Báo cáo khoa học: Molecular characterization of H2O2-forming NADH oxidases from Archaeoglobus fulgidus potx

Molecular characterization of H
2
O
2
-forming NADH oxidases
from
Archaeoglobus fulgidus
Serve
´
W. M. Kengen, John van der Oost and Willem M. de Vos
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, the Netherlands
Three NADH oxidase encoding genes noxA-1, noxB-1
and noxC were cloned from the genome of Archaeoglobus
fulgidus, expressed in Escherichia coli, and the gene products
were purified and characterized. Expression of noxA-1 and
noxB-1 resulted in active gene products of the expected size.
The noxC gene was expressed as well but the protein pro-
duced showed no activity in the standard Nox assay. NoxA-
1 and NoxB-1 are both FAD-containing enzymes with
subunit molecular masses of 48 and 69 kDa, respectively.
NoxA-1 exists predominantly as homodimer, NoxB-1 as
monomer. NoxA-1 and NoxB-1 showed pH optimum of
8.0 and 6.5, with specific NADH oxidase activities of
5.8 UÆmg
)1
and 4.1 UÆmg
)1
, respectively. Both enzymes
were specific for NADH as electron donor, but with different
apparent K
m

values (NoxA-1, 0.13 m
M
; NoxB-1, 0.011 m
M
).
The apparent K
m
values for oxygen differed significantly
(NoxA-1, 0.06 m
M
; NoxB-1, 2.9 m
M
). In contrast with all
mesophilic homologues, both enzymes were found to pro-
duce predominantly H
2
O
2
instead of H
2
O. Despite apparent
similarities, NoxB-1 is essentially different from NoxA-1.
Whereas NoxA-1 resembles typical H
2
O-producing Nox
enzymes that are expected to have a role in oxidative stress
defence, NoxB-1 belongs to a small group of enzymes that
is involved in catalysing the reduction of unsaturated acids
and aldehydes, suggesting a role in fatty acid oxidation.
Moreover, NoxB-1 contains a ferredoxin-like motif, which

is absent in NoxA-1.
Keywords: Archaeoglobus; flavoprotein; NADH oxidase;
oxygen stress.
Archaeoglobus fulgidus is a strictly anaerobic hyperthermo-
philic archaeon that has been isolated from marine hydro-
thermal environments as well as subsurface oil fields. This
sulfate reducer can grow organoheterotrophically with
a variety of carbon sources, or lithoautotrophically on
hydrogen, thiosulfate and CO
2
[1]. Besides its ability to grow
at extremely high temperatures, this organism is unusual in
that it is evolutionary unrelated to other sulfate reducers.
Recently, the sequence of the entire genome of A. fulgidus
was completed [2]. The sequencing revealed the presence of
eight putative NADH oxidase genes, which were designated
noxA-1 to noxA-5, noxB-1, noxB-2 and noxC, according to
their homology to other NADH oxidase encoding genes.
NADH oxidases (EC 1.6.99.3) catalyse the two-electron
reduction of oxygen to peroxide or the four-electron
reduction of oxygen to water. Although all so-called
NADH oxidases share the ability to reduce oxygen, their
physiological role may differ or is often not known.
Moreover, some homologues have been shown not to
reduce oxygen and to catalyse somewhat different reactions,
such as NADH peroxidase (EC 1.11.1.1) and disulfide
reductase (EC 1.8.1.14). The noxA homologues from
A. fulgidus code for a group of typical H
2
O-forming NADH

oxidases of  49 kDa, found in various prokaryotes, and
including the well-studied NADH oxidase from Enterococ-
cus faecalis [3] (Fig. 1). This group also contains NADH
peroxidases and coenzyme A disulfide reductases [4,5]. The
noxB homologues encode a small group of enzymes of
 72 kDa, that are involved in the reduction of unsaturated
acids and aldehydes, or whose function is not known. In the
presence of oxygen, they also produce H
2
O, except for the
enzyme from Thermoanaerobacter brockii that forms H
2
O
2
and some superoxide (O
2
)
)[6].NoxC codes for a small
20-kDa protein, and it was designated as NADH oxidase,
due to its homology to the NADH oxidases from the
thermophilic bacteria Thermus aquaticus and Thermus
thermophilus [7,8]. Some putative NAD(P)H oxidoreduc-
tases from thermophilic archaea also belong to this group
(Fig. 1).
The physiological role of the putative NADH oxidases in
A. fulgidus is still enigmatic. Common NADH oxidases of
mesophilic origin are assumed to protect the cells from
oxidative stress by reducing oxygen to water, without the
formation of harmful reactive oxygen species. Alternatively,
NADH oxidases may recycle oxidized pyridine nucleotides

during catabolism. Moreover, some homologues were
shown to have functions other than NADH oxidase (see
above). Concerning the homologues from (hyper)thermo-
philic species, only a few have been studied in more detail. A
recently purified NADH oxidase from A. fulgidus (NoxA-2)
was proposed to be involved in electron transfer reactions
Correspondence to S. W. M. Kengen, Laboratory of Microbiology,
Hesselink van Suchtelenweg 4, 6703 CT Wageningen, the Netherlands.
Fax: +31 317 483829, Tel.: +31 317 483748,
E-mail:
Abbreviations: NoxA-1, NADH oxidase A-1; NoxB-1, NADH
oxidase B-1; NoxC, NADH oxidase C; DCPIP, 2,6 dichlorophenol-
indophenol; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid).
Enzymes: NADH oxidases (EC 1.6.99.3); NADH peroxidase
(EC 1.11.1.1); disulfide reductase (1.8.1.14).
(Received 28 February 2003, revised 25 April 2003,
accepted 14 May 2003)
Eur. J. Biochem. 270, 2885–2894 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03668.x
during sulfate respiration [9]. The NADH oxidase of
Pyrococcus furiosus (Nox1; PF1430634) purified from an
overproducing Escherichia coli,wasanticipatedtoplaya
role in protection against oxidative stress [10]. The function
of the NADH oxidase of T. brockii is still unknown [6]. In
several hyperthermophilic archaea and bacteria, other than
A. fulgidus, various putative NADH oxidase genes have
been identified, whose function remains to be established.
Independent of their physiological role, H
2
O
2

