Báo cáo khoa học: Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species pptx

REVIEW ARTICLE
Structure, regulation and evolution of Nox-family NADPH
oxidases that produce reactive oxygen species
Hideki Sumimoto
Medical Institute of Bioregulation, Kyushu University, Fukuoka
CREST, Japan Science and Technology Agency, Tokyo, Japan
Keywords
Duox; Nox; Noxa1; Noxo1; p22
phox
; p40
phox
;
p47
phox
; p67
phox
; Rac; Rboh
Correspondence
H. Sumimoto, Medical Institute of
Bioregulation, Kyushu University, 3-1-1
Maidashi, Higashi-ku, Fukuoka 812-8582,
Japan
Fax: +81 92 642 6807
Tel: +81 92 642 6806
E-mail:
(Received 8 January 2008, revised 2 April
2008, accepted 30 April 2008)
doi:10.1111/j.1742-4658.2008.06488.x
NADPH oxidases of the Nox family exist in various supergroups of
eukaryotes but not in prokaryotes, and play crucial roles in a variety of
biological processes, such as host defense, signal transduction, and hor-

mone synthesis. In conjunction with NADPH oxidation, Nox enzymes
reduce molecular oxygen to superoxide as a primary product, and this is
further converted to various reactive oxygen species. The electron-transfer-
ring system in Nox is composed of the C-terminal cytoplasmic region
homologous to the prokaryotic (and organelle) enzyme ferredoxin reduc-
tase and the N-terminal six transmembrane segments containing two
hemes, a structure similar to that of cytochrome b of the mitochondrial bc
1
complex. During the course of eukaryote evolution, Nox enzymes have
developed regulatory mechanisms, depending on their functions, by insert-
ing a regulatory domain (or motif) into their own sequences or by obtain-
ing a tightly associated protein as a regulatory subunit. For example, one
to four Ca
2+
-binding EF-hand motifs are present at the N-termini in
several subfamilies, such as the respiratory burst oxidase homolog (Rboh)
subfamily in land plants (the supergroup Plantae), the NoxC subfamily in
social amoebae (the Amoebozoa), and the Nox5 and dual oxidase (Duox)
subfamilies in animals (the Opisthokonta), whereas an SH3 domain is
inserted into the ferredoxin–NADP
+
reductase region of two Nox enzymes
in Naegleria gruberi, a unicellular organism that belongs to the supergroup
Excavata. Members of the Nox1–4 subfamily in animals form a stable hete-
rodimer with the membrane protein p22
phox
, which functions as a docking
site for the SH3 domain-containing regulatory proteins p47
phox
, p67

phox
,
and p40
phox
; the small GTPase Rac binds to p67
phox
(or its homologous
protein), which serves as a switch for Nox activation. Similarly, Rac acti-
vates the fungal NoxA via binding to the p67
phox
-like protein Nox regula-
tor (NoxR). In plants, on the other hand, this GTPase directly interacts
with the N-terminus of Rboh, leading to superoxide production. Here I
describe the regulation of Nox-family oxidases on the basis of three-dimen-
sional structures and evolutionary conservation.
Abbreviations
AIR, autoinhibitory region; Duox, dual oxidase; FNR, ferredoxin–NADP
+
reductase; Fre, ferric reductase; FRO, ferric-chelate reductase;
Noxa1, Nox activator 1; Noxo1, Nox organizer 1; NoxR, Nox regulator; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PMA,
4b-phorbol 12-myristate 13-acetate; PPII, polyproline II; PRR, proline-rich region; PtdIns(3)P, phosphatidylinositol 3-phosphate; PtdIns(3,4)P
2,
phosphatidylinositol 3,4-bisphosphate; PX domain, phagocyte oxidase domain; Rboh, respiratory burst oxidase homolog; ROS, reactive
oxygen species; TPR, tetratricopeptide repeat.
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3249
Introduction
Reactive oxygen species (ROS) are conventionally
regarded as inevitable deleterious byproducts of aero-
bic metabolism. On the other hand, there exist
enzymes dedicated to ROS production. The first exam-

ple of such enzymes is an NADPH oxidase expressed
in mammalian professional phagocytes [1–10]. During
engulfment of invading microbes, the phagocyte
NADPH oxidase becomes activated to reduce molecu-
lar oxygen to superoxide anion (O
2
)
), a precursor of
microbicidal ROS, in conjunction with oxidation of
NADPH. As the rapid increase in oxygen consumption
during phagocytosis is known as the respiratory burst,
this enzyme is also called respiratory burst oxidase.
The significance of the phagocyte oxidase in host
defense is exemplified by recurrent and life-threatening
infections that occur in patients with chronic granu-
lomatous disease, whose phagocytes genetically lack
the superoxide-producing activity [11,12].
The catalytic core of the phagocyte NADPH oxidase
(phox) is gp91
phox
, a membrane-integrated glycoprotein
with an apparent molecular mass of about 91 kDa.
gp91
phox
contains two hemes in the N-terminal trans-
membrane region, and NADPH-binding and FAD-
binding domains in the C-terminal cytoplasmic region
(Fig. 1A), forming a complete apparatus that trans-
ports electrons from NADPH via FAD and two hemes
to molecular oxygen. In the mid-1990s, homologs of

the flavocytochrome gp91
phox
were discovered in land
plants; these have been designated respiratory burst
oxidase homolog (Rboh) [13–15]. Subsequent searches
in genome databases led to the identification of novel
homologs of gp91
phox
in animals, which are presently
known as Nox (NADPH oxidase) or Duox (dual oxi-
dase) [1–10]. The human genome contains seven genes
encoding gp91
phox
homologs: Nox1–Nox5, where
gp91
phox
is renamed Nox2, and the distantly related
oxidases Duox1 and Duox2. It is currently known that
a wide variety of eukaryotes express superoxide-pro-
ducing NADPH oxidases that harbor a gp91
phox
-like
electron-transferring system; the enzymes constitute the
Nox family. Recent studies on Nox-family enzymes
have increasingly clarified the importance of deliberate
ROS production in various biological events, including
signal transduction, development, and hormone bio-
synthesis, in addition to well-established roles in host
defense [1–10]. It is likely that individual Nox enzymes
have developed regulatory systems, according to their

respective special functions, during the course of
eukaryote evolution by inserting a regulatory domain
(or motif) into their own sequences or by obtaining a
tightly associated protein as a regulatory subunit. Reg-
ulation by these proteins includes multiple protein–
protein and protein–lipid interactions. In this review, I
describe post-translational regulation of Nox-family
enzymes on the basis of three-dimensional structures
and evolutionary conservation.
Structure of Nox-family enzymes
The phagocyte NADPH oxidase gp91
phox
⁄ Nox2 (570
amino acid residues) exists in not only the plasma
membrane but also the membrane of the specific gran-
ule in neutrophils: the latter contains a higher amount
of Nox2 [1–10]. Although the phagosomal membrane
is considered to primarily derive from the plasma
membrane, the specific granule is fused to the phago-
some during phagocytosis, and thus gp91
phox
⁄ Nox2 is
further enriched in the phagosomal membrane. For
killing engulfed microbes, superoxide (and microbicidal
ROS derived from superoxide) must be produced from
molecular oxygen within the phagosome. The intra-
phagosomal production requires electrons to be trans-
ported from the cytoplasmic NADPH across the
membrane into the interior of the phagosome. Such
Fig. 1. (A) A model for the structure of gp91

phox
. Cylinders repre-
sent six transmembrane a-helices. (B) Bis-heme ligation in
gp91
phox
. Heme-coordnating His residues are numbered according
to their localization in gp91
phox
. (C) Intramembrane bis-heme motifs
in various cytochromes. The numbers of intervening amino acids
that separate a pair of His residues in a transmembrane segment
are indicated.
Structure, regulation and evolution of Nox H. Sumimoto
3250 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
transmembrane electron transport very often involves
di-heme membrane proteins.
gp91
phox
⁄ Nox2 can be divided into two parts of sim-
ilar size (Fig. 1A). The C-terminal half is a cytoplasmic
domain homologous to ferredoxin–NADP
+
reductase
(FNR), bearing the NADPH-binding and FAD-bind-
ing sites [16,17], whereas the N-terminal moiety com-
prises six predicted a-helical transmembrane segments
(Fig. 1A). The bipartite structure is common not only
to all the Nox-family enzymes but also to the family
of fungal ferric reductases (Fre) [18]. Fre enzymes,
which are expressed in the plasma membrane, reduce

Fe
3+
and Cu
2+
for iron and copper uptake, but fail
to use molecular oxygen as a substrate [19,20].
Among the conserved transmembrane segments of
gp91
phox
⁄ Nox2, the third and fifth helices each contain
two invariant His residues, which are considered to
provide the axial and distal ligands for binding to the
irons of two nonidentical hemes, thereby placing one
heme towards the cytoplasmic face and the other
towards the outer face (Fig. 1B). As the hemes are ori-
ented perpendicular to the surface of the membrane,
electrons are transferred from the cytosolic NADPH,
through FAD, and across the membrane via the hemes
to molecular oxygen, leading to superoxide production.
Thus, transmembrane electron transport in gp91
phox
⁄
Nox2 is considered to occur in the N-terminal
bis-heme-containing region [18,21].
This model was initially proposed on the basis of a
similar motif consisting of two pairs of spaced His resi-
dues that was predicted to be linked to heme coordina-
tion in a class of organelle and bacterial b-type
cytochromes, such as the bis-heme cytochrome b of the
mitochondrial cytochrome bc

