Báo cáo khoa học: Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2O pot

Hexameric ring structure of a thermophilic archaeon
NADH oxidase that produces predominantly H
2
O
Baolei Jia
1,
*, Seong-Cheol Park
1,
*
,
, Sangmin Lee
1
, Bang P. Pham
1
, Rui Yu
1
, Thuy L. Le
1
, Sang
Woo Han
2,3
, Jae-Kyung Yang
4
, Myung-Suk Choi
4
, Wolfgang Baumeister
5
and Gang-Won Cheong
1,3
1 Division of Applied Life Sciences (BK21 Program), Gyeongsang National University, Jinju, Korea
2 Department of Chemistry, Research Institute of Natural Science, Gyeongsang National University, Jinju, Korea

3 Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Korea
4 Division of Environmental Forest Science and Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Korea
5 Department of Molecular Structural Biology, Max-Planck-Institute for Biochemistry, Martinsried, Germany
Thermococcus profundus is a thermophilic anaerobic
archaeon belonging to the Thermococcaceae family
that also includes Thermococcus kodakaraensis KOD1,
a model thermophilic organism whose whole genome
sequence has been reported [1]. As an anaerobe living
in deep-vent environments, it seems likely that Thermo-
coccus encounters high levels of oxygen stress in the
water surrounding the vent [2]. In anaerobes, flavin-
dependent NAD(P)H oxidases play an important role
protecting organisms from oxidative stress [3].
NADH oxidase (NOX) is a member of the flavo-
protein disulfide reductase family that catalyzes the
pyridine-nucleotide-dependent reduction of various
substrates, including O
2
,H
2
O
2
and thioredoxin [4].
There are two types of NOX: those that catalyze the
two-electron reduction of O
2
to H
2
O
2

and those that
catalyze the four-electron reduction of O
2
to H
2
O [5].
The physiological role of NOX is diverse, depending
on its substrates and products in different organisms.
In anaerobic mesophiles, NOX enzymes, such as those
Keywords
electron microscopy; H
2
O-producing;
hexameric ring structure; NADH oxidase;
thermophilic archaeon
Correspondence
G W. Cheong, Division of Applied Life
Sciences, Gyeongsang National University,
Jinju 660-701, Korea
Fax: +82 55 752 7062
Tel: +82 55 751 5962
E-mail:
Present address
Research Center for Proteineous Materials,
Chosun University, Kwangju 501-759, Korea
*These authors contributed equally to this
work
(Received 7 August 2008, revised 31 August
2008, accepted 3 September 2008)
doi:10.1111/j.1742-4658.2008.06665.x

An NADH oxidase (NOX) was cloned from the genome of Thermococ-
cus profundus (NOXtp) by genome walking, and the encoded protein was
purified to homogeneity after expression in Escherichia coli. Subsequent
analyses showed that it is an FAD-containing protein with a subunit
molecular mass of 49 kDa that exists as a hexamer with a native molecular
mass of 300 kDa. A ring-shaped hexameric form was revealed by electron
microscopic and image processing analyses. NOXtp catalyzed the oxidiza-
tion of NADH and NADPH and predominantly converted O
2
to H
2
O, but
not to H
2
O
2
, as in the case of most other NOX enzymess. To our knowl-
edge, this is the first example of a NOX that can produce H
2
O predomi-
nantly in a thermophilic organism. As an enzyme with two cysteine
residues, NOXtp contains a cysteinyl redox center at Cys45 in addition to
FAD. Mutant analysis suggests that Cys45 in NOXtp plays a key role in
the four-electron reduction of O
2
to H
2
O, but not in the two-electron
reduction of O
2

to H
2
O
2
.
Abbreviations
CoADR, coenzyme A disulfide reductase; GR, glutathione reductase; Nbs
2
, 5,5¢-dithiobis-(2-nitrobenzoic acid); NOX, NADH oxidase
(EC 1.6.99.3); NOXtp, Thermococcus profundus NADH oxidase.
FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5355
of Clostridium aminovalericum [6], Enterococcus (Strep-
tococcus) and Lactococcus [3], are considered to be
important enzymes in protecting against oxidative
stress and in regenerating oxidized pyridine nucleo-
tides through their capacity to reduce O
2
to H
2
O
without the formation of harmful reactive oxygen spe-
cies. Some NOX proteins have also been purified and
studied in (hyper)thermophilic organisms. NOX from
Archaeoglobus fulgidus may be involved in electron
transfer in sulfate respiration [7]. An H
2
O
2
-forming
NOX functions as an alkyl hydroperoxide reductase in

Amphibacillus xylanus [8]. Some NOX enzymes, such
as those of Pyrococcus furiosus [9] and Thermotoga
maritima [10], have been proposed to protect anaer-
obes from oxidative stress. In (hyper)thermophiles,
the roles of some NOX enzymes remain to be
elucidated [11].
NADH oxidase varies with the organism; however,
these proteins generally share similar secondary struc-
tural folding [4,12]. An NOX from Thermus thermophi-
lus is a homodimer as determined by X-ray
crystallography [13]. Gel filtration chromatography
indicated that NADH:flavin oxidoreductase from
Eubacterium is composed of three identical subunits
[14]; NOX in Clostridium thermohydrosulfuricum is
probably made up of six subunits, as demonstrated by
gel filtration [15]. In contrast, a heterogeneous NOX
from Eubacterium ramulus is proposed to have an a
8
b
4
assembly, as revealed by gel filtration and PAGE [16].
Two NOX enzymes from the Thermococcaceae fam-
ily have been described. One is a novel enzyme in
P. furiosus that produces both H
2
O
2
(77%) and H
2
O