-producing
NADH oxidases may be applicable in biosensors, where
they act as mediator between NADH-forming dehydrogen-
ases and the electrode [11]. Concerning this, enzymes from
(hyper)thermophiles may be superior to mesophilic coun-
terparts, because they generally exhibit a higher stability not
only with respect to temperature, but also towards chemical
denaturants, like detergents or organic solvents [12,13].
In order to analyse the biochemical properties, to unravel
their physiological role and to test the potential stability, the
noxA-1 gene, the noxB-1 gene and the noxC gene were
cloned and expressed in E. coli and the overproduced
NADH oxidases were purified and characterized.
Experimental procedures
Materials
3,3¢-Dimethoxybenzidine, coenzyme A (oxidized), glutathi-
one (oxidized), horseradish peroxidase and 2,3-dimethyl-
1,4-naphthoquinone were from Sigma Chemie.
Q-Sepharose, Superdex 200 HR 10/30, and Mono-Q HR
5/5 were from Amersham Pharmacia Biotech. Hydroxy-
apatite (Bio-Gel HT), SDS/PAGE calibration proteins
(broad range), and the protein assay kit were from Bio-Rad.
The pET9d expression vector was from Novagen Inc.
E. coli BL21(DE3) and Pfu DNA polymerase was from
Stratagene. A. fulgidus (DSM 4304) was from the
German Collection of Microorganisms and Cell Cultures
(Braunschweig, Germany). Polysulfide was prepared by
adding 12 g Na
2
S to 1.6 g elemental sulfur in 100 mL

anoxic water [14]. Cytochrome c was purified from the
mesophilic Syntrophobacter fumaroxidans and was a gift of
Frank de Bok (Wageningen University, the Netherlands).
Cloning of the NADH oxidase genes
In the genome sequence of A. fulgidus [2] various putative
NADH oxidase genes have been identified, of which three
were selected for further research (noxA-1, AF0254; noxB-1,
AF0455; noxC:, AF0226). The following primer sets were
designed to amplify the selected Nox open reading frames:
for noxA-1 primer BG852 (5¢-CGCGTCATGAAGGTT
GCAATTATAGGCGGT-3¢, sense) and primer BG853
(5¢-CGCGGGATCCCTACGGCAATCCGAGCTTC-3¢,
antisense), with BspHI and BamHI restriction sites in bold;
for noxB-1 primer BG854 (5¢-CGCGCCATGGCCAAG
CTTTTCGAGCCAATCGAG-3¢, sense) and BG855
(5¢-CGCGGGATCCCTAAACCTTCAAAGCCAGAT-3¢,
antisense), with restriction sites NcoIandBamHI in bold;
for noxC primer BG831 (5¢-GCGCGTCATGATGGAAT
GCCTTGACTTGCTGTTC-3¢,sense)andBG832(5¢-CG
CGCGGATCCTCACCATTTTTCGAAGTGCGTGAG-3¢,
antisense), with BspHI and BamHI restriction sites in bold.
The 50 lL PCR reaction mixture contained 200 ng A. ful-
gidus SL-5 genomic DNA, isolated as described previously
[15], 100 ng each primer, 0.3 m
M
dNTPs, Pfu polymerase
buffer, and 2.5 U Pfu DNA polymerase and was subjected
to 35 cycles of amplification (15 s at 94 °C, 30 s at 50 °C
and 2 min at 68 °C) on a DNA Thermal Cycler (Perkin
Elmer Cetus). The PCR product was digested with the

appropriate enzymes and cloned into an NcoI/BamHI-
digested pET9d vector, resulting in pWUR66, pWUR67,
and pWUR68, for noxA-1, noxB-1 and noxC, respectively.
The plasmids were transformed into E. coli TG1 and E. coli
BL21(kDE3) by heat-shock. Sequence data were analysed
using the computer program
DNASTAR
.
Expression of the NADH oxidase genes in
E. coli
Ten millilitres of Luria–Bertani medium with 50 lgÆmL
)1
kanamycin was inoculated (1%) from overnight cultures
of E. coli BL21(DE3) containing either pWUR66,
pWUR67 or pWUR68. After growth at 37 °Cto
D
600
¼ 0.8, 0.5 m
M
isopropyl thio-b-
D
-galactoside was
added to induce expression. After overnight growth, 2-mL
of each culture was centrifuged (10 min at 20 000 g)and
cells were resuspended in 50 m
M
Tris/HCl buffer pH 7.8.
Cells were sonicated and the supernatants were subse-
quently heated for 20 min at 70 °CtodenatureE. coli
proteins. The supernatants were analysed by SDS/PAGE

and by activity measurements.
For enzyme purification 2-L cultures were grown in
essentially the same way as described above. Cells (5–6 g
wet weight) were harvested by centrifugation (2200 g for
15 min at 10 °C) and resuspended in 28 mL 50 m
M
Tris/
HCl buffer pH 7.8. The suspension was passed twice
Fig. 1. Phylogenetic tree of NADH oxidases and related enzymes. The
tree was constructed from alignments using the
CLUSTAL
method [20]
of the Megalign program (
DNASTAR
, London, UK) and Nox sequences
available at the NCBI data base. The units at the bottom indicate the
number of substitution events. Genbank indentifiers are indicated in
parentheses.
2886 S. W. M. Kengen et al.(Eur. J. Biochem. 270) Ó FEBS 2003
through a French press (110 MPa) and the resulting crude
cell extract was used for purification of the recombinant
NADH oxidases.
Purification of recombinant NoxA-1 and NoxB-1
The E. coli cell extract was heated for 30 min at 70 °C
(NoxA-1) or for 30 min at 50 °C (NoxB-1), and denatured
proteins were pelleted by centrifugation (17 200 g for
15 min at 10 °C). This pellet fraction was washed with
10 mL Tris/HCl buffer pH 7.8 and the centrifugation step
was repeated. The supernatants of both centrifugation steps
were combined, filtered through a 0.45-lm filter and loaded

