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

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

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

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

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

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

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,

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

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

(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

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

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

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

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

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

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

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

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

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

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

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

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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