(23%) [9]. The other NOX, in Pyrococcus horikoshii
OT3, may function as a CoA disulfide reductase (CoA-
DR) [17]; however, the function and structure of NOX
in Thermococcus, a genus of the Thermococcaceae fam-
ily, has not been clarified. In this study, we have
cloned, overexpressed and purified a NOX that is com-
posed of two cysteine moieties from T. profundus.We
report its biochemical characterization and structure,
and also used mutants to analyze its catalytic mecha-
nism.
Results
Cloning and sequencing of the nox gene from
T. profundus
In order to clone the T. profundus nox gene (NOXtp),
we utilized a PCR-based DNA-walking method using
the ClonTech genome-walker cloning kit, as described
in Experimental procedures; the resulting DNA
sequence comprised an ORF of 1329 bp, predicting a
protein composed of 442 amino acids with a molecular
mass of 48 611 Da. Figure 1 shows the nucleotide
sequence of NOXtp and its flanking regions, together
with the translated amino acid sequence. The 5¢-flank-
ing region of NOXtp contained a putative Archaea
promoter with a TATA box and ribosome-binding site.
The 3¢-flanking region did not match with other
Archaea genes, as judged by homolog searches in the
NCBI database. Unlike NOX, which has only one
conserved cysteine residue (Cys45) in its N-terminus
[4], the amino acid composition of NOXtp revealed
the presence of two cysteine residues, Cys45 and

Cys122. Additionally, two conserved cofactor-binding
domains were also identified in NOXtp. One was a
FAD-binding domain containing the AMP-binding
and FMN-binding motifs observed in enzymes belong-
ing to the glutathione reductase (GR) family [18]. The
other domain was a glycine-rich NAD-binding motif
located between the AMP-binding and FMN-binding
motifs (two FAD-binding domains) (Fig. 1). We pro-
pose that NOXtp belongs to the GR family, because
of the high sequence identity of the cofactor-binding
domains described above.
Multiple sequence alignment (Fig. S1) revealed that
Cys45 is located at a similar position to that of the
cysteine residue in the conserved active site of NOX
from P. horikoshii (also called CoADR) [17] and NOX
and NADH peroxidase from Enterococcus faecalis
[19,20]. Sequence analysis by clustal w showed that
NOXtp shared a significant level of identity with NOX
(CoADR) from P. horikoshii (80%) [17], NADH per-
oxidase from E. faecalis (28%) [20], and NOX from
P. furiosus (36%) [9], Lactococcus lactis (30%) [21],
Lactococcus sanfranciscensis (26%) [11] and E. faecalis
(27%) [19] (Fig. S1). These proteins are generally com-
posed of two identical subunits related by two-fold
symmetry. Each subunit can be divided into a C-termi-
nal dimerization domain and an N-terminal pyridine
nucleotide disulfide oxidoreductase domain, which is
actually a small NADH-binding domain with a large
FAD-binding domain [4,12]. NOXtp has similar
primary structure architecture to these proteins as

determined by NCBI protein blast analysis.
Purification of native and recombinant NOXtp
In order to understand the oxygen detoxification mecha-
nism of anaerobic microbes, we purified NOXtp from
T. profundus by several chromatographic methods. The
purified protein revealed a subunit with a molecular
mass of approximately 50 kDa (Fig. S2). The N-termi-
nal amino acid sequence of purified NOXtp from T. pro-
fundus was determined to be MERKRVVIIGGGAAG,
Hexameric thermophilic NADH oxidase-producing H
2
O B. Jia et al.
5356 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS
which is highly similar to that of NOX in T. kodakaren-
sis KOD1, P. furiosus, Pyrococcus abyssi and P. horiko-
shii OT3, belonging to the pyridine nucleotide disulfide
oxidoreductase family. The purification of recombinant
NOXtp from Escherichia coli was performed by ion
exchange chromatography as described in Experimental
procedures. SDS ⁄ PAGE analysis of recombinant
NOXtp revealed a molecular mass of approximately
50 kDa, which is close to that of the purified protein
from T. profundus (Fig. S2). However, gel filtration
analysis under nondenaturing conditions showed that
the purified NOXtp had a molecular mass of
approximately 300 kDa (Fig. 2). These results indicated
that NOXtp is a hexamer of 50 kDa subunits, in
contrast to NOX proteins from thermophilic archaeans,
which have been reported to be dimers or tetramers
[13,17].