onto a Q-Sepharose column (1.6 · 10 cm) equilibrated with
20 m
M
Tris/HCl buffer pH 7.8. Bound proteins were eluted
by a 200-mL linear gradient of NaCl (0–1
M
in Tris/HCl
buffer pH 7.8). NoxA-1 eluted in a single peak at 0.38
M
NaCl. In a similar purification NoxB-1 eluted at 0.15
M
NaCl. Active fractions were pooled and applied to a
hydroxyapatite column (Bio-Gel HT; 1.6 · 10 cm) equili-
brated with 10 m
M
sodium phosphate buffer pH 7.0.
Elution was performed with a 200-mL gradient from 10
to 500 m
M
sodium phosphate (pH 7.2). The NoxA-1 as well
as the NoxB-1 eluted right after the flow-through fraction.
Active fractions were pooled and concentrated by ultrafil-
tration (Filtron; 10 kDa cut-off). A 200-lL aliquot of the
concentrated samples was loaded onto a Superdex-200 HR
column, equilibrated in 20 m
M
Tris/HCl pH 7.8 containing
150 m
M
NaCl.NoxA-1aswellasNoxB-1elutedastwo

overlapping activity peaks.
PAGE
The purity of the various purification fractions was regularly
checked by SDS/PAGE according to the procedure of
Laemmli using 15% (w/v) gels [16]. Protein samples were
denatured by heating in SDS-sample buffer for 5 min at
100 °C. SDS/PAGE was also used to determine the subunit
molecular mass. Calibration was performed using a set of
calibration proteins: myosin (200 kDa), b-galactosidase
(116.25 kDa), phosphorylase b (97.4 kDa), serum albumin
(66.2 kDa), ovalbumin (45 kDa) and carbonic anhydrase
(31 kDa). Protein bands were stained with Coomassie
brilliant blue R250.
Enzyme assays
NADH oxidase activity was measured spectrophoto-
metrically in 1-mL quartz cuvettes on a Hitachi U-2010
spectrophotometer equipped with a thermostatted cuvette
holder. Initially, one standard method was used for
measuring NADH oxidase activity of the recombinant gene
products. The standard assay mixture contained 100 m
M
potassium phosphate buffer (pH 7.0), 0.06 m
M
FAD,
0.29 m
M
NADH and an appropriate amount of enzyme.
The activity was determined by monitoring the oxidation of
NADH at 334 nm and at 70 °C(e
334

¼ 6.18 m
M
)1
Æcm
)1
)
[17]. In addition, separate assays were used for determining
NoxA-1 and NoxB-1 activity, which were performed at
80 °Cand70°C, respectively. NoxB-1 was less stable than
NoxA-1, and therefore the assays were performed at 70 °C
instead of 80 °C.
For NoxA-1 the assay mixture contained potassium
phosphate/sodium citrate buffer (50 m
M
each; pH 8.0),
0.06 m
M
FAD, 0.29 m
M
NADH and an appropriate
amount of enzyme. For NoxB-1 the same assay mixture
was used, except that the pH was adjusted to 6.5. Specific
activities were calculated from the initial linear change in
absorbance. Absorbance changes were corrected for non-
enzymatic NADH conversion. Where indicated FAD was
omitted from the assay mixture.
When electron acceptors other than oxygen were tested,
all assay constituents were made anoxic by repeated
evacuation and gassing with N
2

gas in stoppered serum
bottles. The stoppered cuvettes were also evacuated and
gassed with N
2
, and the different components were added
by syringe. The following extinction coefficients were used
to calculate the specific activities: ferricyanide, 1.00 m
M
)1
at
420 nm; 2,6 dichlorophenolindophenol (DCPIP), 20 m
M
)1
at 600 nm; 5,5¢-dithiobis(2-nitrobenzoic acid) (DTNB),
13.6 m
M
)1
at 412 nm, benzyl viologen, 8.6 m
M
)1
at
578 nm; cytochrome c, 21.1 m
M
)1
at 550 nm. For menadi-
one and 2,3-dimethyl-1,4-naphthoquinone extinction coef-
ficients were determined as 2.72 and 2.39 m
M
)1
at 334 nm,

respectively, based on a 1 : 1 stoichiometry with NADH.
To investigate whether the NADH oxidases produced
either H
2
OorH
2
O
2
, an activity assay without FAD was run
to completion at 50 °Canda50-lL sample from the
reaction mixture was tested in a separate assay at 22 °Cfor
the presence of H
2
O
2
. This second mixture (1 mL) con-
tained potassium phosphate/sodium citrate buffer (50 m
M
each, pH 6.5), 0.5 m
M
3,3¢-dimethoxybenzidine, and 7 U
horseradish peroxidase. The increase in absorbance
(460 nm) was compared to a standard curve, which was
prepared separately using known amounts of H
2
O
2
.The
assay was not disturbed by residual NADH, which might
have remained in the Nox assay. The decrease in NADH

in the first assay was related to the amount of H
2
O
2
found
in the second assay.
Protein was determined according to Bradford [18] using
the Bio-Rad protein assay kit, with BSA as standard.
Analysis of catalytic properties
Kinetic parameters of NoxA-1 and NoxB-1 were deter-
mined in the specific assay systems, by measuring the initial
rate at different starting concentrations of NADH in the
presence of ambient dissolved oxygen concentrations. K
m
(for NADH) and V
max
values were obtained by a
computer-aided direct fit to the Michaelis–Menten curve
(
TABLE CURVE 2D
). The K
m
values for O
2
were determined
from one single assay, in which the stoppered cuvettes were
completely filled with the specific assay buffers. The buffers,
which were equilibrated at 60 °C were calculated to contain
0.135 m
M