1
complex (complex III)
and cytochrome b
6
of the chloroplast cytochrome b
6
f
complex [22–24] (Fig. 1C). The model for two bis-hist-
idyl heme ligation was subsequently verified by determi-
nation of crystal structures of protein complexes
containing these cytochromes [25–28]. In cytochrome b
of the cytochrome bc
1
complex, containing eight a-heli-
cal transmembrane segments, the two b-type hemes are
bound within a four helix bundle formed by the first
four segments: His residues ligated to both hemes are
located in the second and fourth helices [25,26,29], and a
pair of the His residues in each helix are separated by 13
intervening amino acids (Fig. 1C). A similar coordina-
tion occurs in cytochrome b
6
of the cytochrome b
6
f
complex in cyanobacteria and chloroplasts [27,28]: cyto-
chrome b
6
comprises four a-helical transmembrane
segments [30,31], and the two hemes are bis-His-coordi-

nated by imidazole side chains separated by 13 and 14
residues in the second and fourth helices, respectively
(Fig. 1C). In the fungal Fre-family enzymes, both His
pairs are separated by 13 amino acids, as in cyto-
chrome b of the cytochrome bc
1
complex (Fig. 1C).
Although gp91
phox
⁄ Nox2, as well as other Nox-family
enzymes, contains a pair of His residues in the third
transmembrane a-helix with 13 intervening amino acids
(His101 and His115 in human gp91
phox
⁄ Nox2), the other
pair in the fifth helix (His209 and His222 in human
gp91
phox
⁄ Nox2) are separated by 12 amino acids
(Fig. 1). It should be noted that the imidazoles sepa-
rated by 12–14 amino acids are likely to face the same
side of the helix. Substitution of any of these four His
residues results in disrupted insertion of hemes into
gp91
phox
⁄ Nox2 [32], supporting the view that the two
bis-histidyl heme ligation also occurs in gp91
phox
⁄ Nox2.
Reduction of molecular oxygen to superoxide in

gp91
phox
⁄ Nox2 requires both heme groups to be in the
low-spin (hexacoordinate) state, which implies that
electron transfer to oxygen occurs via the outer heme
in a pocket near the heme edge, rather than through
direct coordination of oxygen to the heme iron [33,34].
This outer sphere (or peripheral) mechanism is consis-
tent with the ‘two bis-histidyl heme ligation’ structure,
and explains well why gp91
phox
⁄ Nox2-catalyzed super-
oxide production is not inhibited by cyanide or carbon
monoxide. Taken together, electron transfer from
NADPH to molecular oxygen occurs in a module des-
ignated the Nox superdomain. The Nox superdomain
comprises two moieties, the N-terminal bis-heme cyto-
chrome b, composed of six a-helical transmembrane
segments, and the C-terminal FNR, which contains
FAD-binding and NADPH-binding domains. Thus,
gp91
phox
⁄ Nox2 and its relatives (the Nox family) are
flavocytochromes [16,17,35].
It is known that members of the Duox subfamily in
animals, in contrast to oxidases of the other subfa-
milies, release H
2
O
2

without forming detectable
amounts of superoxide [36]. However, they are also
expected to produce superoxide as an initial product,
as Duox has the superoxide-producing Nox super-
domain that comprises the bis-heme-containing trans-
membrane region and the FNR-like moiety. Indeed, it
has been reported that Duox in an immature form is
capable of producing superoxide [37]. In mature Duox,
superoxide produced by the Nox superdomain may be
rapidly converted to H
2
O
2
via intramolecular dismuta-
tion, possibly by a peroxidase-like ectodomain; this
module is located on the outer surface of the mem-
brane, where superoxide is expected to be released.
In addition to the Nox and Fre families, both the
bis-heme transmembrane segment and FNR-related
moiety are present in ferric-chelate reductase (FRO) of
land plants [38]. To acquire iron from soils of low iron
availability, land plants such as Arabidopsis thaliana
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3251
reduce Fe
3+
to Fe
2+
by FRO in the plasma mem-
brane of root epithelial cells. Four His residues in

FRO that lie on two predicted, similarly orientated,
transmembrane a-helices are in equivalent locations to
the His residues in Nox enzymes that coordinate the
two hemes: the 13 and 12 amino acids separating
heme-liganding His residues exist in the helices
(Fig. 1C). The FRO-family enzymes contain eight or
10 transmembrane segments, which is different from
the situation in members of the Nox and Fre families;
the precise membrane topology of FRO enzymes
remains controversial [39,40].
Origin of Nox-family enzymes
There is no evidence for the presence of Nox, Fre or
FRO in prokaryotes: a superfamily of flavocyto-
chromes that transport electrons across membranes. On
the other hand, members of this superfamily are pres-
ent in a variety of eukaryotes. The shared bis-heme
binding motif raises the possibility of an evolutionary
and functional relationship between the eukaryotic cell
surface membrane proteins Nox, Fre and FRO and the
prokaryotic (or organelle) b-type cytochromes. On the
other hand, the C-terminal moiety of the flavocyto-
chrome superfamily is homologous to FNR, a prokary-
otic (organelle) protein that is made up of two
structural domains, each containing about 150 amino
acids: the C-terminal region includes most of the resi-
dues involved in NADPH binding, whereas the large
cleft between the two domains accommodates the FAD
group [41,42]. It is tempting to postulate that a gene
encoding a protein containing two di-heme transmem-
brane helices was fused to an FNR gene in eukaryote

evolution, leading to a common ancestor of the Nox
and Fre families (Fig. 2). In this context, it seems inter-
esting that FNR directly interacts with the cyto-
chrome b
6
f complex of plant chloroplasts, albeit in a
noncovalent manner, and participates in electron trans-
fer [43]; cytochrome b
6
in the complex has two b -type
hemes across the membrane, as expected for Nox.
A similar fusion of the FNR gene with a gene
encoding an electron-transporting protein is also con-
sidered to have occurred during evolution: eukaryotic
diflavin reductases such as NADPH–cytochrome P450
reductase (CPR or P450R), methionine synthase reduc-
tase and novel reductase 1 probably arose from the
fusion of the ancestral genes for FNR and flavodoxin,
a prokaryotic FMN-containing protein that transfers
electrons in a variety of photosynthetic and nonphoto-
synthetic reactions in prokaryotes [43–48] (Fig. 2). In
turn, a diflavin reductase is likely to be the precursor
of further fusion products such as nitric oxide reduc-
tase, which consists of a C-terminal CPR-like domain
and an N-terminal, heme-containing oxygenase domain
[48,49].
Distribution of Nox-family enzymes
in eukaryotes
In contrast to the absence of Nox in prokaryotes,
genes encoding Nox-family enzymes are found in a

wide variety of eukaryotes. Eukaryotes can be divided
into several major supergroups, including the Opi-
sthokonta, the Amoebozoa, the Plantae, the Excavata,
the Rhizaria, and the Chromalveolata (the Hetero-
konta plus the Alveolata): animals and fungi belong to
the Opisthokonta; the social amoeba Dictyostelium dis-
coideum is a member of the Amoebozoa; land plants
and red algae belong to the Plantae; and diatoms and
oomycetes are members of the Heterokonta, a group
that belongs to the Chromalveolata (Fig. 3) [50–52].
Fig. 2. A putative common ancestor of the
Nox family. CPR, NADPH–cytochrome P450
reductase.
Structure, regulation and evolution of Nox H. Sumimoto
3252 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
The relationships between these supergroups, however,
remain to be determined, and thus the root of eukary-
otes is presently uncertain [53,54].
Recent expansion of information available in gen-
ome databases has revealed that Nox-family enzymes
are present in all the eukaryotic supergroups except
the Rhizaria (Fig. 3). This suggests that a common
ancestor of Nox genes emerged at an early stage in the
evolution of eukaryotes; it diverged well in some lin-
eages (e.g. in the Opisthokonta and Plantae), whereas
it was often lost in some other lineages. The loss of
Nox genes appears to have occurred at multiple stages
in eukaryote evolution. For example, in the super-
group Amoebozoa, the social amoeba Di. discoideum
contains three Nox genes [55], whereas they are absent

in Entamoeba histolytica [56]. In the supergroup Excav-
ata, the heterolobosa Naegleria gruberi has at least two
Nox genes, as found by the present search using the
database of the DOE’s Joint Genome Institute (http://
genome.jgi/psf.org/euk_home.html) (Fig. 3); on the
other hand, no Nox gene has been found in the kineto-
plastid Leishmania major or Trypanosoma brunei,or
the diplomonad Giardia lamblia [56]. In the Chromal-
veolata, Nox genes are present in genomes of the
oomycete Phytophthora sojae [56,57] and the diatom
Thalassiosira pseudonana [58], although they have not
been found in the genomes of Plasmodium falciparum
and Theileria parva, both of which belong to the same
supergroup [56]. Even in the Opisthokonta, Nox genes
have been lost independently in several fungal lineages;
for example, a Nox gene is absent in budding and
fission yeasts [56,57,59].
Regulation of Nox-family enzymes
by Ca
2+
Superoxide production by Nox is regulated by various
mechanisms. Several subfamilies of Nox enzymes
appear to be directly regulated by Ca
2+
. It is well
established that mammalian thyroid oxidase and sea
urchin NADPH oxidase, both of which belong to the
Duox subfamily, are reversibly activated by Ca
2+
.In

addition to the Nox superdomain comprising the bis-
heme-containing transmembrane region and the FNR-
homologous moiety, Duox-subfamily oxidases feature
an N-terminal peroxidase-like ectodomain that is sepa-
rated from two EF-hands by an additional transmem-
brane segment (Fig. 4). Biosynthesis of thyroid
hormones in humans requires Duox2, which is highly
expressed in the thyroid gland: mutations in Duox2
are associated with a loss of thyroid hormone synthesis
and can lead to permanent and severe congenital hypo-
thyroidism [60]. With the H
2
O
2
produced by Doux2,
thyroid peroxidase catalyzes conjugation of iodide ions
to Tyr residues on thyroglobulin in the thyroid folli-
cles, an essential step for the synthesis of the active
hormone [61]. On the other hand, during fertilization
of sea urchins, a rapid increase in H
2
O
2
generation
occurs, which is catalyzed by the sea urchin Duox
homolog Udx1, leading to formation of the fertiliza-
Fig. 3. Distribution of Nox-family enzymes
in eukaryotes and animals. Upper panel:
eukaryotes can be divided into six major
supergroups, including the Opisthokonta,

the Amoebozoa, the Plantae, the Excavata,
the Rhizaria, and the Chromalveolata. All the
supergroups except the Rhizaria are known
to contain Nox genes. Lower panel: M. b.,
Mo. brevicollis; N. v., Ne. vectensis; L. g.,
Lot. gigantea; C. sp. I, Capitella sp. I; C. e.,
Ca. elegans; D. m., Dr. melanogaster; S. p.,
S. purpuratus; B. f., B. floridae; C. i., Ci.
intestinalis; D. r., Da. rerio; X. t., X. tropical-
is; G. g., Ga. gallus; H. s., Homo sapiens.
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3253
tion envelope as the physical block to polyspermy [62].
It is also known that Duox1 of Caenorhabditis elegans
in the phylum Nematoda is involved in cross-linking
of Tyr residues of extracellular matrix proteins,
thereby facilitating cuticle formation [63], and that
Duox plays a critical role in innate immunity in the
gut of the fruit fly Drosophila melanogaster in the
phylum Arthropoda [64].
Regulation of Duox by Ca
2+
probably occurs via its
paired EF-hand motif. It has been reported that lim-
ited proteolysis with a-chymotrypsin renders thyroid
NADPH oxidase fully and irreversibly active indepen-
dently of Ca
2+
[65]. This implies that the Ca
2+