Structure of NOXtp
Gel filtration analysis under nondenaturing conditions
revealed that purified NOXtp has a molecular mass
of  300 kDa, corresponding to a hexamer with
50 kDa subunits (Fig. 2). This structure is different
from that of other homologous NOX proteins, which
consist of dimers or tetramers as revealed by X-ray
crystallographic studies [12,13,22]. In order to clarify
the oligomeric structure of NOXtp, we performed
electron microscopy using purified NOXtp. The elec-
tron micrographs of the negatively stained NOXtp
oligomers showed a uniform distribution of the ring-
shaped structure in the top-view orientation
(Fig. 3A). In total, 939 well-stained particles were
translationally aligned, and were subjected to multi-
variate statistical analysis [23]. The eigenimages
Fig. 1. Nucleotide sequence of the noxtp
gene and predicted amino acid sequence
of the gene product from Thermo-
coccus profundus. The putative TATA-box
and ribosome-binding site are underlined
and in bold letters, respectively. The resi-
dues involved in FAD binding are shadowed
in gray. The NAD-binding site is boxed. The
cysteine residues are in bold italic.
B. Jia et al. Hexameric thermophilic NADH oxidase-producing H
2
O
FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5357
obtained from the translationally, but not rotation-

ally, aligned images revealed a six-fold rotational
symmetry (Fig. 3Ba). Using the 10 most significant
eigenvectors, nine classes were discriminated on the
basis of similarity of features after rotational align-
ment without symmetrization. Most class averages
showed a star-shaped structure with six-fold symme-
try with heavy stain accumulation in its center
(Fig. 3Bb). In particular, the two class averages
shown in panels 3 and 6 of Fig. 3Bb exhibited an
obvious deviation from the star-like structure. This
could result from incomplete stain embedding of the
particle or from an unintentional inclination during
preparation or microscopy. In order to analyze
further the rotational symmetry of the top-on-view
images, the same dataset was separated into many
classes (10–30) using different eigenimages (10–20).
The dataset was also aligned with an arbitrarily
chosen reference and separated according to the simi-
larity of features in the eigenimages. The resulting
class averages revealed no other statistically signifi-
cant symmetry (data not shown). We found no
Fig. 2. Gel filtration chromatography profile of NOXtp purified from
Es. coli. The purified protein was subjected to Superdex-200 gel fil-
tration chromatography. Absorbance was measured at 280 nm. The
x-axis shows the elution time. The standard proteins are ferritin
(440 kDa), catalase (232 kDa), albumin (67 kDa) and ovalbumin
(43 kDa).
A
B
a

C
D
b
Fig. 3. Electron micrograph and structural
analysis of NOXtp. (A) Purified NOXtp was
absorbed onto the grids as described in
Experimental procedures. The electron
micrograph of the protein was then obtained
by negative staining with 2% uranyl acetate.
(B) Multivariate statistical analysis of NOXtp.
(a) The average (AV) of 939 translationally,
but not rotationally, aligned particles with
end-on orientation and the 10 most signifi-
cant eigenimages (numbers 1–10) are
shown. In (b), the nonsymmetrized class
averages (numbers 1–9) were derived from
rotationally aligned images using the 10
most significant eigenvectors. The numerals
shown in the top right corner of the class
averages are the number of particles seen
in each class. (C) The average of the side-on
view of NOXtp (939 particles). (D) A sche-
matic model for the assembly of NOXtp
complexes. The diameters of the cavity,
middle ring and outer ring are 4, 15 and
19 nm, respectively.
Hexameric thermophilic NADH oxidase-producing H
2
O B. Jia et al.
5358 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS

evidence for the existence of a NOXtp protein with
intrinsically lower symmetry, at least at the resolution
employed.
The average of the 939 top-on views revealed a star-
shaped structure (Fig. 3C), which contained a middle
region of high density with heavy stain accumulation
in its center. The average view also revealed that the
density of the complex was not homogeneous; the den-
sity increased towards the middle, such as seen in the
valosine-containing protein-like ATPase from Thermo-
plasma acidophilum complex, which is composed of
two stacked ring structures of different diameters [24].
The upper (or middle) ring of the NOXtp complex has
a region that is denser than that of the outer ring, indi-
cating the presence of a cavity in the complex with a
width of approximately 4 nm. The diameters of the
outer ring and the middle ring (Fig. 3D) were approxi-
mately 19 and 15 nm, respectively. These projected
images as well as the gel filtration analysis indicated
that NOXtp predominantly exhibits a hexameric star-
shaped structure, in contrast to the structure recently
reported by Kuzu et al. [22], which suggested a tetra-
meric structure for NOX from Lactobacilluus brevis.
NOXtp is an FAD-dependent NADH and NADPH
oxidase
On the basis of the amino acid sequence, NOXtp con-
tains two FAD-binding domains. The isoalloxazine
ring system in FAD has been suggested to induce light
absorbance in the UV and visible spectral range, giving
rise to the yellow appearance of flavin and flavopro-

teins [25]. We performed light absorbance analysis to
confirm NOXtp binding to FAD. Purified NOXtp
from Es. coli has absorption maxima at 378 and
456 nm, with a shoulder at 480 nm, which are charac-
teristic spectral features of proteins with bound flavin
cofactors (Fig. 4A). The absorbance behavior also
allowed the determination of the number of flavin mol-
ecules bound per mole of NOXtp subunit [17,25,26]. A
stoichiometry of 0.7–0.9 mol FAD per mol NOXtp
subunit was determined from the absorbance at
460 nm.
As NOXtp contains FAD as a prosthetic group,
apo-NOXtp was prepared by hydrophobic interaction
chromatography under acidic conditions (pH 3.5)
with saturated NaBr buffer [26,27], in order to
0
0.04
0.08
0.12
0.16
0.2
300
400 500 600 700 800 (nm)
Absorbance
0
10
20
30
40
50

60
70
80
90
100
0 2 4 6 8 10 12 (min)
NAD(P) amount (μM)
0
1
2
3
4
5
6
7
8
20 30 40 50 60 70 80 90 100 (ºC)
Specific activity (U·mg
–1
)
0
1
2
3
4
5
6
7
8
Specific activity (U·mg