of dissolved oxygen [19]. The reaction was
started upon addition of anoxic NADH. The decrease in
oxygen concentration was calculated from the decrease in
NADH, assuming that for each mole of NADH one mole
of O
2
was required (in case only H
2
O
2
was produced). The
reaction rates at the various O
2
concentrations were
subsequently fitted using the Michaelis–Menten equation.
It was assumed that the NADH concentration was
saturating, meaning that only apparent K
m
values were
obtained.
Ó FEBS 2003 H
2
O
2
-forming NADH oxidases from Archaeoglobus fulgidus (Eur. J. Biochem. 270) 2887
The temperature dependence of NoxA-1 and NoxB-1
was determined in the range 20–90 °C. The pH-dependence
was determined in the standard potassium phosphate/
sodium citrate buffer at 80 °Cor70°C, for NoxA-1 or
NoxB-1, respectively.

Stability analysis
The thermostability of the enzymes was tested by incubating
the purified enzyme in potassium phosphate buffer
(100 m
M
, pH 7.0) in a closed vial in a water bath at
80 °C. At regular time intervals a sample was taken and
tested in the standard assay. For NoxB-1 the stability was
determined also in the presence of 2 m
M
dithiothreitol.
Half-life values were calculated from a fit of the data
(exponential decay: y ¼ aÆe
–bx
).
Results
Characterization based on amino acid sequence
The sequences of the three NADH oxidases from A. fulgi-
dus that were investigated here were aligned with various
other NADH oxidase sequences, available at the NCBI
database. The resulting phylogenetic tree (Fig. 1) clearly
showed that the three NADH oxidases belong to different
phylogenetic clusters. NoxA-1 falls within a group of typical
NADH oxidases of  49 kDa, including the well-studied
NADH oxidases from Enterococcus feacalis and Strepto-
coccus mutans [3,21]. This group also contains a NADH
peroxidase, which performs a NADH-dependent reduction
of H
2
O

2
to H
2
O, and a coenzyme A disulfide reductase,
which catalyses the NADPH-dependent reduction of
CoA-S-S-CoA to CoA-SH [4,5]. The NADH oxidase and
-peroxidase are H
2
O-forming enzymes and are believed to
play a role in oxidative stress defence or in the recycling of
oxidized pyridine nucleotides. The CoA disulfide reductase
is involved in maintaining a sufficiently high intracellular
thiol concentration [5]. The alignment (not shown) revealed
conserved binding motives for FAD or NAD(P) and,
moreover, most sequences in this group contain a redox
active cysteine, which is regarded essential for the reduction
of O
2
to H
2
O and for the disulfide reduction [22]. The
NoxA-4 from A. fulgidus (GI:11498556), a NADH oxidase
gene from P. horikoshii (GI:14590747), and three genes
from Sulfolobus solfataricus (GI:15899854, GI:15897884,
GI:15899669) do not contain this cysteine.
NoxB-1 belongs to a small group of 72-kDa proteins, of
which several are involved in the reduction of unsaturated
acids or aldehydes. For instance, enoate reductase (enr)
from Clostridium tyrobutyricum, 2,4 dienoyl-CoA reductase
(fadH) from E. coli, and NADH:flavin oxidoreductase

(baiH) from Eubacterium sp. strain VPI 12708, all perform a
NAD(P)H-dependent reduction of a carbon–carbon double
bond [23–25]. This group also contains the NADH oxidase
of T. brockii, but no physiological role has been ascribed to
it [7]. The alignment revealed potential ligands for an iron–
sulfur cluster and FAD or NAD(P) binding domains
(Fig. 2).
NoxC belongs to a group of small 20-kDa proteins, that
form H
2
O
2
instead of H
2
O. The NADH oxidases from
Thermus aquaticus and Thermus thermophilus also belong to
this group [7,8]. The physiological role of these enzymes is
not known.
Cloning and expression
All three nox genes gave gene products when expressed in
E. coli BL21 (DE3) as judged by SDS/PAGE (Fig. 3).
NoxA-1 gave a clear band of the expected size (48 kDa).
Expression of noxB-1 was less clear, but still visible on the
gel. The expected molecular mass based on the sequence of
NoxB-1 is 68 kDa. NoxC appeared as two proteins of
36 kDa and 20 kDa. The larger protein may represent
undenatured NoxC, because upon prolonged boiling in
SDS-sample buffer, it disappeared and the expected 20-kDa
protein increased (data not shown). Cell-free extracts of
E. coli producing NoxA-1 and NoxB-1 showed significant

NADH oxidase activity (see below). For NoxC, however,
no NADH oxidase activity was measured. Also, in the
presence of FMN instead of FAD, or NADPH instead of
NADH no NoxC activity was found For this reason,
further purification and characterization of NoxC was
abandoned.
Purification of the recombinant enzymes
NoxA-1 and NoxB-1 were purified essentially by the same
procedure. The first step that capitalized on their thermo-
stability was a heat treatment, resulting in the removal of
E. coli proteins. Whereas this treatment worked fine with
the NoxA-1, NoxB-1 remained contaminated with E. coli
proteins. Four subsequent chromatographic steps were
necessary to obtain a homogeneous preparation (Tables 1
and 2). Gelfiltration of NoxA-1 on Superdex 200 resulted in
two activity peaks, corresponding to molecular masses of
the native NoxA-1 of approximately 94 and 178 kDa,
which suggests that the NoxA-1 exists as dimer and to some
extent as tetramer. Most homologous NADH oxidases
from mesophiles (Enterobacter feacalis, Streptococcus
mutans and Serpulina hyodysenteria) are monomers or
dimers [3,21,26]. A NADH oxidase from P. furiosus,which
was recently described, also existed as dimer [10]. Gelfiltra-
tion of NoxB-1 resulted in a major activity peak with a
shoulder, corresponding to molecular masses of approxi-
mately 70 and 152 kDa. These data suggested that the
NoxB-1 exists as monomer and for a minor part as dimer.
Other homologous enzymes have been shown to have
different quaternary structures, like a trimer (Eubacterium
sp.), hexamer (T. brockii) or dodecamer (Clostridium

