C of about 12 and
2 h, respectively.
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FIG. 1.
SDS-12.5% polyacrylamide gel electrophoresis of
P. furiosus NROR. Lanes 1 and 4 contain molecular mass
markers. Lanes 2 and 3 contain 1 and 2 µg of NROR, respectively.
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|
Catalytic properties of NROR.
Purified NROR catalyzed the
NADPH-dependent reduction of various electron carriers. These included
(where 100% activity equals 51 µmol reduced/min/mg at 50°C with
the indicated concentration of electron carrier) methyl viologen (6%,
1 mM), benzyl viologen (100%, 1 mM), FAD (42%, 150 µM), flavin
mononucleotide (44%, 150 µM), menadione (72%, 200 µM),
2,6-dichlorophenol indophenol (365%, 100 µM), cytochrome
c (8%, 50 µM), and iron (III) citrate (2%, 100 µM).
P. furiosus ferredoxin (40 µM) was not utilized as an electron acceptor by NROR with either NADPH or NADH as the electron donor. The most efficient electron acceptor was rubredoxin (1,160%, 9.5 µM). The kinetics obtained with this protein are summarized in
Table 2. The apparent
Vmax at 80°C was approximately 20,000 µmol/min/mg, which corresponds to a
kcat/Km value of 300,000 mM1 s1. These data suggest that rubredoxin
is the physiological substrate of NROR, while NADPH
(Km, 5 µM) rather than NADH
(Km, 34 µM) is the preferred electron donor
(Table 2). Surprisingly, NROR exhibited high catalytic activity even at
a low temperature. For example, the apparent
Vmax at 25°C was approximately 300 µmol/min/mg. However, as shown in Table 2, upon an increase in the
temperature to 80°C, the apparent Km increased
fivefold and the apparent Vmax increased almost
2 orders of magnitude. Table 2 also shows that NROR catalyzed the
NADPH-dependent reduction of DTNB (5,5'-dithiobis[2-nitrobenzoic acid]) (45%, 2.5 mM) at 80°C, indicating that this enzyme can function as a disulfide reductase (8). The pH optima for the reduction of DTNB and rubredoxin were similar (7.6 and 7.0, respectively). NROR also functioned as an NADPH peroxidase and
catalyzed the NADPH-dependent reduction of peroxide (Table 2).
Physiological role of NROR.
While the reduction by NROR of
both a disulfide (DTNB) and a peroxide occurred at significant rates at
80°C (>10 µmol reduced/min/mg), these rates are about 3 orders of
magnitude less than the rate of rubredoxin reduction under the same
conditions (Table 2). Therefore, it seems unlikely that the primary
role of NROR in vivo is either as an NADPH peroxidase or as a disulfide
reductase. The measured NADPH peroxidase activity probably occurred by
a nonspecific reaction, perhaps with the flavin group, but
DTNB-dependent disulfide reductase activity of NROR was inhibited by
55% when the enzyme (0.6 mg/ml in 50 mM Tris, pH 7.8) was treated with iodoacetate (9 mM) at 25°C for 10 h. This result indicates that two cysteinyl residues of NROR may be the catalytic site for the disulfide reductase activity. Disulfides, such as thioredoxin (7) and glutaredoxin (19), have been identified
in P. furiosus, but whether these can be reduced by NROR
remains unknown.
On the other hand, the fact that NROR is such a remarkably efficient
enzyme for reducing rubredoxin
(
kcat/
Km = 3 × 10
5 mM
1 s
1 at 80°C) suggests
that this is its sole physiological function
and that reduced
rubredoxin plays an important role in vivo. The
question is, what is
the purpose of the rapid reduction of rubredoxin?
Unfortunately, the
function of this protein in
P. furiosus or
in any other of
the anaerobic organisms from which it has been
purified is not known.
Rubredoxin has been proposed to play a
role in nitrate reduction
(
22), methanogenesis from H
2 and CO
2 (
16), O
2 reduction (
5,
6), and CO
oxidation (
18), but
the nature of the enzymes involved in
oxidizing and reducing the
protein either is not specific or is not
consistent among the
various
organisms.
Two enzymes that catalyze the NADH-dependent reduction of rubredoxin
have been characterized for mesophilic anaerobes. That
from
Clostridia acetobutylicum (
17), which is a
FAD-containing
monomer with a molecular mass of 41 kDa, closely
resembles
P. furiosus NROR in its molecular properties. In
contrast, NROR from
Desulfovibrio gigas consists of two
different subunits with molecular
masses of 27 and 32 kDa, and it
contains both FAD and flavin mononuclotide
(
5).
Interestingly, the specific activities of the two mesophilic
enzymes in
the NADH-dependent reduction of rubredoxin were 46
and 12 U/mg,
respectively (
5,
17). A value of 294 U/mg was
obtained for
the
P. furiosus enzyme when assayed under comparable
conditions (at 25°C), which approach 80° below the optimum growth
temperature of the organism. At present it is not clear why
P. furiosus NROR is so active at such a "low" temperature.
Obviously,
much more is to be learned about the role of this enzyme and
of
rubredoxin in
P. furiosus and other
microorganisms.
| |
ACKNOWLEDGMENTS |
We thank H.-S. Huang and Z.-H. Zhou for providing P. furiosus rubredoxin.
This research was supported by a grant (BCS-9632657) from the National
Science Foundation.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, Life Sciences Building, University of Georgia, Athens, GA
30602. Phone: (706) 542-2060. Fax: (706) 542-0229. E-mail: adams{at}bmb.uga.edu.
| |
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Journal of Bacteriology, September 1999, p. 5530-5533, Vol. 181, No. 17
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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