–1
)
5678910 (pH)
A
B
C
D
Fig. 4. Activity assays of NADH and NADPH oxidase. (A) Visible
spectra of NOXtp (solid line), apo-NOXtp (dashed line) and the
C45A mutant (dotted line). The absorbance was measured in
50 m
M sodium phosphate buffer (pH 7.2) at 25 °C. (B) FAD effect
on NAD(P)H oxidase activity. An activity assay was performed as
described in Experimental procedures. The solid line shows the
NADH oxidase activity of NOXtp purified from Es. coli (h), reconsti-
tuted NOXtp (s), and apo-NOXtp (4). The dashed line shows the
NADPH oxidase activity of NOXtp from Es. coli (h), reconstituted
NOXtp (s), and apo-NOXtp (4). (C) Optimal temperature of NAD(P)H
oxidase activity. The assay was performed at the indicated temper-
atures in 50 m
M potassium phosphate buffer (pH 7.2). NADH and
NADPH oxidase activity are shown by a solid line and a dashed
line, respectively. The squares show the measured temperature
points. (D) Optimal pH of NAD(P)H oxidase activity. Different buffers
were used in this assay. Sodium phosphate was used at pH 6.0,
6.6, 7.2 and 7.7; Hepes buffer and Tris buffer were used at pH 8.0
and 8.5; sodium borate buffer was used at pH 9.0. These buffers
were used at a concentration of 50 m
M. NADH and NADPH oxi-
dase activity are shown by a solid line and a dashed line, respec-

tively. The squares show the measured pH points.
B. Jia et al. Hexameric thermophilic NADH oxidase-producing H
2
O
FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5359
confirm the function of FAD. The absorption spec-
trum of apo-NOXtp did not show any significant
absorbance in the visible region, revealing that FAD
was indeed absent (Fig. 4A). To determine whether
FAD was required for the enzymatic activity of
NOXtp, holoprotein and apoprotein activities were
assayed. The NADPH oxidase activity of NOXtp
was also measured, as described previously for NOX
(CoADR) from P. horikoshii [17] and NOX from
L. sanfranciscensis [12], which show high similarity to
NOXtp and accept both NADH and NADPH as
cofactors. The activity of the reconstituted enzyme,
which was accomplished by incubating equimolar
concentrations of apomonomers and FAD at room
temperature for 5 min [26], was also measured. These
assays revealed that NADH oxidase activity was
slightly higher than that of NADPH oxidase, and
FAD significantly restored the oxidase activity of
apo-NOXtp (Fig. 4B). These results clearly indicated
that NOXtp is an FAD-dependent NADH and
NADPH oxidase, in contrast to NOX enzymes from
other thermophilic archaeons, which only exhibit
activity towards NADH [9–11].
To further determine the function of NOXtp, the
steady-state kinetic parameters of NOXtp with either

NADH or NADPH as the reducing substrate were
measured at pH 7.2. NOXtp could catalyze NADH
and NADPH oxidization with k
cat
values of
6.2 ± 0.5 ⁄ s and 2.5 ± 0.3 ⁄ s, respectively. The steady-
state kinetic parameters of NOXtp were similar to
those of NOX (CoADR) from P. horikoshii (Table 1).
On the basis of the K
m
, both enzymes preferred
NADPH as the substrate for oxidase activity, indicat-
ing that NOX (CoADR) from P. horikoshii and NOX-
tp belong to similar enzyme families. The optimal
temperature for the NADH and NADPH oxidase
activity of NOXtp was near 70 °C (Fig. 4C), which is
lower than the optimal growth temperature (80 °C) of
this organism. The optimal pH was between 7.5 and
8.0 for both NADH and NADPH oxidase activity
(Fig. 4D).
NOXtp preferentially produces H
2
O
The product of O
2
reduction is an important factor in
evaluating the physiological function of NOXs [10].
For instance, NOX from P. furiosus, which may pro-
tect anaerobic thermophiles against oxidative stress,
can produce both H

2
O
2
and H
2
O [9]. In order to
determine the product of the NAD(P)H oxidase activ-
ity of NOXtp, reactions containing 100 lm NAD(P)H
were performed [all NAD(P)H consumed] according to
the published method [9], and H
2
O
2
was quantified
using a peroxi-DETECT kit from Sigma (St Louis,
MO, USA). When NADPH oxidation was performed
at 80 °C, approximately 7% of the NADPH supplied
was used to produce H
2
O
2
, and 2% of the NADH was
recovered as H
2
O
2
under the same conditions
(Fig. 5D). These results demonstrated that NOXtp
produces predominantly H
2