tyrobutyricum) [6,23,25].
Catalytic properties
The NADH oxidase activity of NoxA-1 and NoxB-1 was
stimulated upon addition of FAD (60 l
M
) to the assay
mixture. For NoxA-1 this stimulation was  2.5 fold, for
NoxB-1 3.7-fold. Addition of FMN instead of FAD did not
stimulate NADH oxidase activity. This result suggested that
both Nox enzymes contain FAD as prosthetic group, and
that part of the protein had apparently lost its cofactor.
Indeed, during the purification of especially NoxA-1, it was
observed that the yellow colour of the enzyme fractions
gradually disappeared. From the UV/visible spectrum of
2888 S. W. M. Kengen et al.(Eur. J. Biochem. 270) Ó FEBS 2003
NoxA-1 (and NoxB-1), with an absorbance maximum at
450 nm, it could be concluded that a flavin is present (data
not shown).
For NoxA-1 and NoxB-1 different pH optima of 8.0 and
6.5 were found, respectively (Fig. 4). At their pH optimum,
apparent V
max
values were found of 8.7 ± 0.5 UÆmg
)1
for
NoxA-1 and 1.5 ± 0.03 UÆmg
)1
for NoxB-1. The latter
activity was found to be strongly influenced by the
presence of mercaptans. Dithiothreitol (DTT; 2 m

M
)or
b-mercaptoethanol stimulated NoxB-1 activity up to two-
fold. In contrast, NoxA-1 activity was inhibited by DTT
(2 m
M
), e.g. in its presence the activity rapidly decreases to
<10% of the activity without DTT. The affinity for NADH
was the highest for NoxB-1 (apparent K
m
¼ 0.011 ±
0.001 m
M
) (Fig. 5). NoxA-1 showed a rather low affinity
for NADH (apparent K
m
¼ 0.13 ± 0.014 m
M
)compared
to NoxB-1 and to other thermoactive NoxA homologues
from P. furiosus (K
m
<4l
M
)orA. fulgidus (NoxA-2:
K
m
¼ 3.1 l
M
). NoxA-1 and NoxB-1 did not show activity

with NADPH.
For NoxA-1 an apparent K
m
for oxygen of 0.06 ± 0.03
was determined. The K
m
value for oxygen of NoxB-1
appeared to be much higher and for this reason difficult to
assess. The fit-program resulted in an apparent K
m
of
2.9 m
M
, far above the maximum dissolved oxygen concen-
tration (0.086 m
M
at a pO
2
of 0.2 10
5
Pa at 80 °C).
Fig. 2. Multiple alignment of NoxB-1 homologues. Conserved and moderately conserved residues are shaded black or grey. Putative NAD- or
FAD-binding motifs are boxed. Cysteine residues of a putative ferredoxin-like motif are indicated by arrowheads. The abbreviations used are as
follows (Genebank identifier in parentheses): Nox Tbro, NADH oxidase of Thermoanaerobacter brockii (GI:48123); DienoylCoA, 2,4-dienoyl-CoA
reductase of E. coli (GI:1176118); NADH:flav, NADH: flavin oxidoreductase of Eubacterium sp. (GI:416702); Enoate red, enoate reductase of
Clostridium acetobutylicum (GI:15026455); Nox Sso, NADH oxidase (SSO2025) of Sulfolobus solfataricus (GI:15898816).
Ó FEBS 2003 H
2
O
2

-forming NADH oxidases from Archaeoglobus fulgidus (Eur. J. Biochem. 270) 2889
Addition of EDTA to the assay mixture did not cause a
decrease in activity of NoxA-1 or NoxB-1, suggesting that
divalent cations are not required for activity.
Effect of temperature on activity
For NoxA-1and NoxB-1 an identical temperature optimum
of 80 °C was found, corresponding to the physiological
growth optimum of the organism (Fig. 6). Up to 70 °C, the
increase in NADH oxidase activity followed a linear
Arrhenius plot (ln k vs. 1/T; data not shown), from which
activation energies of 76.6 kJÆmol
)1
and 52.2 kJÆmol
)1
could be calculated for NoxA-1 and NoxB-1, respectively.
Despite the identical temperature optima, the thermostabi-
lity of both Nox enzymes at this temperature was consid-
erably different. Whereas NoxA-1 showed a half-life of
 40 h at 80 °C (data not shown), NoxB-1 showed a half-
life of only 40 min. For NoxB-1, the temperature stability
was investigated in the presence and absence of DTT. In
addition to the stimulating effect on the absolute activity,
DTT also raised the stability about twofold (half-life
83 min) (Fig. 7).
The product of the NADH oxidase reaction
NADH oxidases can perform the bivalent reduction of
oxygen to H
2
O
2

or the tetravalent reduction of oxygen to
H
2
O. Production of H
2
O
2
was tested by analysing the assay
mixture in a separate peroxidase assay, using horseradish
peroxidase and 3,3¢-dimethoxybenzidine as electron donor.
In the NoxA-1 assay between 71% and 95% of the amount
Fig. 3. SDS/PAGE of extracts of recombinant E. coli containing
NoxA-1, NoxB-1 or NoxC from A. fulgidus. M, Calibration proteins.
The molecular mass of the calibration proteins is indicated (kDa).
Table 1. Purification scheme of NoxA-1 from A. fulgidus. Activities were determined in phosphate/citrate buffer pH 8.0.
Total volume
(mL)
Protein
(mgÆmL
)1
)
Total protein
(mg)
Specific activity
(UÆmg
)1
)
Total activity
(U)
Purification

