From the Department of Immunology, Lerner Research
Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, § Medical Biotechnology Center, University of Maryland
Biotechnology Institute, Baltimore, Maryland 21201, and
¶ Department of Pharmaceutical Sciences, University of Maryland
School of Pharmacy, Baltimore, Maryland 21201
Nitric-oxide synthases
(NOS,1 EC 1.14.13.39) are
hemeproteins that catalyze oxidation of L-arginine to
NO· and L-citrulline. Three main isozymes exist in
mammals that are regulated by distinct genes (1-6): a constitutive
neuronal NOS (nNOS or NOS I), (7, 8), an endotoxin- and
cytokine-inducible NOS (iNOS or NOS II) (9, 10), and a constitutive
endothelial NOS (eNOS or NOS III) (11). Although NOS have some unique
features that distinguish them from other hemeprotein monooxygenases
such as cytochrome P-450s, there are, nevertheless, a number of
similarities that allow comparisons to be made (12). One such finding
is that both enzymes inadvertently secrete O In NOS, electrons from NADPH are used to reduce and activate
O2 at the heme, with H2O generated as a
co-product. Synthesis of NO· requires the enzyme to cycle twice
(Fig. 1). In the first step, NOS consumes
1 mol of NADPH to hydroxylate L-arginine to
N
INTRODUCTION
Relevant NOS Enzymology
-hydroxyl-L-arginine, which is
an enzyme-bound intermediate. Thereafter, NOS consumes 0.5 mol of NADPH
to oxidize N
-hydroxyl-L-arginine
to citrulline and NO· (19). Given that NOS must bind and
activate O2 twice to generate NO· from
L-arginine, the enzyme would need to carefully control the quantity and tempo of electron transfer to minimize uncoupled O2 reduction during the reaction.
View larger version (11K):
[in a new window]
Fig. 1.
Stepwise NO synthesis by NOS.
Symbols ( , *) trace the sources of nitrogen and oxygen in
the products.
NOS subunits are comprised of two domains connected by a central
Ca2+/calmodulin-binding region (20). Fig.
2 illustrates the electron transport
pathway as proposed for a NOS II dimer. To accomplish O2
activation, electrons from NADPH transfer into a reductase domain that
contains FAD and FMN and then pass from the FMN to an oxygenase domain
that contains bound heme, H4B, and substrate. The reduced
heme can then bind and activate O2 for NO·
synthesis. A similar electron transfer sequence has been shown for
cytochrome P-450 (13, 21), although in that case the flavoprotein and
hemeprotein components are typically separate monomeric entities.
|
The reductase domain of NOS actually catalyzes three separate electron transfer reactions: (a) NADPH reduction of bound FAD via hydride transfer, (b) subsequent distribution of single electrons between FAD and FMN (disproportionation), and (c) electron transfer from reduced FMN to either the NOS heme or to an artificial acceptor like ferricytochrome c. Calmodulin binding to NOS speeds electron transfer at points a, b, and c in the reductase domains of NOS I and NOS III (22-25). As such the rates become equivalent to those seen in related flavoproteins like sulfite oxidase and cytochrome P-450 reductase (26). Calmodulin may function by relieving a repression brought on by two unique negative control elements that are present in the NOS reductase domains (27-32). One control element is located in the FMN module and the other at the C terminus of the reductase domain. Calmodulin may cause the control element in the FMN module to interact with the C-terminal element and relieve its repression of electron transfer from NADPH to FAD. Because NOS II contains tightly bound calmodulin and is missing the FMN module control element, its flavoprotein domain is never repressed regarding the three electron transfer reactions noted above (26, 27). Each flavin in NOS can shuttle between its fully oxidized, semiquinone, or two-electron reduced form (e.g. FAD, FADH·, and FADH2). However, thermodynamic measurements on the NOS I reductase domain indicate that only fully reduced FMN (e.g. FMNH2) is capable of reducing the ferric heme of the enzyme (33).
Oxygen activation by NOS heme appears to occur in steps and involves
transfer of two electrons singly to heme (Fig.
3). The first electron transfer to heme
can only come from the reductase domain (i.e.
H4B and L-arginine are not donors) (34).
However, the second electron provided to heme can derive either from
the reductase domain, H4B, or
N-hydroxyl-L-arginine (34-38).
Recent reports suggest that H4B is kinetically preferred
over the reductase domain as a source of the second electron (34-36)
and may discount electron donation by
N
-hydroxyl-L-arginine, although
its potential to donate cannot be ruled out based on theoretical
studies (39).
|
Importantly, NOS heme-oxy intermediates are unstable and will either
release O
![]() |
NOS-catalyzed Production of O![]() |
---|
NOS has been known to generate O 90 M
1 s
1,
physiological pH, Refs. 46 and 47) compared with self-dismutation (k = 3.0 × 105
M
1 s
1,
pH 7.4, Ref. 48) plus an inherent instability of the corresponding spin
trapped adducts.