O using NADH and
NADPH as electron donors.
Cys45 but not Cys122 functions as the nonflavin
redox center
NADH oxidase in members of the Thermococcaceaee
family, such as T. kodakaraensis KOD1, P. horrikoshii,
P. abyssi and P. furiosus, have only one conserved cys-
teine residue, Cys45; however, the sequence of NOXtp
(Fig. 1) revealed that it contains two cysteine residues,
Cys45 and Cys122. As cysteines are important residues
for NOX enzyme activity, we replaced Cys45 and
Cys122 with alanines to analyze the function of these
two residues. After purification using the same method
as that used for the wild-type enzyme, the number of
cysteines in the three mutant enzymes (NOXtpC45A,
NOXtpC122A and NOXtpC45A⁄ C122A) was exam-
ined using Ellman’s method (Table 2). The single
mutants, NOXtpC45A and NOXtpC122A, contained
about one cysteine, and the double mutant contained
no cysteines. These data confirmed the identity of the
mutants and also indicated that the nonmutated cyste-
ine remained in its native state. The visible absorption
spectra showed that the three mutants contained
tightly bound FAD (Fig. 4A, NOXtpC45A only
shown – the other two mutants produced similar
absorbance spectra). Electron microscopy and native
PAGE showed no significant difference between wild-
type NOXtp and its mutants (Fig. 5A,B). All of the
data indicated that the disulfide bond was not respon-
sible for hexameric oligomerization and that substitu-

tion of Cys45 and Cys122 with alanine did not result
in major changes in NOXtp quaternary structure.
In order to determine the catalytic mechanism of
NOXtp, NAD(P)H oxidase assays were performed
Table 1. Steady-state kinetic parameters of NOXtp and NOX (CoA-
DR) from Pyrococcus horikoshii (50 m
M potassium phosphate buf-
fer, pH 7.2, 75 °C). Data shown are means of triplicate
determinations ± SD.
Parameter
NOXtp-
NADH
oxidase
NOXtp-
NADPH
oxidase
CoADR-
NADH
oxidase
CoADR-
NADPH
oxidase
K
m
(lM) 53.1 ± 2.8 12.1 ± 1.1 73
a
13
a
k
cat

(s
)1
) 6.2 ± 0.5 2.5 ± 0.3 8.2
a
2.0
a
a
From reference [17].
Hexameric thermophilic NADH oxidase-producing H
2
O B. Jia et al.
5360 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS
with the three mutants under the same conditions as
used for the wild-type. The results showed that the
C122A mutant had similar NADH and NADPH oxi-
dase activity to that of the wild-type protein; however,
the C45A mutant and the double C45A ⁄ C122A
mutant had < 10% of the NAD(P)H oxidase activity
of the wild-type protein (Fig. 5C). These results are
similar to those obtained with a NOX from E. faecalis,
where a serine substitution of its active site residue
Cys42 (C42S) resulted in approximately 3% of the
activity of the wild-type under the same conditions
[4,28]. Considering these results, Cys45 may provide
the essential second redox center in addition to the fla-
vin. We further examined the products of NOXtp and
its mutants. NAD(P)H oxidation was allowed to go to
completion, and the amount of H
2
O

2
formed in the
reaction was quantified using the peroxi-DETECT kit.
The NOXtpC122A mutant produced a similar amount
of H
2
O as the wild-type under the same conditions
and with the same substrates (Fig. 5D). Oxidation of
NADH and NADPH by NOXtpC45A and NOX-
tpC45A ⁄ C122A led to the formation of about one
equivalent of H
2
O
2
(Fig. 5D), demonstrating that
H
2
O
2
production by these two mutants is stoichiome-
tric with NADH and NADPH oxidation. The activity
and product assays using the wild-type and mutants
clearly demonstrated that Cys45 participates in the
direct four-electron reduction of O
2
to H
2
O, and the
Cys45 mutation alters the reaction to produce H
2

O
2
instead of H
2
O.
Discussion
In this study, we have demonstrated that NOXtp has a
hexameric ring-shaped structure. Gel filtration under
nondenaturing conditions revealed that NOXtp is com-
posed of six subunits. Moreover, upon electron micro-
scopic analysis, NOXtp was found to predominantly
exhibit a hexameric structure that contained a middle
region of high density with heavy stain accumulation
in its center. However, the crystal structure of NOX
from L. sanfranciscensis revealed a dimeric form with
an N-terminal oxidoreductase domain and a C-termi-
nal dimerization domain [12]. NPX from Streptococ-
cus faecalis, catalyzing the conversion of H
2
O
2
to
H
2
O, was reported to be a homotetrameric structure
[29]. These two mesophilic proteins show different
types of subunit oligomerization and low sequence
identity (Fig. S1), but each of their subunits shows
high structural similarity and their folding patterns are
similar to that of GR [12,29]. In contrast, NOX from

Thermoanaerobium brockii was found to have a hexa-
meric quaternary structure by gel filtration [15]. Elec-
tron microscopic analysis has revealed that NOXtp has
a hexameric ring-shaped structure composed of two
0
1
2
3
4
5
6
7
8
Specific activity (U·mg
–1
)
12
dcbadcba
100 nm 100 nm
100 nm100 nm
a
b
c
d
0
20
40
60
80
100

H
2
O
2
production (µmol)
12
dcbadcba
123 45
669 kDa
440
67
140
232
ABC
D
Fig. 5. Comparisons of activity, products and structures between NOXtp and the mutants. (A) Electron micrographs of NOXtp (a), NOX-
tpC45A (b), NOXtpC122A (c) and NOXtpC45A ⁄ C122A (d). The bar represents 100 nm. (B) Native PAGE of wild-type NOXtp and its mutants.
Lanes 1–4 correspond to (a), (b), (c) and (d) in (A), respectively; lane 5 is the molecular weight marker. The lower part shows the correspond-
ing proteins determined by SDS ⁄ PAGE. (C) Specific activity of wild-type NOXtp (a), NOXtpC45A (b), NOXtpC122A (c) and NOX-
tpC45A ⁄ C122A (d) with NADH (bar 1) and NADPH (bar 2) as substrates. (D) The amount of H
2
O
2
produced by NOXtp (a), NOXtpC45A (b),
NOXtpC122A (c) and NOXtpC45A ⁄ C122A (d) when 100 l
M NADH (bar 1) or 100 lM NADPH (bar 2) was oxidized.
Table 2. Determination of the sulfhydryl contents of wild-type and
mutant NOXtp using Ellman’s reagent. Data shown are means of
triplicate determinations ± SD.
Protein