(fold)
Recovery
(%)
Crude extract 28 21.5 602 0.59 355.2 1.0 100
Heat-treatment 30.4 3.55 108 3.29 355 5.57 100
Q-sepharose 47.54 1.32 62.75 4.69 294 7.94 82.7
Hydroxyapatite 44.9 1.06 47.61 4.69 223.3 7.94 62.8
Macrosep 6.92 6.62 45.79 5.07 232 8.59 65.3
Superdex 200 93.38 0.298 27.83 5.82 161.9 9.86 45.6
Table 2. Purification scheme of the NoxB-1 from A. fulgidus. Activities were determined in phosphate/citrate buffer (pH 6.5).
Total volume
(mL)
Protein
(mgÆmL
)1
)
Total protein
(mg)
Specific activity
(UÆmg
)1
)
Total activity
(U)
Purification
(fold)
Recovery
(%)
Crude extract 28 20.84 583.5 1.139 664.6 1.0 100
Heat-treatment 28.5 6.16 175.56 2.35 412.56 2.06 62.1

Q-sepharose 60.16 2.09 125.7 2.52 316.8 2.21 47.7
Hydroxyapatite 83.8 0.67 56.13 3.43 192.5 3.01 29
Macrosep 7.74 8.2 63.5 2.27 144.16 1.99 21.7
Superdex 200 196.9 0.153 30.1 4.05 122 3.55 18.3
Fig. 4. pH dependence of purified NoxA-1 (s)andNoxB-1(d).
2890 S. W. M. Kengen et al.(Eur. J. Biochem. 270) Ó FEBS 2003
of NADH which was converted was recovered as H
2
O
2
.In
the NoxB-1 assay, this value amounted to 97%. FAD was
omitted from these assays, because unbound FAD may
facilitate H
2
O
2
production via nonenzymatic oxidation of
FADH
2
[27]. These results indicate that both enzymes
probably produce exclusively H
2
O
2
. The fact that the
recovery of H
2
O
2

was sometimes less than 100% (70–80%),
could be explained by the observation that the amount of
H
2
O
2
measured, was influenced by the time period between
the NADH conversion and the actual measurement of
H
2
O
2
. Apparently, the amount of H
2
O
2
in the assay mixture
slowly decreased, despite the absence of NADH, which was
already completely converted at that moment.
Electron acceptors other than oxygen
Because the physiological role of NoxA-1 and NoxB-1
is not known, it was of interest to test various electron
acceptors other than oxygen. The results of these assays,
which were performed in the absence of oxygen, are
summarized in Table 3. Flavines, like FAD, are known to
be able to react with various one- or two-electron acceptors.
In accordance, NoxA-1 and NoxB-1 show activity with
several of the e-acceptors tested. DCPIP, ferricyanide,
menadione and 2,3-dimethyl-1,4-naphthoquinone are
e-acceptors commonly used in the detection of membrane

bound dehydrogenases. However, the activities were rather
low (see Discussion). NoxA-1 also showed some activity
with a cytochrome c, but again the activity is low.
One of the homologues of NoxA-1 has been identified as
NADH peroxidase [4]. For this reason H
2
O
2
was tested as
electron acceptor under anoxic conditions. Neither NoxA-1
nor NoxB-1 showed convincing NADH peroxidase activity.
Another NoxA-1 homologue has recently been recog-
nized as CoA disulfide reductase, an enzyme that performs a
Fig. 5. Rate dependence of NoxA-1 and NoxB-1 on the NADH con-
centration. Data points were fitted according to the Michaelis–Menten
equation.
Fig. 6. Temperature dependence of purified NoxA-1 (s) and NoxB-1
(d).
Fig. 7. Thermal stability of NoxB-1. The purified enzyme was incu-
bated at 80 °Cin100 m
M
phosphate buffer pH 7.0 in the presence (d)
and absence (s)of2m
M
DTT.
Table 3. Specific activity of NoxA-1 and NoxB-1 with different electron
acceptors.
Presence
of FAD
a

NoxA-1
(UÆmg
)1
)
NoxB-1
(UÆmg
)1
)
Oxygen + 5.8 4.1
H
2
O
2
+/– 0 0
DCPIP – 4.2 1.0
Ferricyanide – 5.8 1.8
Menadione – 1.29 1.93
2,3-Dimethyl-1,4-naphthoquinone – 1.56 1.29
Cytochrome c – 0.03 0
Benzyl viologen + 4.5 1.3
DNTB + 3.7 0.52
Coenzyme A (oxidized) +/– 0 0
Glutathione (oxidized) +/– 0 0
Cystine +/– 0 0
Polysulfide + 0 0.65
b
Tiglic acid +/– NT 0
Cinnamic acid +/– NT 0
Crotonate +/– NT 0
Fumarate +/– NT 0

a
Activity was determined in the presence (+) or absence (–) of
FAD. +/–, Substrates were tested with and without FAD.
b
Only
in 100 m
M
Tris/CL buffer pH 7.8 at 60 °C. NT, not tested; tiglic
acid, trans-2-methyl-2-butenoic acid; cinnamic acid, 3-phenyl-
2-propenoic acid; crotonate, 2-butenoate; menadione, 2-methyl-
1,4-naphthoquinone.
Ó FEBS 2003 H
2
O
2
-forming NADH oxidases from Archaeoglobus fulgidus (Eur. J. Biochem. 270) 2891
disulfide reductase activity via a single cysteine residue [5].
Forthisreason,NoxA-1aswellasNoxB-1weretested
using DTNB as e-acceptor. An activity of 3.68 UÆmg
)1
was
determined for NoxA-1. NoxB-1 also showed a significant
disulfide reductase activity of 0.52 UÆmg
)1
. Both activities
were determined in the presence of FAD. In the absence of
FAD, the reduction of DTNB was substantially less. Other
disulfides of more physiological nature like oxidized Coen-
zyme A, glutathione or cystine did not cause a NADH
oxidation in the absence of oxygen, nor did they stimulate