-bind-
ing EF-hands of Duox serve as an autoinhibitory
domain, whereas those of Nox5 function as an activa-
tion domain [66]. The inhibition of Duox by the
EF-hands might be released reversibly by physiological
Ca
2+
-induced conformational change and irreversibly
by proteolytic removal of the autoinhibitory domain
[37]. A recent study has shown that ectopically
expressed Duox produces ROS without cell stimulants,
and the production is enhanced two-fold by the addi-
tion of ionomycin [67], suggesting that elevation of
cytoplasmic concentrations of Ca
2+
is dispensable.
Besides direct regulation by Ca
2+
, protein kinase C
(PKC) may modulate Duox in a Ca
2+
-independent
manner, as ROS production in thyrocytes is triggered
by 4b-phorbol 12-myristate 13-acetate (PMA), an
agent that activates PKC without elevating the cyto-
plasmic concentration of Ca
2+
[68]. A PKC-dependent
pathway may also function in H
2

O
2
generation at
fertilization in the sea urchin [62].
In addition to the animal Duox subfamily, oxidases
of other two subfamilies have been shown to be
directly regulated by elevations in cytoplasmic Ca
2+
concentrations: the Nox5 subfamily in animals
[66,69,70], and the Rboh subfamily in land plants
[71,72] (Fig. 4). The regulation by Ca
2+
appears to be
consistent with the presence of the Ca
2+
-binding
EF-hand motif in the cytoplasmic region N-terminal to
the Nox superdomain (Fig. 4).
Human Nox5 is abundantly expressed in T and
B cells of spleen and lymph nodes, and also in the sperm
precursors of testis [69]. Although the role for mamma-
lian Nox5 remains unknown, Drosophila Nox5 has been
reported to mediate smooth muscle contraction [70].
Oxidases of this subfamily build on the basic structure
of the Nox prototype, adding an N-terminal extension
that contains four EF-hands: three canonical motifs
and one noncanonical motif [63] (Fig. 4). Biochemical
analysis has shown that activation of Nox5 is directly
regulated by Ca
2+

: superoxide production by Nox5-
containing membrane fractions is dependent on the
presence of Ca
2+
[66]; and cells ectopically expressing
Nox5 produce superoxide in response to the Ca
2+
iono-
phore ionomycin [69]. The Ca
2+
-binding domain of
Nox5, in contrast to that of Duox, may function as an
activator module: the binding of Ca
2+
causes a confor-
mational change, which leads to intramolecular interac-
tion of the N-terminal Ca
2+
-binding domain with the
Fig. 4. Models for structures of various sub-
types of Nox-family enzymes. Cylinders rep-
resent six transmembrane a-helices. EF,
Ca
2+
-binding EF-hand motif.
Structure, regulation and evolution of Nox H. Sumimoto
3254 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
C-terminal Nox superdomain, culminating in Nox5 acti-
vation [66]. On the other hand, the EC
50

for calcium of
about 1 lm, determined in a cell-free activation system
for Nox5 [66], is relatively high and unlikely to be
achieved in most cells treated with physiological stimu-
lants. Two mechanisms for the elevation of the Ca
2+
sensitivity have recently been proposed [73,74]. First,
PKC phosphorylates Ser ⁄ Thr residues in the FAD-bind-
ing domain of Nox5, which increases the Ca
2+
sensitiv-
ity of the Nox5 activity regulated by the N-terminal
Ca
2+
-binding domain [73]. Second, a consensus calmod-
ulin-binding site is present in the NADPH-binding
domain of Nox5; calmodulin interacts with the site at a
lower concentration of Ca
2+
, thereby elevating the
Ca
2+
sensitivity for Nox5 activation [74].
The Rboh subfamily of NADPH oxidases is respon-
sible for ROS formation associated with plant defense
responses, and also plays a crucial role in plant devel-
opment [13,14,75]. Ten and nine members are present
in A. thaliana [14] and the rice Oryza sativa [76],
respectively. The Rboh-subfamily enzymes carry an
N-terminal extension with two EF-hand motifs

(Fig. 4). It has been reported that only about a two-
fold to three-fold increase in superoxide production
occurs in the membrane fraction of tobacco and
tomato upon addition of Ca
2+
; in addition, the effect
requires high concentrations of Ca
2+
(approximately
millimolar) [71]. This observation suggests that the
direct effect of Ca
2+
may not contribute to Rboh acti-
vation to a large extent. A recent study has shown that
elicitor-responsive phosphorylation of the N-terminal
region of Rboh is involved in superoxide production
[77,78], which is mediated by Ca
2+
-dependent protein
kinases [77]. Activation of Rboh is also regulated by
plant homologs of the Rho-family small GTPase Rac
(also known as Rop for Rho-like protein) [79–81]; Rac
in the GTP-bound form functions by directly binding
to the N-terminal region of Rboh, and this is probably
inhibited by Ca
2+
[76]. Thus, regulation of Rboh is
more complicated than previously expected, as
described in detail in a later section.
As in plant Rboh, two copies of EF-hands are pres-

ent in the N-terminal cytoplasmic region of the NoxC-
subfamily members in the Amoebozoa and Nox
enzymes in oomycetes of the eukaryotic supergroup
Chromalveolata [57], whereas enzymes in the fungal
NoxC subfamily contain a single EF-hand in the
N-terminal cytoplasmic region [56,57,59]. These sub-
families of Nox enzymes may be regulated by Ca
2+
;
however, no experimental evidence for Ca
2+
-mediated
regulation has been obtained.
It is presently unknown whether the EF-hand-con-
taining Nox subfamilies originated from a common
ancestor gene. It seems rather likely that EF-hand
motifs have been obtained independently several times
during evolution. The genomes of Monosiga brevicollis
in the choanoflagellates (a sister group of animals) and
Nematostella vectensis in the cnidarians (a basal group
of animals) contain solely Nox2-like enzymes, and not
EF-hand-containing oxidases such as Nox5 and Duox,
although these two families are found in a variety of
species of protostomes and deuterostomes (Fig. 3).
Thus, Nox5 and Duox may have evolved from Nox2-
like prototype oxidases. Similarly, in fungi, the NoxC
subfamily containing an EF-hand is found solely in
more evolved groups, including the Sordariomycetes
and Dothideomycetes, whereas the Nox2-like
EF-hand-free subfamilies NoxA and NoxB are present

also in relatively basal groups such as the Chytridi-
omyceta and Basidiomycota [57]. These features sug-
gest that the NoxC subfamily emerged at a later stage
of fungal evolution. Thus, the classification of the Nox
family in eukaryotes into the two major groups,
depending on the presence or absence of the EF-hand
motif, does not seem to reflect molecular evolution.
Regulation of Nox-family enzymes by
protein–protein interactions
The genome of Na. gruberi ( />euk_home.html), a member of the eukaryotic super-
family Excavata, contains two genes encoding Nox-
family enzymes of 627 and 630 amino acids, both of
which have an SH3 domain and thus are tentatively
designated as NoxSH3 (Fig. 5). The SH3 domain is
inserted into a loop region in the NADPH-binding
domain of the C-terminal FNR-homologous region
(Fig. 5). It is tempting to postulate that the NoxSH3
enzymes in Naegleria are regulated by a protein har-
boring a proline-rich region (PRR); SH3 domains are
generally known as modules that recognize a PRR to
mediate protein–protein interactions [82,83]. Identifica-
tion of a NoxSH3-binding protein will shed light on
our understanding of Nox regulation.
Nox1–Nox4 in animals form a heterodimer with the
nonglycosylated integral membrane protein p22
phox
,
which contains two (or possibly four) putative trans-
membrane segments (Fig. 4). The complex of
gp91

phox
⁄ Nox2 with p22
phox
in phagocytes is known as
flavocytochrome b
558
. In the C-terminal cytoplasmic
region of p22
phox
, there exists a PRR, which serves as
an anchoring site, thereby juxtaposing the catalytic
center gp91
phox
and the SH3 domain-containing regu-
latory proteins p47
phox
, p67
phox
, and p40
phox
; on the
other hand, the small GTPase Rac functions in Nox
activation by interacting with p67
phox
or its homolo-
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3255
gous proteins. Detailed mechanisms for regulation by
these proteins will be described below. Complex forma-
tion of p22

phox
with Nox also contributes to the stabil-
ization of each protein [1–10]. Formation of the
mutually stabilizing complex appears to require the
correct folding of Nox, because heme incorporation
into gp91
phox
⁄ Nox2 is essential for heterodimer forma-
tion [84].
It is known that human Duox2 associates with a
specific maturating protein named DuoxA2, which
contains putative five transmembrane helices [67,85]
(Fig. 6). The membrane protein DuoxA2 allows the
transition from the endoplasmic reticulum to the Golgi
apparatus, maturation and translocation to the plasma
membrane of functional Duox2 [67,85]. Interestingly,
the DuoxA2 gene is arranged head-to-head and coex-
pressed with the Duox2 gene; the gene for DuoxA1, a
paralog of DuoxA2, is similarly linked to the Duox1
gene [85]. Biallelic inactivation of the DuoxA2 gene
has recently been reported as a novel cause of congeni-
tal hypothyroidism [86], confirming its crucial contri-
bution to function of Duox2, which is directly
involved in thyroid hormone synthesis [60].
The Nox subfamilies in animals
The Nox enzymes in animals can be divided into three
subfamilies: one containing Nox1–Nox4 (the Nox1–4
Fig. 5. Structure of the SH3 domain-containing enzyme NoxSH3 in
Na. gruberi. The two NoxSH3 enzymes in Na. gruberi, tentatively
named NoxSH3-1 and NoxSH3-2, contain 627 and 630 amino acids,

respectively. The amino acid sequences of NoxSH3-1 and NoxSH3-2
are shown: the heme-coordinated His residues in transmembrane
segment 3 (TM3) and TM5 are shown in red; residues of FAD-bind-
ing motifs are shown in green; residues of NADPH-binding motifs
are shown in blue; and residues of the SH3 domain are shown in
magenta. A model for the structure of NoxSH3 is also shown. The
SH3 domain is inserted into the C-terminal NADPH-binding domain.
Cylinders represent six transmembrane a-helices.
Fig. 6. Models for the structure of Duox1 ⁄ 2 complexed with Duox-
A1 ⁄ 2. Cylinders represent six and five transmembrane a-helices of
Duox1 ⁄ 2 and DuoxA1 ⁄ 2, respectively. EF, Ca
2+
-binding EF-hand
motif.
Structure, regulation and evolution of Nox H. Sumimoto
3256 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
subgroup), which form a heterodimer with p22
phox
[87–
93]; the Nox5 subfamily; and the Duox subfamily
(Fig. 4). Although the Nox5 and Duox subfamilies are
not found in the sea anemone Ne. vectensis of the
Cnidaria (the basal group of animals) (http://geno-
me.jgi/psf.org/euk_home.html) [94] (Fig. 3), they were
probably present at the protostome–dueterostome diver-
gence. This is because the Nox5 and Duox subfamilies
exist in both extant protostomes and deuterostomes,
with the exception that Nox5 has been lost in the lineage
of the phylum Nematoda and in that of the order Rod-
entia in mammals: Nox5 is absent in the nematode