No. cysteines
per protein
NOXtp 1.84 ± 0.31
NOXtpC45A 0.85 ± 0.34
NOXtpC122A 0.84 ± 0.18
NOXtpC45A ⁄ C122A 0.14 ± 0.07
B. Jia et al. Hexameric thermophilic NADH oxidase-producing H
2
O
FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5361
stacked rings of different diameters (19 and 15 nm
respectively) that encompass a central opening; this is
the first hexameric NOX determined by electron
microscopy. Significantly, this structural feature of
NOXtp is highly similar to that of valosine-containing
protein-like ATPase from Th. acidophilum, an archaeal
member of the AAA family (ATPases associated with
a variety of cellular activities) [24]. In addition, the
structure of the cysteine mutants, NOXtpC45A, NOX-
tpC122A and NOXtpC45A ⁄ C122A, was the same as
that of the wild-type. Thus, it appears that a disulfide
bond does not participate in the oligomerization and
quaternary structure of NOXtp.
NADH oxidase catalyzes the transfer of electrons
from reduced pyridine nucleotides to O
2
[2,4]. Here we
have demonstrated that NOXtp can efficiently reduce
O
2

to produce H
2
O using both NADH and NADPH
as electron donors. In addition, the activity and prod-
uct assays of the wild-type and mutants showed that
Cys45 is the active site residue and that Cys122 does
not function in the NADH and NADPH oxidase
activity. These results indicate that Cys45 participates
in the direct four-electron transfer reduction of O
2
to
H
2
O, and that the Cys45 mutant alters the reduction
to produce H
2
O
2
instead of H
2
O, similar to NOX in
E. faecalis [28]. NOX in E. faecalis belongs to a group
of enzymes that use a cysteine sulfenic acid as the non-
flavin redox center. These enzymes are found in
Enterococcus and Streptococcus, which are aerotolerant
anaerobes, where they play an important role in O
2
tolerance [4]. For example, H
2
O-forming NOX-defi-

cient mutants of Streptococcus pyogenes are unable to
grow under high-O
2
conditions, revealing the impor-
tance of NOX-scavenging activity against harmful O
2
[30]. We therefore propose that NOXtp may decom-
pose O
2
in the anaerobe T. profundus.
The predominant production of H
2
O by NOXtp is
in contrast to the exclusive production of H
2
O
2
by
most NOXtp homologs in thermophiles, such as NOX
in A. fulgidus, Desulfovibrio gigas, Thermot. maritima
and Thermoanaerobium brockii [10,11,15,31]. Previously,
the production of H
2
O
2
was considered to be the dis-
tinctive property of NOX proteins from thermophiles
[10,11], with the exception of NOX from P. furiosus,
which produces both H
2

O
2
(77%) and H
2
O (23%) [9].
To our knowledge, NOXtp is the first NOX to be
purified from thermophilic microorganisms that can
catalyze electron transfer from NADH and NADPH
to O
2
and predominantly produce H
2
O. NOXtp is
therefore better for removing O
2
than other reported
O
2
-scavenging systems, which must employ intermedi-
ates to reduce H
2
O
2
produced by NAD(P)H oxidases,
such as in D. gigas, where rubredoxin and neelaredoxin
act as intermediates [31]. As NOXtp and the mesophil-
ic enzymes that decompose injurious O
2
belong to the
same group (discussed above), and NOXtp reduces O

2
to H
2
O directly, we propose that NOXtp may play an
important role in O
2
removal or aerobic tolerance in
thermophilic anaerobes.
Experimental procedures
Purification of NOXtp from T. profundus
Thermococcus profundus cells (8 L) were grown at 80 °Cas
reported previously [32]. After harvesting, the cells were dis-
solved in 20 mm potassium phosphate buffer (pH 6.5), con-
taining 5 mm MgCl
2
, 0.5 mm EDTA, 1 mm dithiothreitol
and 10% glycerol (PMEDG buffer), and disrupted by soni-
cation. The homogenates were centrifuged at 10 000 g for
30 min. The supernatant was loaded on a phosphocellulose
column that had been equilibrated with PMEDG buffer.
After being washed completely, the proteins were eluted by
100, 200, 300, 400, 500 and 1000 mm NaCl in a stepwise
gradient, and the eluates in 200 mm NaCl were dialyzed
with 50 mm Tris buffer (pH 8.0) containing 400 mm NaCl.
The sample was then loaded on an amino-benzimide col-
umn equilibrated with the same buffer. Unabsorbed pro-
teins on the resin were collected and dialyzed with PMEDG
buffer, concentrated using a centricon (Millipore, Billerica,
MA, USA), and stored at )80 °C. Eluates in all steps were
checked by transmission electron microscopy. The protein