the DTNB reduction.
A special type of electron acceptor tested here was
polysulfide. Polysulfide is a soluble form of elemental sulfur,
which has been shown to act as electron acceptor by various
hyperthermophiles. NoxB-1, but not NoxA-1, showed a
significant NADH-dependent polysulfide reductase activity
of 0.65 UÆmg
)1
. However, this activity is again low when
compared to other polysulfide reductases, like the NADPH-
dependent sulfide dehydrogenase (7.0 UÆmg
)1
)from
P. furiosus [28].
Because NoxB-1 showed homology to enzymes involved
in the reduction of unsaturated acids or aldehydes (Fig. 2),
a few model substrates were tested as potential electron
acceptors (Table 3). However, none of these caused an
oxidation of NADH when added to the anoxic reaction
mixture.
Discussion
The noxA-1 and the noxB-1 gene from the hyperthermo-
philic archaeon A. fulgidus were successfully cloned and
expressed in E. coli. The alignment to homologous genes in
the database revealed that NoxA-1 and NoxB-1 belong to
different phylogenetic groups (Fig. 1). Nevertheless, the
similarity to other NADH oxidase gene sequences does not
simply lead to their physiological function. For instance,
NoxA-1 belongs to the family of pyridine nucleotide
disulfide oxidoreductases (Pfam), which contains enzymes

that may function as NADH oxidase, NADH peroxidase
or as CoA disulfide reductase. The various electron
acceptors tested here did not indicate an obvious function
(Table 3). The reduction of DCPIP and ferricyanide
suggests that NoxA-1 may have a role as NADH
dehydrogenase as part of the electron transport chain for
sulfate reduction. Moreover, it has recently been suggested
that NoxA-2 of A. fulgidus may also function in electron
transport for sulfate reduction, because the enzyme copu-
rified with
D
-lactate dehydrogenase and both enzymes
colocalized to the periplasmic side of the membrane [9,30].
However, the activities found here for NoxA-1 towards
menadione, 2,3-dimethyl-1,4-naphthoquinone and cyto-
chrome c are rather low, and thus do not support this
hypothesis. For example, the F
420
H
2
: quinone oxido-
reductase from A. fulgidus showed specific activities of
96 UÆmg
)1
and 92 UÆmg
)1
with 2,3-dimethyl-1,4-naphtho-
quinone and menadione, respectively [29]. A novel type
menaquinone, present in the membrane fraction of
A. fulgidus, probably acts as the physiological electron

acceptor [31].
NoxA-1 showed substantial disulfidereductase activity
(3.7 UÆmg
)1
), but this DNTB-reducing activity was not
stimulated by disulfides like oxidized coenzyme A, gluta-
thione or cystine. The activity was, however, strongly
stimulated upon addition of FAD, which indicated that free
FAD was involved in the reduction of DNTB. This suggests
that the observed disulfide reduction may not be the
physiological role of NoxA-1. Moreover, when we compare
the disulfide reductase activity of NoxA-1 with that of a true
disulfide reductase (CoA disulfide reductase from Staphylo-
coccus aureus; Spec. activity ¼ 4570 UÆmg
)1
), the latter is at
least 1000-fold more active [5]. In this respect we also tested
whether NoxA-1 exhibited polysulfide reductase activity.
Polysulfide is a soluble form of sulfur consisting of
predominantly tetrasulfide (S
4
2–
) and pentasulfide (S
5
2–
),
and it is assumed that an S–S bond is cleaved similar to the
disulfide reduction. This activity was of special interest
because a similar NADH oxidase (NoxA-2) of P. furiosus
was shown by DNA microarray analysis to be strongly

up-regulated (7.4-fold) when cells were grown in the
presence of sulfur [32]. The expression of two other ORFs
in the P. furiosus genome increased more than 25-fold, and
their products termed SipA and SipB are proposed to be
part of an S-reducing protein complex. Although A. fulgidus
is not able to grow by sulfur reduction, its genome contains
homologues of the SipA and SipB encoding genes. Unfor-
tunately, NoxA-1 did not show polysulfide reductase
activity. Nevertheless, the similarity to the S-upregulated
NoxA-2 of P. furiosus and to the membrane associated
NoxA-2 of A. fulgidus, suggests some respiratory role.
Alternatively, the function of NoxA-1 may actually be
that of an NADH oxidase, using the reducing power of
NADH to remove traces of oxygen that otherwise may
lead to harmful oxygen species like O
2
–
,H
2
O
2
,orOHÆ.
The K
m
for oxygen of NoxA-1 is  60 l
M
, which is not
very low compared to the amount of oxygen that can
maximally dissolve at 80 °C(102l
M

at ambient oxygen
concentrations and average marine salinity). However,
because A. fulgidus is a strict anaerobe, it will most likely
have to deal with much lower oxygen concentrations. A
role as detoxicant has also been proposed for the NADH
oxidase (NOX1) from the hyperthermophile P. furiosus
[10]. The affinity for oxygen of the latter enzyme,
however, is even lower (a K
m
of at least 110 l
M
has
been reported), which makes this enzyme also not very
efficient if it is assumed to remove small amounts of
oxygen in an anaerobic environment. Unfortunately,
oxygen affinity data of true NADH oxidases are not
available in the literature, making a comparison impos-
sible. The most plausible argument against a role as
detoxicant, however, is the fact that the NoxA-1 and also
theenzymefromP. furiosus produce predominantly
H
2
O
2
. Thus, instead of preventing oxidative stress through
O
2
removal, these Nox enzymes aggravate the problem by
producing H
2

