Karen Bedard and Karl-Heinz Krause
Karen Bedard and Karl-Heinz Krause
Karen Bedard and Karl-Heinz Krause
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250 KAREN BEDARD AND KARL-HEINZ KRAUSE<br />
of other cytosolic factors, hence its designation as “organizer<br />
subunit.” The localization of p47 phox to the membrane<br />
brings the “activator subunit” p67 phox into contact<br />
with NOX2 (342) <strong>and</strong> also brings the small subunit p40 phox<br />
to the complex. Finally, the GTPase Rac interacts with<br />
NOX2 via a two-step mechanism involving an initial direct<br />
interaction with NOX2 (214), followed by a subsequent<br />
interaction with p67 phox (476, 508). Once assembled, the<br />
complex is active <strong>and</strong> generates superoxide by transferring<br />
an electron from NADPH in the cytosol to oxygen on<br />
the luminal or extracellular space.<br />
NOX2 can be regarded as a transmembrane redox<br />
chain that connects the electron donor (Fig. 1), NADPH<br />
on the cytosolic side of the membrane with the electron<br />
acceptor, oxygen on the outer side of the membrane. It<br />
transfers electrons through a series of steps involving a<br />
flavin adenine dinucleotide (FAD), binding to amino acids<br />
337HPFTLSA <strong>and</strong> 355IRIVGD (917) <strong>and</strong> two assymetrical<br />
hemes found in transmembrane domains III <strong>and</strong> V, with<br />
the inner heme binding to histidines H101 <strong>and</strong> H209 <strong>and</strong><br />
the outer heme binding to histidine H115 <strong>and</strong> H222 (262).<br />
In the first step, electrons are transferred from<br />
NADPH to FAD, a process that is regulated by the activation<br />
domain of p67 phox (658). NOX2 is selective for<br />
NADPH over NADH as a substrate, with K m values of<br />
40–45 �M versus 2.5 mM, respectively (160). In the second<br />
step, a single electron is transferred from the reduced<br />
flavin FADH 2 to the iron center of the inner heme. Since<br />
the iron of the heme can only accept one electron, the<br />
inner heme must donate its electron to the outer heme<br />
before the second electron can be accepted from the now<br />
partially reduced flavin, FADH. The force for the transfer<br />
of the second electron, while smaller (31 vs. 79 mV), is<br />
Physiol Rev VOL 87 JANUARY 2007 www.prv.org<br />
FIG. 3. Assembly of the phagocyte NADPH oxidase<br />
NOX2. The phagocyte NADPH oxidase was the first identified<br />
<strong>and</strong> is the best studied member of the NOX family. It<br />
is highly expressed in granulocytes <strong>and</strong> monocyte/macrophages<br />
<strong>and</strong> contributes to killing of microbes. In resting<br />
neutrophil granulocytes, NOX2 <strong>and</strong> p22 phox are found primarily<br />
in the membrane of intracellular vesicles. They<br />
exist in close association, costabilizing one another. Upon<br />
activation, there is an exchange of GDP for GTP on Rac<br />
leading to its activation. Phosphorylation of the cytosolic<br />
p47 phox subunit leads to conformational changes allowing<br />
interaction with p22 phox . The movement of p47 phox brings<br />
with it the other cytoplasmic subunits, p67 phox <strong>and</strong><br />
p40 phox , to form the active NOX2 enzyme complex. Once<br />
activated, there is a fusion of NOX2-containing vesicles<br />
with the plasma membrane or the phagosomal membrane.<br />
The active enzyme complex transports electrons from<br />
cytoplasmic NADPH to extracellular or phagosomal oxygen<br />
to generate superoxide (O 2 � ).<br />
still energetically favorable. However, the transfer of the<br />
electron from the inner heme to the outer heme is actually<br />
against the electromotive force between these two<br />
groups. To create an energetically favorable state, oxygen<br />
must be bound to the outer heme to accept the electron<br />
(175, 223, 917).<br />
NOX2 was first described in neutrophils <strong>and</strong> macrophages<br />
<strong>and</strong> is often referred to as the phagocyte NADPH<br />
oxidase. NOX2 is still widely considered to have a very<br />
limited, essentially phagocyte-specific tissue expression<br />
(e.g., Ref. 844), yet when tissue distribution of total mRNA<br />
from various organs is investigated, NOX2 appears to be<br />
among the most widely distributed among the NOX isoforms<br />
(Table 2). It is described in a large number of<br />
tissues, including thymus, small intestine, colon, spleen,<br />
pancreas, ovary, placenta, prostate, <strong>and</strong> testis (143).<br />
Mostly this wide tissue distribution is due to the presence<br />
of phagocytes <strong>and</strong>/or blood contamination in the tissues<br />
from which total mRNA has been extracted. However,<br />
there is now also increasing evidence at both the message<br />
<strong>and</strong> the protein level for expression of NOX2 in nonphagocytic<br />
cells, including neurons (806), cardiomyocytes<br />
(372), skeletal muscle myocytes (426), hepatocytes<br />
(739), endothelial cells (313, 434, 538), <strong>and</strong> hematopoietic<br />
stem cells (704).<br />
In phagocytes, NOX2 localizes to both intracellular<br />
<strong>and</strong> plasma membranes in close association with the<br />
membrane protein p22 phox (97, 394). In resting neutrophils,<br />
most of the NOX2 localizes to intracellular compartments,<br />
in particular secondary (i.e., specific) granules (26,<br />
97, 439) <strong>and</strong> tertiary (i.e., gelatinase-containing) granules<br />
(465). Upon phagocyte stimulation, there is a translocation<br />
of NOX2 to the surface as the granules fuse with the<br />
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