concentration was determined by the Bradford method, and
BSA was used as standard.
SDS/PAGE and N-terminal sequencing
The purified enzyme was subjected to SDS ⁄ PAGE and elec-
troblotted onto poly(vinylidene difluoride) membranes. The
visible band was excised and applied to a protein sequence
analyzer (Korea Basic Science Institute, Daejeon, Korea).
Cloning of NOXtp from T. profundus
Polymerase chain reaction experiments with T. profundus
genomic DNA as a template were performed using degener-
ate oligonucleotides (sense primer, 5¢-GTA GTA ATA
ATA GGA GGA GGA GCN GCN GGN ATG-3¢; anti-
sense primer, 5¢-TAN ACT TTY TCN CAN SWN GTY
TGC AT-3¢; N = A, G, C and T; Y = C and T; W = A
and T). The sense primer was designed from the known
N-terminal sequence, and the antisense primer was from
the conserved C-terminal sequence of NOX. The experi-
ment using the two oligonucleotides afforded an amplificate
of  1.3 kb, which was ligated into the pTOPO vector
(Invitrogen, Carlsbad, CA, USA), transformed, and
confirmed by sequencing. The resulting sequence was used
Hexameric thermophilic NADH oxidase-producing H
2
O B. Jia et al.
5362 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS
for subsequent cloning. Full gene cloning of NOXtp was per-
formed using the Universal Genomewalker kit (ClonTech,
Mountain View, CA, USA). Briefly, the genomic DNA was
digested with EcoRV, DrabI, PvuII and SspI separately, and
ligated to the adaptor provided by the kit. PCR was per-

formed with the adaptor primers (provided by the kit) and
tail-specific primers (N-terminal genomewalker primer,
5¢-TGG AGG TCT TTG CCG CGC TTT TTG AT-3¢;
C-terminal genomewalker primer, 5¢-GGT GTG CAG
GCT GTA AAT GCC GAG AT-3¢), which corresponded
to the known sequence detected by the degenerate primers.
The PCR products were ligated into the pTOPO vector,
transformed, and sequenced.
Expression and purification of NOXtp in Es. coli
The new primers (sense primer, 5¢-CGCGCG CCA
TGG AGAGGAAACGCGTTGTTAT-3¢; antisense primer,
5¢-CGCG
AAGCTT TAAAACTTTAGAACCCTG-3¢)were
designed on the basis of the sequence of the Genomewalker
result (the underlined bases indicate the restriction enzyme
site). The PCR product and pET28-(a) were digested by
NcoI and HindIII and ligated. The ligation product was
transformed into Es. coli BL21(DE3) by electroporation.
Finally, the recombinant vector (pENOXtp) was confirmed
by sequencing.
Recombinant Es. coli cells (2 L) were cultured in LB
broth to a D
600 nm
of 1.0, and then induced with 1 mm iso-
propyl-thio-b-d-galactoside for 4 h. After harvesting, the
cells were resuspended in PMEDG buffer and disrupted by
sonication. After centrifugation (3000 g, 30 min), the super-
natants were heated at 65 °C for 30 min, and then the
denatured proteins were removed by centrifugation (3000 g,
30 min). The supernatants were loaded onto a phosphocel-

lulose column that had been equilibrated with the same
buffer. After being washed completely, the proteins were
eluted with 200 mm NaCl. The purified protein was
checked by SDS ⁄ PAGE and dialyzed with PMEDG buffer,
concentrated, and stored at )80 °C.
Mutagenesis
The primers used for the single cysteine to alanine mutants
were as follows: C45A, forward primer, 5¢-ACG GAA
TGG GTG AGC CAC GCT CCC GCC GGT ATC CCC
TAC GTA GTT GAG GGT-3¢; C45A, reverse primer,
5¢-ACC CTC A AC TAC GTA GGG G AT ACC GGC GGG
AGC GTG GCT CAC CCA TTC CGT-3¢; C122A, for-
ward primer, 5¢-CCG CAG GTT CCG GCG ATA GAG
GGC GCC CAC CTG GAA GGA GTA TTC ACA GCA-
3¢; and C122A reverse primer, 5¢-TGC TGT GAA TAC
TCC TTC CAG GTG GGC GCC CTC TAT CGC CGG AAC
CTG CGG-3¢. The PCR was performed using Pfu polymer-
ase (Takara, Kyoto, Japan), and the cycling parameters
were: 95 °C for 5 min (one cycle), 95 °C for 30 s, and 68 ° C
for 12 min (12 cycles). After amplification, the PCR mixture
was digested with DpnI and then transformed into Es. coli
BL21(DE3) by electroporation. The mutants were confirmed
by DNA sequencing. The double cysteine mutants were
produced by the same method, except that pENOXtpC45A
was used as the template and C122A primers were used for
the amplification. The mutant proteins were purified using
the same method as used for wild-type purification.
Gel filtration chromatography
The sample (1 mgÆmL
)1