Ca. elegans and the rodents mouse and rat (Fig. 3).
On the other hand, Nox2, a member of the Nox1–4
subgroup, was present before the divergence of the
Choanoflagellata and Metazoa (equivalent to animals):
Nox2 is found not only in the Cnidaria but also in the
choanoflagellate Mo. brevicollis ( />psf.org/euk_home.html) [94] (Fig. 3). During the evolu-
tion of protostomes, one of the two major groups of
animals, Nox2 has been lost in the lineage of the clade
Ecdysozoa, including the phyla Arthropoda and Nem-
atoda: Nox2 is absent in the fruitfly Drosophila and
the nematode Ca. elegans. On the other hand, Nox2 is
present in the Lophotrochozoa, another major proto-
stomian clade, including the phyla Mollusca and
Annelida: this oxidase exists in the limpet Lottia gigan-
tea (Mollusca) and the leech Capitella species (Annel-
ida) (Fig. 3). Thus, it is likely that Nox2 is the closest
to the ancestral Nox in animals. In contrast, Nox1,
Nox3 and Nox4 are not found in protostomes or the
Echinodermata, a phylum that belongs to the deuter-
ostomes (a sister group of protostomes). These three
Nox enzymes appear to have diverged from Nox2 at
distinct stages of evolution of the phylum Chordata in
deuterostomes.
The Chordata is divided into three subphyla: the
Vertebrata, Urochordata (also known as the Tunicata),
and the Cephalochordata. Although tunicates were
long considered to be the earliest offshoot of the chor-
date lineage, and cephalochordates (such as amphi-
oxus) as the closest group to vertebrates, recent
analyses have reversed their positions: amphioxus is

now viewed as the ‘basal chordate’ [95], and tunicates
as the sister group, or closest relatives, of the verte-
brates [96] (Fig. 3). Thus, our understanding of the
evolution of a certain protein in the Chordata requires
information on the corresponding protein in the sub-
phylum Cephalochordata. The present search for the
database of the amphioxus (lancelet) Branchiostroma
floridae ( rev-
ealed that, in addition to Nox2, Nox5, and Duox,
Nox4 exists in the Cephalochordata. It is known that
Nox4 is present in Ciona intestinalis (the Urochordata)
[97] but not in the sea urchin Strongylocentrotus purpu-
ratus of the phylum Echinodermata, another major
group of the Chordata [90]. Therefore, Nox4 appears
to have branched from a root close to Nox2 with the
emergence of the Chordata. Nox1 is found from fishes
to mammals, but not in the Urochordata or Cephalo-
chordata, suggesting that this oxidase probably arose
with the emergence of the Vertebrata. Nox3 has
emerged most recently, probably from a common
ancestor of birds and mammals, because it is found
solely in mammals and birds, but not in fishes or
amphibians [98] (Fig. 3).
The membrane protein p22
phox
has been demon-
strated to be complexed with mammalian Nox1–Nox4.
Consistent with this, p22
phox
is absent in the Ecdyso-

zoa, where the Nox1–4 subfamily is absent, but widely
distributed in species that have Nox2, including the
choanoflagellate Mo. brevicollis and the cnidarian
Ne. vectensis. The known exceptions are the leech Cap-
itella species (the Annelida) and the sea urchin S. pur-
puratus (the Echinodermata). Nox2 of these species or
groups might be stable without p22
phox
, leading to loss
of the p22
phox
gene. It may also be possible that the
p22
phox
gene escaped cloning or correct sequencing for
unknown reasons, which is known to often occur in
various genome projects.
Regulation of the Nox1–4 subfamily
in animals
The phagocyte oxidase gp91
phox
⁄ Nox2 in mammals is
dormant in resting cells, but becomes activated during
phagocytosis to produce superoxide, a precursor of
microbicidal ROS. The oxidase activity is spatially and
temporally restricted to the phagosome, as inappropri-
ate or excessive production of ROS results in damage
to surrounding cells and severe inflammation. Activa-
tion of gp91
phox

⁄ Nox2 requires stimulus-induced mem-
brane translocation of p47
phox
, p67
phox
, p40
phox
, and
Rac, i.e. formation of the active oxidase complex at
the membrane (Fig. 7A). The essential role of these
regulatory proteins is evident from the following two
lines of evidence. First, the phagocyte NADPH oxi-
dase activity can be reconstituted in a cell-free system
with gp91
phox
, p22
phox
, p47
phox
, p67
phox
, and Rac, using
an anionic amphiphile, e.g. arachidonic acid, as an
in vitro stimulant. Second, defects in any of the four
genes encoding gp91
phox
, p22
phox
, p47
phox

and p67
phox
cause the primary immunodeficiency chronic granu-
lomatous disease [11,12].
In the cytoplasm of resting cells, p47
phox
, p67
phox
and p40
phox
form a ternary complex, whereas Rac is
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3257
complexed with Rho GDP dissociation inhibitor.
Upon cell stimulation, the three phox proteins are
en bloc recruited to the membrane; on the other hand,
Rac translocates independently but without Rho GDP
dissociation inhibitor, which remains in the cytoplasm.
Although activation of gp91
phox
⁄ Nox2 complexed with
p22
phox
in cells absolutely requires p47
phox
, p67
phox
and
Rac as cytosolic regulators, p47
phox

is dispensable for
cell-free activation in the presence of excess amounts
of p67
phox
and Rac [99,100]. It is thus considered that
p47
phox
functions as an organizer, whereas p67
phox
serves as an activator that directly participates in
gp91
phox
⁄ Nox2 activation.
Nox1 is abundantly expressed in colon epithelial
cells and also in vascular smooth muscle cells
[101,102], and seems to be involved in angiotensin II-
mediated hypertension [103–105]. Nox1, as well as
Nox2, forms a complex with p22
phox
[88,106].
Although Nox1 is also inactive without an organizer
or an activator, it generates superoxide in the presence
of the p47
phox
paralog Noxo1 (Nox organizer 1) and
the p67
phox
paralog Noxa1 (Nox activator 1) [106–109]
(Fig. 7B). Rac is directly involved in Nox1 activation
as well [110–112].

In the inner ear of mice, Nox3 plays a crucial role in
formation of otoconia, tiny mineralized structures that
are required for perception of balance and gravity
[113]. Although Nox3 also forms a functional heterodi-
mer with p22
phox
, this oxidase is capable of producing
superoxide in the absence of an organizer or an activa-
tor [88]. The superoxide-producing activity can be
strongly enhanced by p47
phox
, Noxo1, and p67
phox
[89,110,114,115]. In the presence of p67
phox
or Noxa1,
Nox3 activity is upregulated by Rac [110,116].
Although it is well known that Nox4 is highly
expressed in epithelial cells of the adult and fetal kid-
ney [117,118] and vascular endothelial cells [91,119], its
function remains to be elucidated. Nox4 is complexed
with p22
phox
as well [80,82,84], and constitutively gen-
erates superoxide in an NADPH-dependent manner
[120]. The mechanism of Nox4 regulation is largely
unknown at present: Nox4-mediated superoxide gener-
ation appears to be independent of p47
phox
, Noxo1,

p67
phox
, or Noxa1 [89,117,118], whereas the role of
Rac remains controversial [79,121].
The organizer p47
phox
is required for
Nox2 activation
The oxidase organizer p47
phox
is a 390 amino acid pro-
tein that contains, from the N-terminus, a phagocyte
oxidase (PX) domain, tandem SH3 domains, and a
PRR (Fig. 7). The two SH3 domains cooperatively
interact with the PRR in the C-terminal cytoplasmic
region of p22
phox
, an interaction that is essential for
both membrane translocation of p47
phox
and oxidase
activation [122–124]. The tandem SH3 domains sand-
wich a short PRR of p22
phox
(amino acid resi-
dues 151–160), containing a polyproline II (PPII) helix
[125–127] (Fig. 8). Pro152, Pro156 and Arg158 in the
human p22
phox
PRR are indispensable for the inter-

action with p47
phox
: Pro152 and Pro156 are recognized
by the N-terminal SH3 domain, whereas Arg158
directly contacts with the C-terminal one [127] (Fig. 8).
On the other hand, Pro151, Pro155, Pro157 and
Pro160 are also involved in binding to p47
phox
but to a
lesser extent [127].
The gene encoding p22
phox
exists in a wide variety of
animals and also in the Choanoflagellata, a sister
group of the Metazoa (Animalia) [128,129]; the p22
phox
gene is absent in the Ecdysozoa, which is consistent
with the absence of the Nox1–4 subfamily in this clade
(Fig. 9). The p22
phox
region comprising the N-terminal
cytoplasmic region and the two transmembrane seg-
ments is functionally important [130] and well con-
served in the Metazoa (animals) and Choanoflagellata,
whereas the C-terminal cytoplasmic region is highly
variable except for the PRR (Fig. 8). The three resi-
dues indispensable for binding to p47
phox
(Pro152,
Pro156 and Arg158 in human p22

phox
) are invariant in
all known animal p22
phox
proteins (Fig. 8), although
identifiable p47
phox
exists solely in the phylum Chorda-
ta, as described later (Fig. 9).
Fig. 7. Activation of gp91
phox
⁄ Nox2 and Nox1. (A) Interactions
required for activation of gp91
phox
⁄ Nox2. Interactions in a resting
state are indicated by blue arrows, stimulus-induced interactions by
arrows in magenta, and constitutive interactions by green arrows.
(B) Interactions required for activation of Nox1. Interactions in a
resting state are indicated by blue arrows; and stimulus-induced
interactions by arrows in magenta. T1, T2, T3 and T4, tetratricopep-
tide repeats 1, 2, 3 and 4, respectively; AD, activation domain.
Structure, regulation and evolution of Nox H. Sumimoto
3258 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
In the resting state, the two SH3 domains of p47
phox
are inaccessible to the target protein, as they are masked
via an intramolecular interaction with the C-terminally
flanking region called the autoinhibitory region (AIR)
[125,131–133] (Fig. 7). During phagocytosis of invading
microbes or with soluble stimuli such as N-formyl che-

motactic peptide and PMA, p47
phox
undergoes phos-
phorylation at multiple Ser residues, several of which
are present in the AIR [134,135]. Simultaneous phos-
phorylation of Ser303, Ser304 and Ser328 in the AIR, in
cooperation with other agonists such as arachidonic
acid, induces unmasking of the SH3 domains [136]; as a
result, the bis-SH3 domain interacts with the PRR of
p22
phox
[125–127]. The SH3-mediated interaction with
p22
phox
also participates in p47
phox
-dependent enhance-
ment of Nox3 activation [88].
The domain architecture of p47
phox
(PX, bis-SH3,
AIR, and a p67
phox
-binding PRR) is conserved from
fishes to mammals (Fig. 10). In addition, the genome
of the amphioxus B. floridae, which belongs to the sub-
phylum Cephalochordata, a basal group of the Chor-
data [96], contains the gene for p47
phox
(http://

genome.jgi/psf.org/euk_home.html), in which protein
the whole domain structure is duplicated (Fig. 10). The
presence of the p47
phox
gene in the Cephalochordata
indicates that it emerged early in chordate evolution.
On the other hand, a typical p47
phox
is absent in the
ascidian Ci. intestinalis of the Urochordata, suggesting
that the p47
phox
gene has been lost in the ascidian line-
age. This organism contains a typical p22
phox
carrying
the conserved PRR [97], which may imply the presence
of its interacting protein. Although Ci. intestinalis has
been reported to possess a protein harboring a PX
domain and three SH3 domains, but lacks an AIR and
a PRR [97] (Fig. 10), there is no experimental evidence
showing that this protein participates in oxidase activa-
tion. The residues in the AIR that strongly interact
with the SH3 domains are all conserved in chordate
p47
phox
proteins (Fig. 11): in human p47
phox
, the Pro–
Pro–Arg sequence at the AIR N-terminus adopts a

