section 14.7
Other Reducing-Equivalent Transport and Oxygen-Consuming Systems
271
D-Amino acid oxidase is a flavoprotein located in per-
oxisomes. D-Amino oxidases catalyze the oxidation of a
D-amino acid to the corresponding keto acid:
c o o -
o
2
H
2
0
2
R
o -A m in o a c id
O
I
R— C — C O O " + N H
4
+
K eto acid
The Oj and H
2
O
2
produced in the above two reactions
are potentially toxic. Superoxide anion is also produced
by oxygen-reducing enzymes of phagocytes (neutrophils,
eosinophils, and mononuclear phagocytes) that defend the
host against invading organisms by producing reactive oxi-
dants from oxygen. The reducing equivalents are provided
by NADPH derived from the hexose monophosphate shunt
(Chapter 15).
Neutrophils are the most active participants in
phago-
cytosis.
Unstimulated neutrophils circulate in the blood-
stream with a life span of a few days. Upon bacterial in-
fection, neutrophils actively migrate to the infected site,
where they kill bacteria by the process of phagocytosis.
During phagocytosis a large amount of oxygen is con-
sumed by the neutrophils in a reaction termed the
res-
piratory burst.
The burst was initially thought to repre-
sent an energy requirement for phagocytosis; however, it
was subsequently found to be insensitive to inhibitors of
the mitochondrial respiratory chain (cyanide, antimycin)
and associated with an increased turnover of the hexose
monophosphate shunt (Chapter 15).
The oxygen consumed during phagocytosis is utilized
by a unique enzyme system termed the
respiratory burst
oxidase
or NADPH oxidase. The oxidase generates super-
oxide anion (O j), a one-electron reduced species, driven
by intracellular NADPH,
NADPH + 20
2
-> NADP+ + 20
2
+ H+
Superoxide anion is also the source of a number of other
microbicidal oxidants. It functions as an electron donor
and is converted to hydrogen peroxide by superoxide dis-
mutase:
2 0 2
+
2
H + ^ H
2 0
2
+
0
2
A number of highly reactive oxidizing agents are produced
from H
2
0
2
and 0
2
, such as the conjugate acid of super-
oxide, the hydroperoxy radical (H02), hydroxyl radical
(OH*), and hypochlorite (OC1- ). These reactive agents
are powerful microbicides:
O
2
T H
2 0
2
—
>
OH T OH
+ o
2
and
c r + H
2 0
2
o c r + h 2o
The former reaction is promoted by metal ions (e.g., Fe2+),
and the latter is catalyzed by myeloperoxidase present in
neutrophil granules. By acquiring a single electron, oxy-
gen can give rise to a variety of toxic products. For exam-
ple, tissue destruction is enhanced when x-ray treatment
is used in conjunction with hyperbaric oxygen.
The NADPH oxidase consists of five components. In the
unstimulated cell, two of the components are membrane-
bound (p
2 2
phox and gp91phox) and three components are
present in cytosol (p47phox, p67pho\ and P40phox). The des-
ignations gp, phox, and p represent glycoprotein, phago-
cytic oxidase, and protein, respectively. The membrane-
bound components occur as a heterodimeric flavohemo-
protein known as cytochrome
6 5 5
».
The NADPH oxidase complex is dormant in resting
phagocytes and becomes assembled and activated for su-
peroxide formation upon bacterial invasion. The respira-
tory burst is stimulated
in vitro
as well as
in vivo
by a vari-
ety of reagents, among which are phorbol esters (PMA,
phorbol 12-myristate 13-acetate), heat-aggregated IgG,
unsaturated fatty acids and analogues of bacterial peptides
(FMLP, formylmethyonyl-leucyl phenylalanine).
The importance of NADPH oxidase is highlighted by
chronic granulomatous disease
(CGD), a rare inherited
disease in which affected individuals are incapable of
mounting a sustained
respiratory burst.
As a consequence,
persons with CGD are incapable of generating the reactive
oxygen compounds necessary for the intracellular killing
of phagocytized microorganisms. The CGD patient suf-
fers severe and recurrent bacterial and fungal infections.
Studies using CGD patients’ neutrophils as well as a cell-
free system have helped identify the cellular components
responsible for Oj generation.
Four different mutations cause CGD and these cause
four defects in different components of the NADPH ox-
idase. These are the two membrane bound components
of cytochrome bssg and two cytosolic factors p47phox and
p
6 7
ph°x
|ocj for
four proteins have been mapped in
chromosomes X, 16, 7, and 1, respectively. CGD patients
are therefore classified as being either of an X-linked type
or autosomal recessive. All reported cases of CGD may
be explained by abnormalities in the genes coding for one
or more of these four components of the oxidase complex.
Recent studies have identified an additional cytosolic fac-
tor, p40phox and a small G protein (rac) as being involved
in the activation of the
0
2
generating system.