) was loaded onto a Superdex-200
column (Amersham Biotech, Piscataway, NJ, USA). Stan-
dard proteins included ferritin (440 kDa), catalase
(232 kDa), albumin (67 kDa) and ovalbumin (43 kDa).
Apo-NOX preparation
The purified NOXtp from Es. coli is a holoenzyme with
FAD. The protein was dialyzed with 100 mm phosphate
buffer (pH 7.2) containing 2.4 m (NH
4
)
2
SO
4
,1mm dith-
iothreitol and 0.5 mm EDTA, and then loaded on the
hydrophobic interaction chromatography column equili-
brated with the same buffer. FAD was eluted with equili-
bration buffer saturated with NaBr (pH 3.5). The column
was balanced again with the equilibration buffer, and the
apoprotein was eluted with 100 mm phosphate buffer
[26,27]. Eluates were dialyzed with the PMEDG buffer
described above, and stored at )80 °C.
Enzyme assays
The NADH or NADPH oxidase activity of the recombi-
nant protein was examined by time-dependent removal of
NAD(P)H in aerobic conditions. The assays were per-
formed in 50 mm sodium or potassium phosphate buffer
(pH 7.2), 0.5 mm NAD(P)H and 100 mm NaCl at the indi-
cated temperatures. The reaction was started by adding
NOXtp in the amounts indicated. The rate of NAD(P)H

consumption was measured by monitoring the decrease in
A
340 nm
. One unit of activity was defined as the amount of
enzyme catalyzing the oxidation of 1 lmol NADH per
min at 75 °Cin50mm potassium phosphate buffer
(pH 7.2) and 0.5 mm NADH. To measure kinetic parame-
ters, reaction rates were measured at a series of NAD(P)H
concentrations, and the rates at various substrate concen-
trations were finally fitted by Lineweaver–Burk plots. The
parameters (with standard deviation) were determined by
three separate experiments.
Determination of the sulfhydryl content
The sulfhydryl contents were determined using Ellman’s
reagent in anaerobic conditions according to a published
B. Jia et al. Hexameric thermophilic NADH oxidase-producing H
2
O
FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS 5363
method [17,33]. After the proteins and 5,5¢-dithiobis-
(2-nitrobenzoic acid) (Nbs
2
) were incubated for 15 min at
25 °C, A
412
nm was monitored to estimate the number of
cysteine residues present as protein ⁄ Nbs
2
mixed disulfide.
The sulfhydryl concentrations in these proteins were deter-

mined from a calibration curve created using known con-
centrations of standard l-cysteine solutions.
H
2
O
2
detection
H
2
O
2
was detected using the PeroxiDetect Kit (Sigma).
Briefly, the assay was performed in 50 mm sodium phos-
phate buffer (pH 7.2), 100 lmol NAD(P)H, 1 mm EDTA,
100 mm NaCl and 0.2 nmol NOXtp. The reaction was
allowed to go to completion. Reaction buffer (100 lL) was
added to the kit. Peroxides convert Fe
2+
to Fe
3+
ions
under acidic conditions. Fe
3+
ions will then form a colored
adduct with xylenol orange, which is observed at 560 nm.
NAD(P)H will interfere with the H
2
O
2
assay, so all of the

NAD(P)H must be consumed completely.
Electron microscopy and image processing
Purified NOX was applied to glow-discharged carbon-
coated copper grids. After the proteins had been allowed to
absorb for 1–2 min, the grids were rinsed with droplets of
de-ionized water, and stained with 2% (w ⁄ v) uranyl acetate.
Electron micrographs were recorded with an FEI TECH-
NAI 12 microscope at a magnification of ·51 600 (nominal
magnification of ·52 000) and an acceleration voltage of
120 kV.
Light-optical diffractograms were used to select the
micrographs, to examine the defocus and to verify that no
drift or astigmatism was present. Suitable areas were digi-
tized as arrays of 1024 · 1024 pixels with leaf scan 45 at
a pixel size of 20 lm, corresponding to 0.38 nm at the spec-
imen level. For image processing, the semper [34] and em
[35] software packages were used. From digitized micro-
graphs, smaller frames of 64 · 64 pixels containing individ-
ual particles were extracted interactively. These images were
aligned translationally and rotationally, using standard cor-
relation methods [36,37]. An arbitrarily chosen reference
was used for the first cycle of alignment and averaging, and
the resulting average was used as a reference in the second
refinement cycle. For analysis of the rotational symmetry of
top-on-view images, the individual images were aligned
translationally but not rotationally [38]. These aligned
images were subjected to multivariate statistical analysis
[39]. The resulting eigenimages represent all-important
structural features of the original dataset. If the images had
different rotational symmetries in the original dataset, the

eigenimages would reveal the different symmetry axes.
Moreover, these images can be distinguished and subse-
quently separated on the basis on eigenimages. The rota-
tionally aligned images were classified on the basis of
eigenvector–eigenvalue data analysis, and subsequent aver-
aging was performed for each class separately. The average
was finally symmetrized on the basis of angular correlation
coefficients [40].
Acknowledgements
B. Jia, S. Lee, B. P. Pham, R. Yu and T. L. Le were
supported by scholarships from the Brain Korea21
project in 2008, Korea. This work was supported by a
grant from the MOST ⁄ KOSEF to the Environmental
Biotechnology National Core Research Center (grant
no. R15-2003-012-01003-0), and the Korea Research
Foundation Grant funded by the Korean Government
(MOEHRD) (grant no. KRF-2007-521-C00241), to
G. W. Cheong.
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Supporting information
The following supplementary material is available:
Fig. S1. Multiple sequence alignment of Thermococ-
cus profundus NADH oxidase (NOXtp) with the homo-

log NADH oxidases or NADH peroxidase (NPX).
Fig. S2. Purification of Thermococcus profundus
NADH oxidase (NOXtp) from Escherichia coli and
Thermococcus profundus.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the cor-
responding author for the article.
Hexameric thermophilic NADH oxidase-producing H
2
O B. Jia et al.
5366 FEBS Journal 275 (2008) 5355–5366 ª 2008 The Authors Journal compilation ª 2008 FEBS