PPII helix conformation, which is lined in the groove
formed between the tandem SH3 domains. Among
these residues, Pro199 and Pro200 undergo hydro-
phobic interactions as addition to forming hydrogen
bonds via their backbone carbonyl groups; Arg201
contacts Asp243 and Glu244 via electrostatic inter-
actions. In addition, Ser303 forms a hydrogen bond
with the side chain of Glu241 (Fig. 11). The PPRRS
region, however, is not sufficient to make the SH3
domains inaccessible to p22
phox
[131]; its C-terminal
extension is required for efficient masking of the SH3
domains [131]. Besides Ser303, Ser310 and Ser328 form
hydrogen bonds with the side chains of Glu211 and
Arg267, respectively (Fig. 11). It is known that phos-
phorylation of Ser303 and Ser328 plays a crucial role
in disruption of the intramolecular interaction to acti-
vate the bis-SH3 domain [131,136]. In the p47
phox
autoinhibited structure, furthermore, Arg318 under-
goes an electrostatic interaction with Asp261 [132],
whereas Ile305 is located in a hydrophobic pocket of
SH3(N), and Tyr324 probably points into a hydro-
phobic pocket formed by the linker region and SH3(C)
(Fig. 11). Intriguingly, the amino acids of the AIR and
Fig. 8. (A) A model for the structure of p22
phox
and alignment of
the PRR. Large asterisks indicate residues crucial for interaction

with the p47
phox
bis-SH3 domain, and small asterisks denote resi-
dues involved in the interaction. Hs, H. sapiens; Mm, Mus muscu-
lus; Tr, Ta. rubripes; Dr, Da. rerio; Ci, Ci. intestinalis; Bf, B. floridae;
Lg, Lot. gigantea; Nv, Ne. vectensis; Mb, Mo. brevicollis. (B) The
complex structure of the p47
phox
bis-SH3 domain with the p22
phox
PRR. Crucial residues in the p22
phox
PRR (Pro152, Pro156, and
Pro158) are drawn as red sticks. Secondary structures in the
p22
phox
PRR complexed with the p47
phox
bis-SH3 domain are indi-
cated below the sequence of human p22
phox
. The figure was
drawn using
PYMOL software () and the
Protein Data Bank coordinates 1WLP.
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3259
SH3 domains, which are involved in the intramolecular
interaction as described above, are invariant among
vertebrate and amphioxus p47

phox
: the exceptions are
that Ser328 is replaced by Thr in Branchiostoma and
that Glu241 is replaced by Gln in the puffer fish
Takifugu rubripes and by Ile in amphioxus. Thus,
autoinhibition of the p47
phox
SH3 domains and phos-
phorylation-mediated regulation are probably con-
served in p47
phox
of chordates.
It was recently proposed that the p47
phox
paralog
Noxo1 in the medaka fish Oryzias latipes retains an
AIR [94]. However, it seems unlikely that this region is
functional, as fewer than a half of the crucial residues
described above are conserved in this fish Noxo1. In
addition, an AIR is absent in Noxo1 of the zebrafish
Danio rerio, which belongs to a more basal group than
medaka in the teleost fish [137], suggesting that the
AIR was lost at an early stage after branching from
the p47
phox
gene.
The homology between the N-termini of p47
phox
and
p40

phox
was recognized when the p40
phox
cDNA was
cloned [138]. It was subsequently pointed out that
p47
phox
exhibits a similarity to a region of the yeast
polarity protein Bem1 [139]. This module of about 120
amino acids was later found to exist in a variety of pro-
teins [140,141], and was named the PX domain [140].
The PX domain of p47
phox
is capable of binding to phos-
phoinositides such as phosphatidylinositol 3,4-bisphos-
phate [PtdIns(3,4)P
2
], albeit with a relatively low affinity
and specificity [142,143]. In neutrophils, PtdIns(3,4)P
2
is
predominantly formed from phosphatidylinositol
3,4,5-trisphosphate, a product of type I phosphatidyl-
inositol-3-kinase (PI3K), which is activated upon cell
stimulation. The binding requires Arg43 and Lys90 in
human p47
phox
[144–146] (Fig. 12). Intriguingly, in con-
trast to the complete conservation of Lys90, Arg43 is
replaced by Lys in the mouse and by Thr in the puffer

fish [147]. In contrast, the neighboring residue Arg42 is
well conserved; this residue is required for stability of
p47
phox
[12] but is not directly involved in binding to
phosphoinositides [145]. Like the p22
phox
-interacting
activity of the SH3 domains, the lipid-binding activity of
the PX domain is negatively regulated under resting
conditions [144,145]. The phosphorylation-induced con-
formational change of p47
phox
also renders the PX
domain accessible to membrane phosphoinositides. This
interaction, in cooperation with the binding to p22
phox
,
allows p47
phox
to be targeted to flavocytochrome b
558
,
which is crucial for phagocyte oxidase activation [144].
In addition to the phosphoinositide-binding pocket, the
PX domain may have a binding site for acidic phospho-
lipids such as phosphatidic acid and phosphatidylserine
Fig. 10. Domain structures of p47
phox
and

its related proteins. The total numbers of
amino acid residues of each protein are indi-
cated on the right: human, H. sapiens;
zebrafish, Da. rerio; ascidian, Ci. intestinalis;
amphioxus, B. floridae.
Fig. 9. Distribution of Nox2, p22
phox
,p47
phox
,
p67
phox
and p40
phox
in animals and choano-
flagellates. M. b., Mo. brevicollis; N. v.,
Ne. vectensis; L. g., Lot. gigantea; C. sp.I,
Capitella sp. I; C. e., Ca. elegans; D. m.,
Dr. melanogaster; S. p., S. purpuratus; B. f.,
B. floridae; C. i., Ci. intestinalis; D. r.,
Da. rerio; X. t., X. tropicalis; G. g., Ga. gallus;
H. s., H. sapiens. Note that it remains unclear
whether p22
phox
, p47
phox
, p67
phox
and p40
phox

are present or absent in the annelid Capitella
sp., because sequencing of the whole gen-
ome has not been completed. Amino acid
sequences of p22
phox
,p47
phox
,p67
phox
and
p40
phox
in the Cephalochordata B. floridae are
shown in supplementary Fig. S1.
Structure, regulation and evolution of Nox H. Sumimoto
3260 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
(Fig. 12), which facilitates membrane binding of the PX
domain [145,148]. Another lipid-binding activity may
explain well why the p47
phox
PX domain participates in
membrane translocation even in the absence of the
PI3K pathway products such as PtdIns(3,4)P
2
[144].
The two residues Lys55 and Arg70 in the proposed
second binding site (Fig. 12) are well conserved in
chordates, suggesting a role in oxidase activation.
Noxo1, the organizer for Nox1
activation

Noxo1 plays an essential role in Nox1 activation. This
organizer exhibits a domain architecture similar to that
of p47
phox
, except that it lacks an AIR (Fig. 10).
Noxo1 probably tethers the activator Noxa1 to Nox1:
Noxo1 functions via its PRR by interacting
with Noxa1 and via its SH3 domains by binding to the
Nox1 partner p22
phox
[106,110,111,149]. In cells
expressing Noxo1 and Noxa1, Nox1 generates super-
oxide without stimulants such as PMA, a potent
in vivo activator of gp91
phox
⁄ Nox2, although the super-
oxide production is further enhanced by PMA [106].
The constitutive activity of Nox1 appears to be at least
partially due to the absence of AIR in Noxo1; hence
its SH3 domains are accessible to p22
phox
even in the
resting state, in contrast to the p47
phox
SH3 domains
[106,115]. Regulation of the Noxo1 SH3 domains may
be more complicated than previously expected, as they
appear to interact intramolecularly with the N-terminal
PRR, albeit with low affinity [149]. It is likely that the
bis-SH3 domain is partly accessible to the p22

phox
PRR, and this intermolecular binding is facilitated by
disruption of the intramolecular interaction with the
p47
phox
PRR [149].
The PX domain of Noxo1 exhibits a phosphoinosi-
tide-binding activity, which is also crucial for activa-
tion of Nox1 [109,150,151]. In addition to its essential
role in Nox1 activation, Noxo1 is capable of enhanc-
ing Nox3-catalyzed superoxide production
[88,110,114,115,152]; the enhancement requires the PX
domain [150] and the SH3-mediated interaction with
p22
phox
[88,157].
Interaction of p47
phox
with the
activator p67
phox
The oxidase activator p67
phox
of 526 amino acids
translocates upon cell stimulation to the membrane in
Fig. 11. Intramolecular interaction of the bis-SH3 domain with the
AIR in p47
phox
. Residues crucial for the intramolecular interaction
with the bis-SH3 domain are highlighted (upper panel). The

sequence of the AIR in human p47
phox
and the consensus AIR
sequence among p47
phox
proteins derived from various species are
shown (middle panel). Large asterisks indicate residues crucial for
the intramolecular interaction with the bis-SH3 domain; small aster-
isks denote residues that play a role in the interaction. Secondary
structure elements of the p67
phox
-binding region in p47
phox
are indi-
cated below the sequence. The three Ser residues Ser303, Ser310
and Ser328 directly interact with residues in the bis-SH3 domain
(Gle241, Glu211, and Arg267, respectively) (lower panel). The
figures were drawn using
PYMOL software ()
and the Protein Data Bank coordinates 1UEC.
Fig. 12. Lipid-binding sites in the p47
phox
PX domain. Residues in
the phosphoinositide-binding and second anion-binding pockets are
shown in blue. The sulfates bound in the two pockets (in a crystal
of the p47
phox
PX domain [145]) are colored magenta. The figure
was drawn using
PYMOL software () and the