O
2
. On the other hand, H
2
O
2
which is
produced by the Nox, may be converted further by a
catalase-peroxidase, which has also been demonstrated in
A. fulgidus [33]. But in this case H
2
O
2
is converted back to
O
2
, which combined with the NADH oxidase lowers the
amount of oxygen by only 50%. The K
m
for oxygen of
NoxB-1 is even much higher ( 3m
M
), making a role as
oxygen detoxifying system very unlikely. Moreover, also
NoxB-1 produces H
2
O
2
instead of H
2

O.
In a recent paper Abreu et al. describe a superoxide
scavenging system in A. fulgidus, and propose that
2892 S. W. M. Kengen et al.(Eur. J. Biochem. 270) Ó FEBS 2003
NAD(P)H oxidases may have a role in oxygen detoxifica-
tion, not by directly reducing oxygen, but via intermediate
redox enzymes like rubredoxin and neelaredoxin [34]. A
similar system has been proposed for Desulfovibrio gigas
[35]. This hypothesis certainly deserves further attention, but
requires purification of the rubredoxin and neelaredoxin.
The production of H
2
O
2
is in contrast with the data on
mesophilic homologues of NoxA-1 or NoxB-1, which all
produce H
2
O. Also the NoxA-2 from A. fulgidus,the
NADH oxidase from P. furiosus,andtheNADHoxidase
from T. brockii have been demonstrated to form H
2
O
2
instead of the usual H
2
O [9,10]. Thus, possibly the
production of H
2
O

2
is a thermophilic feature. It has been
put forward that reduction of oxygen to H
2
O
2
may be an
artefact, because in anaerobes the flavin moiety of flavo-
proteins is exposed to the solvent and can easily transfer
electrons to oxygen to form H
2
O
2
. In aerobes the flavin is
protected from this unwanted oxygen reduction, because
the flavin is buried in the protein. Remarkably, for the
NADH oxidase from P. furiosus only 61% of the NADH
was recovered as H
2
O
2
(NADH/H
2
O
2
ratio of 0.61),
suggesting that the enzyme produced both H
2
O
2

and H
2
O
[10]. Occasionally, we also found <100% recovery of H
2
O
2
compared to NADH, but this could be diminished by
shortening the time period between the Nox assay and the
H
2
O
2
-assay. Possibly, this also applies for the enzyme from
Pyrococcus.
Concerning the physiological role of the NoxA-1, the
direct neighbourhood of noxA-1 inthegenomewas
investigated. A CTP synthase, a GMP synthase and several
hypothetical proteins accompany noxA-1, which do not
provide insight as to the physiological role of the NoxA-1. A
STRING
analysis [36] of the surrounding genes, however,
reveals that all NoxA and NoxB homologues have a
predicted redox protein, regulator of disulfide bond forma-
tion (COG0425) in their neighbourhood. This COG belongs
to a functional category involved in post-translational
modification, protein turnover, and chaperones, which also
does not reveal a physiological role of the Nox enzymes.
Recent experiments by Pagala et al. [30] have shown that
whole cell extracts of A. fulgidus exhibit multiple NADH

oxidase activities, as judged by renatured SDS/PAGE gels.
It was concluded that the majority of the Nox enzymes
in A. fulgidus are expressed constitutively under strictly
anaerobic conditions. The fact that the expression of the
Nox enzymes is not regulated, also suggests that they have
some fundamental metabolic role, and not an occasional
role during oxygen stress.
NoxB-1 shows homology to a small group of enzymes
that is involved in the reduction of unsaturated acids or
aldehydes. For instance, enoate reductase (enr) from
Clostridium tyrobutyricum, 2,4 dienoyl-CoA reductase
(fadH) from E. coli, and NADH:flavin oxidoreductase
(baiH) from Eubacterium sp. strain VPI 12708, all perform a
NAD(P)-dependent reduction of a carbon–carbon double
bond [23–25]. However, several commonly used unsatur-
ated compounds, like tiglic acid, cinnamic acid or crotonate
did not show any activity when tested with NoxB-1.
Possibly, the enzyme requires CoA-activated unsaturated
compounds, which have not been tested here. As mentioned
above, the adjacent genes of NoxB-1 do not reveal any
information concerning its function. On the other hand, the
gene encoding NoxB-2 of A. fulgidus, which is 98.9%
identical to NoxB-1, lies upstream of a gene encoding a
medium-chain acyl-CoA ligase, suggesting a role in fatty
acid and phospholipid metabolism.
Thus, despite an extensive analysis of the catalytic
capabilities of NoxA-1 and NoxB-1, no obvious physio-
logical role can be ascribed to them. Further studies, for
instance using Northern blots or DNA microarrays may
indicate conditions at which the enzymes are expressed and

thereby unveil their cellular function.
Because both NADH oxidases produce H
2
O
2
instead of
H
2
O, they may find application in biosensors as mediator
between a dehydrogenase and the electrode. For this
purpose the enzymes should have sufficient stability and
appropriate kinetics. Although, the stability of NoxB-1 is
considerably lower than that of NoxA-1 (stable at 80 °Cfor
1 h and 40 h, respectively), the stability is likely to be
sufficient at more moderate temperatures. Concerning the
catalytic activity, k
cat
/K
m
values of 0.053 · 10
6
M
)1
Æs
)1
and
0.156 · 10
6
M
)1

Æs
)1
can be calculated for NoxA-1 and
NoxB-1, respectively. These catalytic efficiencies are sub-
stantially lower than the value found for the NADH oxidase
of Thermus thermophilus, which was determined at room
temperature (k
cat
/K
m
¼ 1.250 · 10
6
M
)1
Æs
)1
)[37].Thus,
compared to the latter enzyme, which also forms H
2
O
2
and which is also reasonably stable, NoxA-1 and NoxB-1
are less suited for biosensor application.
Acknowledgements
This work was partly funded by the European Community under the
Industrial & Materials Technologies Programme (Brite-Euram III)
(Contract BRPR-CT97-0484).
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