Protein Data Bank coordinates 1O7K.
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3261
a manner dependent on p47
phox
. p67
phox
contains two
SH3 domains (Fig. 7A), both of which play a role in
activation of the phagocyte NADPH oxidase [153].
Although the N-terminal (the first) SH3 domain is the
most conserved region in p67
phox
[154], it is unknown
how this module functions. The C-terminal SH3
domain participates in membrane translocation of
p67
phox
by specifically binding to the C-terminal PRR
of p47
phox
with high affinity [155–157]. The high affin-
ity and specificity are achieved by the following mecha-
nism: in addition to the canonical PxxP binding site of
p67-SH3(C), which is occupied by the eight PRR resi-
dues of p47
phox
(Gln362–Pro369), which adopt a PPII
helix conformation (Fig. 13), p67-SH3(C) makes direct
contacts with the remaining C-terminal segment of the

p47
phox
tail, which comprises two a-helices (Asp372–
Asn376 and Glu380–Ser386), linked by a turn of
Arg377–Ser379 [157] (Fig. 13). Structural analysis and
comparison of p47
phox
proteins from various species
reveal the consensus sequence xPxøPxRP(S ⁄ A)xxøIL-
xRC(S ⁄ T)xxT(K ⁄ R)(R ⁄ K)xØ, where x is any amino
acid and Ø is a hydrophobic residue (Fig. 13). The res-
idue adjacent to the invariant Cys is a Ser or a Thr;
the corresponding residue in human p47
phox
(Ser379) is
known to undergo phosphorylation during cell stimu-
lation [158]. This phosphorylation attenuates the bind-
ing to p67
phox
[158], and thus negatively regulates
oxidase activation [159].
The consensus sequence is also conserved in the
C-terminal region of Noxo1, which is capable of inter-
acting with p67
phox
as well as with Noxa1 [106]. The
interaction of the Noxa1 SH3 domain with the Noxo1
PPR (Fig. 7B) plays an important role in both mem-
brane localization of Noxa1 and activation of Nox1
[110,111].

Interaction of Rac with p67
phox
Rac, a member of the Rho-family small GTPases,
plays an essential role in gp91
phox
⁄ Nox2 activation
[160–162]. Among phagocytes, human neutrophils pre-
dominantly express Rac2, whereas both Rac1 and
Rac2 are present in monocytes ⁄ macrophages. A
patient with abnormal neutrophil function was shown
to have an inhibitory (dominant-negative) mutation in
Rac2, resulting in decreased oxidase activity and other
neutrophil function defects [163,164]. Upon cell stimu-
lation, Rac is recruited to the membrane independently
of p47
phox
or p67
phox
. The recruitment requires gera-
nylgeranylation of Cys189 at the C-terminus. At the
membrane, Rac is converted to the GTP-bound active
form via the function of guanine nucleotide-releasing
factors. Rac in the GTP-bound form directly interacts
with the p67
phox
N-terminal region of about 200 amino
acids, which is crucial for activation of gp91
phox
[165,166]. On the other hand, Cdc42 neither binds to
p67

phox
nor activates gp91
phox
.
In the Rac-binding region, four tetratricopeptide
repeat (TPR) motifs are found (Fig. 14): TPR motifs
are degenerate 34 amino acid repeats that are present
in a variety of organisms, ranging from bacteria to
humans, and are involved in a variety of protein–pro-
tein interactions [167]. The TPR domain of p67
phox
contains an insertion of 19 amino acids between the
a-helices of TPR3 and TPR4: the insertion contains
two short antiparallel b-strands and a 3
10
helical turn
[168,169], with the consensus sequence of LRGNxøID-
YxxLGøx(Y ⁄ F)KLx, where x is any amino acid and Ø
is a hydrophobic residue (Fig. 14). The b-hairpin inser-
tion and the loops connecting TPR1 and TPR2, and
TPR2 and TPR3, create the binding surface for Rac
[168] (Fig. 14). The invariant Arg in the b-hairpin
(position 32 of TPR3; Arg102 in human p67
phox
) plays
a key role in complex formation with Rac and oxidase
activation, and the side chain of this residue undergoes
direct hydrogen bonding interactions with main and
Fig. 13. Interaction of the p67
phox

C-terminal SH3 domain with the
p47
phox
C-terminus. (A) Ribbon diagram of the structure of the com-
plex of the p67
phox
C-terminal SH3 domain with the p47
phox
C-ter-
minal region. Residues involved in binding to the SH3 domains are
drawn as red sticks (residues in the PPII helix) or magenta sticks
(residues in the a-helices). The figure was drawn using
PYMOL
software () and the Protein Data Bank coordi-
nates 1K4U. (B) Secondary structure elements of the p67
phox
-bind-
ing region in p47
phox
are indicated below the sequence. Asterisks
indicate residues crucial for interaction with p67
phox
; the dot
denotes Ser379, which becomes phosphorylated upon cell stimu-
lation.
Structure, regulation and evolution of Nox H. Sumimoto
3262 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
side chain atoms of no less than four residues from
Rac [168]. Asp67 in the loop that connects TPR1 and
TPR2 (position 31 of TPR2) also makes direct con-

tacts with Rac (Fig. 14) and is completely conserved
among p67
phox
and its related proteins such as verte-
brate Noxa1 and fungal NoxR. In addition, the two
residues involved in binding to Rac, Ser37 in the loop
between the a-helices of TPR1 and TPR2 (position 1
of TPR2) and Asn204 in the b-hairpin (position 34 of
TPR3), are highly conserved among p67
phox
and its
related proteins (Fig. 14). In addition, substitution of
Glu for the invariant Arg in human Noxa1 (Arg103)
[106] and in fungal NoxR (Arg101) [170] results in a
loss of binding to Rac. Thus, these proteins probably
recognize Rac in the same way as p67
phox
.
Between the Rac-binding TPR domain and the
N-terminal SH3 domain, human p67
phox
contains a
PRR region, PPPRPKTP (amino acids 227–234),
which is well conserved among mammalian, avian and
amphibian p67
phox
proteins, but not among fish
p67
phox
[147] or mammalian Noxa1 [106]. Cell-free

experiments using a series of C-terminally truncated
mutants of p67
phox
showed that the PRR is dispensable
for oxidase activation in vitro [171]. Intriguingly, these
experiments also revealed that the region of amino
acids 200–212, C-terminal to the Rac-binding TPR
domain, plays an essential role in cell-free activation of
the NADPH oxidase: a truncated p67
phox
of amino
acids 1–212 is fully active, whereas a mutant protein of
amino acids 1–199 is completely inactive [171]. These
results were subsequently confirmed, and the short
region (amino acids 201–210) was named the ‘activa-
tion domain’ [172]. Among amino acids in the activa-
tion domain, Ala substitution for Val204 results in an
almost complete loss of gp91
phox
⁄ Nox2 activation, not
only under cell-free conditions [172], but also in intact
cells [173]. The corresponding mutation in the p67
phox
homolog Noxa1, the V205A substitution, attenuates
Nox1 activation [109]. Thus, both the Rac-binding
TPR domain and the activation domain are probably
minimal prerequisites for p67
phox
function.
Careful alignment of regions C-terminal to the TPR

domain of various Nox activators reveals the con-
sensus sequence of the activation domain: (V ⁄ L)
xxLxxKD(Y ⁄ F)LGKAxVV(A ⁄ S)(S ⁄ A) (amino acids
190–208) (Fig. 14). Leu193 (the third residue in the
consensus), Asp197, Leu199, Gly200 and Val205 (the
amino acid number corresponds to that of human
p67
phox
) are completely conserved in all Nox activator
proteins, including p67
phox
, Noxa1, and NoxR. The
ninth residue, Tyr (equivalent to Tyr198 of human
p67
phox
), is common to p67
phox
proteins, but is
replaced by Phe in Noxa1.
Binding of Rac is believed to induce a conforma-
tional change in p67
phox
, which may allow the activa-
tion domain to act on gp91
phox
⁄ Nox2 [174–177].
However, it is unknown how the activation domain
functions at the atomic level. The extended activation
domain as proposed here (amino acids 190–208)
appears to be flexible or disordered, judging from the

crystal structure of an isolated p67
phox
TPR domain
(amino acids 1–203) complexed with Rac [168], and
that of a TPR domain alone (amino acids 1–213)
[169]; in the latter case, the N-terminus of the activa-
tion domain is a part of an a-helix (amino
acids 187–193) [169]. The activation domain in the
Rac-bound p67
phox
might adopt a defined structure
upon interaction with the gp91
phox
–p22
phox
complex. It
Fig. 14. The complex of GTP-bound Rac with the N-terminal TPR
domain of p67
phox
. (A) The b-hairpin insertion in the TPR domain of
p67
phox
is colored blue. Arg102 and Asp67, each involved in binding
to Rac, are drawn as blue and green sticks, respectively. GTP
bound to Rac is shown in magenta. The figure was drawn using
PYMOL software () and the Protein Data Bank
coordinates 1E96. (B) The amino acid sequences of the b-hairpin
insertion and activation domain (AD) in human p67
phox
are shown;

secondary structure elements are indicated below the sequence of
the b-hairpin insertion. The consensus sequences of the two
regions among p67
phox
proteins derived from various species are
also shown.
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3263
has been also proposed that GTP-bound Rac by itself
induces electron transport from NADPH by directly
interacting with gp91
phox
⁄ Nox2 [178]. In this case, the
insert helix of Rac (amino acids 123–135), a region
specific for the Rho-family GTPases, is suggested to
play a crucial role [174]; however, the importance of
this region remains controversial [179].
Interaction of p40
phox
with p67
phox
p67
phox
contains a PB1 domain [180] between the two
SH3 domains; via PB1 domain-mediated heterodimer-
ization, p67
phox
stably interacts with p40
phox
in phago-

cytes (Fig. 15). Human p40
phox
of 339 amino acids,
comprising PX, SH3 and PB1 domains, is not essential
for oxidase activation. This protein, however, enhances
recruitment of p67
phox
and p47
phox
to the membrane
[181], especially to the phagosomal membrane, and is
therefore considered to play a crucial role in oxidase
assembly at the phagosome [181–183].
The PB1 domains, comprising about 80 amino acid
residues, adopt a ubiquitin-like b-grasp fold, containing
two a-helices and a mixed five-stranded b-sheet [184–
189]. This module is classified into groups harboring an
acidic OPCA motif (also known as a PC motif) (type I),
the invariant Lys residue on the first b-strand (type II),
or both (type I ⁄ II) [180]. Heterodimeric assembly occurs
between type I and II PB1 domains [180]. The dimeriza-
tion involves specific electrostatic interactions involving
a conserved acidic Dx(D ⁄ E)GD region of the OPCA
motif from a type I PB1 domain and an invariant Lys
residue from a type II PB1 domain. The type II PB1
domain in human p67
phox
contains Lys355 on the
b1-strand, which directly participates in binding to
p40

phox
, membrane targeting of p67
phox
, and oxidase
activation [181]. The crystal structures of the p67
phox
–
PB1 and p40
phox
–PB1 complexes [187] demonstrate that
Lys355 of p67
phox
forms direct bonds with Asp289,
Glu291 and Asp293 in the DxEGD sequence of the
OPCA motif, which is presented on the PB1 domain
(type I) of human p40
phox
(Fig. 15); this is also consis-
tent with the findings that a mutant p40
phox
with the
D289A substitution neither enhances p67
phox
transloca-
tion nor supports superoxide production at the phago-
some [181,182]. Importantly, it is evident from the
crystal structure that even a basic residue such as Arg is
incapable of replacing Lys355. Besides the first electro-
static interaction, a contact of Lys382 (of the p67
phox

PB1 domain) with Asp302 (of the p40
phox
OPCA motif)
also contributes to p67
phox
binding to p40
phox
, but to a
much lesser extent (Fig. 15) [190]. Furthermore, Thr361
and Trp425 of p67
phox
and Arg296 of p40
phox
also make
intramolecular contacts, enhancing the interaction
between p67
phox
and p40
phox
[187]. In agreement with
the presence of the PB1 (type II)-containing p67
phox
in
the amphioxus B. floridae of the Cephalochordata
(Fig. 16), its genome contains the gene for p40
phox
of
346 amino acids, which comprises PX, SH3 and PB1
(type I) domains ( />html). Residues that play crucial roles in PB1 heterodi-
merization, especially the two Lys residues in p67

phox
and the five OPCA residues (Asp289, Glu291, Asp293,
Arg296 and Asp302 in human p40
phox
), are all conserved
in the amphioxus proteins, indicating that the p67
phox
–
p40
phox
interaction is maintained in the Chordata. In
contrast, p40
phox
is absent in other phyla, including the
sea urchin S. purpuratus of the Echinodermata, another
phylum that belongs to the deuterostomes. Thus,
p40
phox
appears to have emerged in the Chordata.
Translocation of p40
phox
to the phagosome is proba-
bly mediated via the N-terminal PX domain. This PX
Fig. 15. Upper panel: domain architecture of p40
phox
and p67
phox
.
Middle panel: ribbon diagram of structures of the p40
phox

–p67
phox
PB1 complex. The notations of these secondary structural ele-
ments are indicated, and the OPCA motif p40
phox
is highlighted.
Lower panel: basic residues (p67
phox
) that directly interact with con-
served acidic residues of the OPCA motif (p40
phox
). Secondary
structure elements of the OPCA motif are indicated below the
sequence. The figures were drawn using
PYMOL software (http://
www.pymol.org) and the Protein Data Bank coordinates 1OEY.
Structure, regulation and evolution of Nox H. Sumimoto
3264 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
domain is capable of specifically and strongly binding
to phosphatidylinositol 3-phosphate [PtdIns(3)P]
[142,143,191], a lipid that is produced by type III
PI3K and is highly enriched in the phagosomal mem-
brane [192,193]. The PtdIns(3)P-binding activity, how-
ever, appears to be normally suppressed via an
intramolecular interaction with the PB1 domain
[194,195]. The high affinity and specificity for
PtdIns(3)P requires the following three residues in
human p40
phox
[196]: Arg58 undergoes extensive inter-

actions with the 3-phosphate moiety of the bound
PtdIns(3)P, NH2 and NE in the side chain of this resi-
due forming hydrogen bonds with oxygens of the
3-phosphate; NH1 and NH2 of Arg105 form hydrogen
bonds with the 4-OH and 5-OH of the inositol moiety;
and the aromatic ring of Tyr59 stacks against one face
of the inositol ring (the face with the axial 2-OH)
(Fig. 16). In addition, Lys92 and Arg60 also contribute
to binding to PtdIns(3)P but to a lesser extent
(Fig. 16): these residues make hydrogen bonds with
the nonbridging oxygens of the 1-phosphoryl group,
although the contribution of Arg60 is much less than
that of Lys92 [196]. The p40
phox
residues involved in
binding to PtdIns(3)P, except Arg60, are completely
conserved from amphioxus ( />euk_home.html) to fishes and mammals [147], suggest-
ing that p40
phox
plays the conserved role in recruiting
the p47
phox
–p67
phox
–p40
phox
complex correctly to the
phagosome.
In contrast to the PX and PB1 domains, the role of
the SH3 domain in p40

phox
remains obscure, although
it is capable of binding to p47
phox
very weakly
[158,197].
Origin of p67
phox
-like proteins
functioning via interaction with Rac
Two major subgroups in animals are protostomes and
deuterostomes, the latter of which includes the phyla
Chordata and Echinodermata (Fig. 9). As described
above, human p67
phox
comprises the Rac-binding TPR
domain, the activation domain, the N-terminal SH3
domain, the PB1 domain (type II), and the C-terminal
SH3 domain (Fig. 17). Such a canonical p67
phox
is
found not only in the Vertebrata (from fishes to
mammals) but also in the Cephalochordata, a basal
group of the Chordata, as shown in the present search
for the database of the amphioxus B. floridae (http://
genome.jgi/psf.org/euk_home.html). Thus, the domain
architecture of p67
phox
has arisen with the emergence
of the Chordata. On the other hand, p67

phox
seems to
be absent in the ascidian Ci. intestinalis of the Uro-
chordata, a sister group of the Vertebrata, suggesting
loss of the p67
phox
gene in this lineage. Instead, an
ascidian protein that contains solely a Rac-binding
TPR domain but not the other domains of p67
phox
(Fig. 17) has been considered as a functional p67
phox
homolog [97]; however, its identity as a protein that
supports oxidase activation awaits functional analysis.
Noxa1, a p67
phox
paralog for Nox1 activation, seems
to have emerged in the Vertebrata (Fig. 17), which is
consistent with the appearance of Nox1 and the Nox1
organizer Noxo1 in this subphylum of the Chordata.
The coincidence of the emergence of Nox1 and its reg-
ulatory proteins may be related to two rounds of
whole genome duplication that occurred in early verte-
brate evolution [198,199]. Noxa1 of the zebrafish
Da. rerio almost retains the domain architecture of
p67
phox
but harbors only a remnant of the N-terminal
(the first) SH3 domain (Fig. 17). Its activation domain
is of the Noxa1 type: the Tyr residue conserved in

p67
phox
is replaced by Phe as in other Noxa1 proteins
(Fig. 14). As zebrafish Noxa1 appears to contain an
intact PB1 domain, it might function by interacting
with p40
phox
[94]. On the other hand, Nox1 of the frog
Xenopus tropicalis has an almost complete N-terminal
SH3 domain and a nonfunctional PB1 domain
(Fig. 17): the PB1-like region lacks crucial residues
Fig. 16. Ribbon diagram of structure of the p40
phox
PX domain
complexed with PtdIns(3)P. Residues interacting with PtdIns(3)P
via hydrogen bonds (Arg58, Arg60, Lys92, and Arg105) are drawn
as blue sticks, and Tyr59, which makes hydrophobic contacts with
the inositol ring, is drawn as green sticks. PtdIns(3)P is shown in
magenta. The figure was drawn using
PYMOL software (http://
www.pymol.org) and the Protein Data Bank coordinates 1H6H.
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3265
such as the irreplaceable Lys (corresponding to Lys355
in human p67
phox
), indicating that amphibian Noxa1 is
not expected to bind to p40
phox
. Human Noxa1 does

not contain an N-terminal SH3 domain but has a PB1
domain-like region that lacks the invariant Lys:
indeed, human Noxa1 fails to interact with p40
phox
[106]. In addition, it was reported that the irreplace-
able Lys is substituted with Arg in Noxa1 of the
chicken Gallus gallus [94], and the present analysis has
revealed that no identifiable PB1 domain is found in
avian (chicken) Noxa1 (Fig. 17); these findings indicate
that p40
phox
does not participate in activating Nox1 in
the chicken. Similar to the tail-to-tail interaction
between p67
phox
and p47
phox
, Noxa1 is capable of
binding to Noxo1 via its C-terminal SH3 domain
(Fig. 7B), which plays a crucial role in formation of
the active Nox1 complex [110–112,200].
A protein homologous to p67
phox
exists in the sea
urchin S. purpurtatus, a member of the Echinodermata
(Genebank accession number XP_781983] (Fig. 17).
This protein contains two sets of the p67
phox
core cas-
sette, comprising the Rac-binding TPR domain and

activation domain (Fig. 17): the sequences of the Rac-
binding insert region and the activation domain are
almost completely fitted to the consensus ones
(Fig. 14). The echinodermal p67
phox
harbors an SH3
domain at the C-terminus, like vertebrate homologs,
but lacks a p40
phox
-binding PB1 domain, which seems
to be consistent with the absence of p40
phox
in this
species. In animals, a p67
phox
-like protein is present
also in the limpet Lot. gigantea of the Mollusca, a
member of the protostomes [94]. This protein contains
a Rac-binding TPR domain and an activation domain,
but not other known modular domains or motifs
(Fig. 17). Although the two species S. purpurtatus and
Lot. gigantea do not seem to have a recognizable
p47
phox
homolog, the echinodermal and molluscan
p67
phox
-homologous proteins might participate in
oxidase activation without cooperating with p47
phox

.It
is known that p47
phox
is dispensable for gp91
phox
⁄ Nox2
activation in the presence of large excess amounts of
p67
phox
and Rac in a cell-free system [99,100], and
that, even in a whole cell system, p67
phox
functions in
activation of Nox3 (but not gp91
phox
⁄ Nox2) in a
manner independent of p47
phox
: p67
phox
is capable of
activating Nox3 in cells lacking p47
phox
[88,115] and
also in cells expressing a mutant p22
phox
that is
defective in binding to p47
phox
[88].

The fungi, a major group of the eukaryotic super-
group Opisthokonta, have three Nox-family members:
NoxA and NoxB are close to animal Nox2; and NoxC
has an N-terminal cytoplasmic extension containing a
single EF-hand motif [56,57,59]. The fungal oxidases
are involved in a variety of biological events such as
development of sexual fruiting bodies and ascospore
germination [59,201], and symbiotic interactions such
Fig. 17. Domain structures of p67
phox
and
its related proteins. The total number of
amino acid residues of each protein is indi-
cated on the right: human, H. sapiens;
avian, Ga. gallus; amphibian, X. tropicalis;
zebrafish, Da. rerio; ascidian, Ci. intestinalis;
amphioxus, B. floridae; molluscan, Lot.
gigantea; fungal, Ep. festucae; amoebal,
Di. scoideum. T1, tetratricopeptide repeat 1;
T2, tetratricopeptide repeat 2; T3, tetratrico-
peptide repeat 3; T4, tetratricopeptide
repeat 4; AD, activation domain.
Structure, regulation and evolution of Nox H. Sumimoto
3266 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
as that between the clavicipitaceous fungal endophyte
Epichloe
¨
festucae and its grass host Lolium perenne
[202].
It has recently been shown that a p67

phox
-like oxi-
dase activator, named NoxR (Nox regulator), exists in
fungi possessing the NoxA gene [57,170], whereas
p47
phox
or p22
phox
appears to be absent in fungi or so
divergent that it has escaped identification. NoxR con-
tains a typical core cassette of p67
phox
, comprising a
Rac-binding TPR domain and an activation domain,
suggestive of its role in Nox activation (Fig. 17).
Indeed, NoxR in Ep. festucae is involved in ROS pro-
duction by NoxA; a noxR deletion mutant confers a
defect in fungus–grass mutualistic symbiosis, which is
similar to that observed for a noxA mutant; and the
phenotype in the noxR deletion mutant is comple-
mented by expression of the wild-type NoxR, but not
by a mutant NoxR carrying the R101E substitution,
indicative of the crucial role of Rac [170]. In addition,
NoxR harbors a PB1 domain of type II [170,180]; a
fungal protein carrying a type I PB1 domain might be
involved in oxidase activation, although a p40
phox
-like
protein is not found in fungi. Thus, a Rac-interacting
Nox activator appears to have evolved in the super-

group Opisthokonta and possibly also in the super-
group Amoebozoa, as described below.
The social amoeba Di. discoideum, which belongs to
the Amoebozoa, is known to have three Nox enzymes
[55]. Two of them lack an EF-hand and are close to
fungal NoxA and NoxB [55,57], which may imply the
presence of a Nox activator. The Dictyostelium genome
contains a gene encoding a protein similar to p67
phox
,
but not one homologous to p47
phox
, p40
phox
, or p22
phox
[55]. The p67
phox
-like protein contains four TPR
motifs, a type II PB1 domain, and a WW domain
(Fig. 17), and is expected to bind to Rac, because
amino acids that participate in the interaction with this
GTPase (Fig. 14) are well conserved in the TPR
domain, including Arg101, which is equivalent to
Arg102 of human p67
phox
. However, the Dictyostelium
protein does not harbor an identifiable activation
domain. Future studies should be addressed to deter-
mine whether this protein indeed serves as an oxidase

activator.
Another mechanism whereby Rac
activates Nox: direct binding of Rac
to Rboh
As described above, the three p67
phox
-like proteins in
the Opisthokonta, namely mammalian p67
phox
, mam-
malian Noxa1, and fungal NoxR, have been experi-
mentally proved to collaborate with Rac in activating
Nox enzymes that lack an EF-hand motif; on the other
hand, Rac does not seem to participate in regulation
of the animal EF-hand-containing oxidases Nox5 and
Duox [68,203]. In contrast, in the eukaryotic super-
group Plantae, Rac (also known as Rop) plays a cru-
cial role in activation of Rboh [79–81], which harbors
two EF-hand motifs that directly bind to Ca
2+
[204].
Plants do not appear to have a gene for p67
phox
or its
homologous protein; although there exist proteins con-
taining TPR motifs and a PB1 domain [94,180], they
do not retain conserved Rac-binding residues and are
therefore not expected to function by interacting with
Rac. Instead, Rac directly binds to the N-terminal
region of Rboh containing two EF-hand motifs [76]

(Fig. 18). A current model for Rboh activation is as
follows [76,77]: the stimulus-elicited initial (weaker) ele-
vation in cytoplasmic Ca
2+
may activate Ca
2+
-depen-
dent protein kinases, which phosphorylate the
N-terminal region of Rboh to induce a conformational
change; this change may expose the N-terminus for the
interaction with Rac; the subsequent binding of Rac
activates the superoxide-producing activity of Rboh;
and, in a second (more prolonged) phase of cytoplas-
mic Ca
2+
accumulation, which may be induced by
ROS and⁄ or the initial Ca
2+
elevation, induced bind-
ing of Ca
2+
to the EF-hands suppresses the Rac–Rboh
interaction. Thus, Ca
2+
is considered to play a nega-
tive role in Rboh regulation via direct interaction with
the EF-hands, which is in contrast to the positive roles
of Ca
2+
–EF-hand interaction in the activation of

Nox5 and Duox in animals [65,66].
Perspectives
About a decade has passed since the discovery of the
gp91
phox
⁄ Nox2 homologs in a variety of eukaryotes,
and increasing attention has been paid to the Nox
family. However, many unanswered questions still
remain. The mechanism of electron transfer in Nox
Fig. 18. Mechanisms whereby Rac activates animal Nox2 and plant
Rboh.
H. Sumimoto Structure, regulation and evolution of Nox
FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS 3267
molecules is largely unknown at present. In particular,
there is no information at the atomic level about how
electron transfer is induced by interaction of Nox with
regulatory proteins such as a p67
phox
-related protein
and Rac. Studies of the crystal structures of full-length
Nox proteins and their complexes will help immensely
to extend our understanding of the molecular mecha-
nisms that govern oxidase activation.
The Nox family exists in a wide variety of eukary-
otes (Fig. 3), but oxidases from only a limited number
of species have been experimentally investigated. As
shown in Fig. 1, members of the Nox family contain a
pair of His residues in the third transmembrane a-helix
with 13 intervening amino acids (His101 and His115 in
human gp91

phox
⁄ Nox2), with the other pair in the fifth
helix (His209 and His222 in human gp91
phox
⁄ Nox2)
being separated by 12 amino acids. However, proposed
Nox members in the red algae Cyanidioschyzon mero-
lae, Cyanidioschyzon crispus, and Porphyra yezoensis,
which belong to the eukaryotic supergroup Plantae
[58,98], contain 13 intervening amino acids between
His residues in both the third and the fifth transmem-
brane segments, like the fungal Fre family (Fig. 1). It
thus seems interesting to test whether these oxidases
(called NoxD) indeed reduce molecular oxygen to
superoxide, or instead use Fe
3+
as a substrate. In
addition, it was reported that flavocytochromes, carry-
ing 14 and 13 intervening amino acids, are present in
the diatom Phaeodactylum tricornutum (supergroup
Chromalveolata), although typical Nox enzymes (with
13 and 12 intervening amino acids) exist in another
diatom, Tha. pseudonana [58]. Biochemical studies of
Nox enzymes derived from various eukaryotes are now
required to establish their identities as real Nox
enzymes or other distinct oxidoreductases and to clar-
ify their regulatory mechanisms.
p22
phox
, the membrane partner of the Nox1–4

subfamily, is present in various animals and in the
choanoflagellate Mo. brevicolli, and retains a conserved
PRR in the C-terminal cytoplasmic region (Figs 8 and
9). On the other hand, p47
phox
or Noxo1, a protein
that specifically binds via its bis-SH3 domain to the
PRR of p22
phox
, is found solely in an animal group of
the Chordata (Figs 9 and 10). Although it was pro-
posed that proteins with single PX domain and multi-
ple SH3 domains might function as p47
phox
[94,97],
experimental evidence supporting this hypothesis has
not yet been provided. Identification of a novel
p22
phox
-binding protein will improve our understand-
ing of regulation of the Nox1–4 subfamily enzymes.
In contrast to the finding that the presence of
p47
phox
or its homolog is restricted to the Chordata,
proteins homologous to p67
phox
exist in a variety of
eukaryotes (Figs 9 and 17). However, ta role in Nox
activation has been shown for only three types of

p67
phox
homolog: p67
phox
and Noxa1 in mammals, and
NoxR in fungi. Thus, it is worth testing whether pro-
teins carrying a Rac-binding TPR domain but not an
activation domain, like those found in the ascidian
Ci. intestinalis and the social amoeba Di. discoideum
(Fig. 17), are indeed involved in Nox regulation. It
would be also interesting to know how a p67
phox
-like
protein of the mollusc Lot. gigantea functions; this
protein contains both the Rac-binding TPR domain
and the activation domain, but lacks other known
modular interaction domains (Fig. 17).
As discussed in the present review, it remains
unknown whether the EF-hand-containing Nox sub-
families are descended from a single ancestor oxidase
that initially obtained EF-hand motifs. It seems more
likely that an EF-hand motif has been obtained inde-
pendently several times during evolution. Nox2, but
not Nox5 or Duox, has been found in the Choanofla-
gellata (a sister group of animals) and Cnidaria (a
basal group of animals), whereas these three oxidases
are widely distributed in protostomes and deuterosto-
mes (Fig. 3), suggesting that Nox5 and Duox, contain-
ing four and two EF-hands, respectively, diverged
after the emergence of a p22

phox
-binding Nox2-like oxi-
dase in animal evolution. Similarly, in fungi, the NoxC
subfamily containing a single EF-hand is found solely
in more evolved groups, such as the Sordariomycetes
and Dothideomycetes, whereas the Nox2-like
EF-hand-free subfamilies NoxA and NoxB are present
also in relatively basal groups such as the Chytridi-
omyceta and Basidiomycota [57], suggesting that the
NoxC subfamily has emerged at a later stage of fungal
evolution. Thus, the classification of the Nox family in
eukaryotes into the two major groups, depending on
the presence or absence of the EF-hand motif, does
not seem to reflect molecular evolution. Although
many studies using phylogenetic trees of the Nox fam-
ily have been performed, it should be noted that this
approach is sometimes misleading, e.g. due to ‘long
branch attraction’, when distantly related genes derived
from a wide variety of species are used [54,205–207].
Further increases in genomic data for various eukary-
otes will help to clarify the origins of Nox subfamilies.
The function of EF-hands is variable between Nox
subfamilies, which may be in agreement with their
sequence diversity [98]. The EF-hands of the animal
enzyme Nox5, in the Ca
2+
-bound state, appear to
interact with the Nox superdomain, leading to super-
oxide production [66]. In the resting state of Duox,
another EF-hand-containing Nox in animals, the

EF-hands may exert an inhibitory effect by normally
Structure, regulation and evolution of Nox H. Sumimoto
3268 FEBS Journal 275 (2008) 3249–3277 ª 2008 The Author Journal compilation ª 2008 FEBS
associating with the Nox superdomain; for Duox acti-
vation, the association is probably disrupted by bind-
ing of Ca
2+
to the EF-hands [65]. In plants, the
EF-hands of Rboh localize within the Rac-binding
region at the N-terminus, and binding of Ca
2+
to the
EF-hands prevents Rac from binding to the N-termi-
nal region, resulting in inactivation of this oxidase
[76]. Structural studies of EF-hands complexed with a
Nox superdomain or Rac will help to extend our
understanding of the molecular mechanisms that
govern oxidase regulation.
Acknowledgements
This work was supported in part by CREST of JST
(Japan Science and Technology Agency) and by
Grants-in-Aid for Scientific Research and Targeted
Proteins Research Program (TPRP) from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan.
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