![]() |
Electron Acceptors and Drugs That Boost NOS Release of
O![]() |
---|
One of the first studies that tested O
![]() |
Superoxide from NOS-containing Cells |
---|
The versatility of spin trapping allows free radicals to be
detected in cell suspensions (56). Not surprisingly,
NOS-dependent cellular secretion of NO· and
O. These findings
complement results with pure enzymes and show how a low intracellular
L-arginine concentration can predispose NOS to generate
O
![]() |
Oxygen Reduction Catalyzed by NOS Prosthetic Groups |
---|
How well NOS prosthetic groups transfer electrons to
O2 depends on the surrounding protein structure and on the
thermodynamics and kinetics of the electron transfer reactions. Such
biochemical data are available and can help explain NOS O
![]() |
Autooxidation of Bound Prosthetic Groups |
---|
NOS reductase domains belong to a class of flavoprotein
dehydrogenases that are sterically constrained against binding
O2 to their reduced flavins (60). This severely limits
two-electron reduction of O2 by the flavins to generate
H2O2, but typically it still allows
one-electron transfer to generate O1 (Table I),
which is about 1000 times slower than free FADH2 in aerated
solution (61). One of the two negative control elements present in the
NOS reductase domain appears to help protect the flavins from
autooxidation (31).
|
A similar situation exists for H4B in NOS. Free
H4B oxidizes in solution to generate O
![]() |
NOS Electron Transfer Kinetics |
---|
To understand how electron transfer into NOS flavins and heme relates to their capacity to reduce O2, Table I summarizes available kinetic data on flavin and heme reduction rates for the various NOS isozymes. Flavin reduction appears as a biphasic process that involves both FAD reduction by NADPH and electron disproportionation into FMN (23, 24, 27, 32, 34). These steps are relatively fast and are markedly enhanced upon calmodulin binding. Although differences exist between the three NOS isozymes, their rates of flavin reduction far exceed rates of flavin autooxidation in all cases. Interestingly, calmodulin binding has a negligible effect on flavin autooxidation, despite calmodulin increasing the rate of flavin reduction and causing conformational change in the FMN module of the reductase domain (32). These data establish the slow step in NOS flavin autooxidation to be their electron transfer to O2 and underscore the role of protein in minimizing this process.
Flavins in all three NOS isozymes transfer electrons more slowly to
their hemes than they receive electrons from NADPH (Table I). However,
heme reduction is still faster than flavin autooxidation. When
H4B is bound, the flavins reduce ferric heme more slowly than autooxidation of the ferrous heme-O2 species (Table I
and Fig. 3). This means that heme reduction should limit the rate of
reduced oxygen species production by substrate-free, calmodulin-bound NOS.
![]() |
New Role for H4B in O2 Activation |
---|
Recent evidence suggests that H4B may provide an
electron to the ferrous heme-O2 species during stepwise
O2 activation (35-37) (Fig. 3). The rate of this reaction
in NOS II (Table I) is faster than flavoprotein reduction of the ferric
heme. Thus, by quickly donating an electron H4B may
minimize oxidative decay of the ferrous heme-O2 species,
which is a process that competes kinetically and generates O
![]() |
Electron Flux from NOS to O2 |
---|
Rates of O2 reduction by substrate-free NOS relate
directly to their rates of NADPH oxidation when these are measured in
the absence of any other electron acceptor besides O2. The
relative contributions of NOS flavins and heme to the O2
reduction rate, as well as the effect of calmodulin binding, can be
discerned by comparing NADPH oxidation by isolated reductase
domains, heme-free NOS, or NOS whose heme reduction is inhibited with
sodium cyanide, imidazole, or
N-nitro-L-arginine.
Table II shows that rates of NADPH
oxidation are slow when only the NOS flavins are allowed to transfer
electrons to O2, even with calmodulin bound. Rates in NOS I
and II are substantially less than 0.1 s1,
but in NOS III those rates are reported to be 0.1 s
1. Although the reason for this difference
is unclear, it might be related to FAD and FMN molecules in solution
catalyzing electron transfer from NOS to O2 (42). Table II
also shows that NADPH oxidation rates are enhanced when NOS heme is
allowed to receive electrons and catalyze O2 reduction.
NADPH oxidation rates in substrate-free NOS isoforms vary
according to their rates of heme reduction (NOS I > NOS II > NOS III), consistent with this step being rate-limiting.
|
In general, these steady-state data are consistent with slow
autooxidation rates observed for the NOS flavoproteins (Table I) and
confirm that NOS flavins are relatively poor O
Although flavin autooxidation in NOS is expected to generate
O
![]() |
Summary |
---|
Four conclusions derive from the studies exploring NOS-generated
O2 µM) or
L-arginine (
100 µM) fall below levels
required to saturate the enzyme. In these circumstances, O
formation (57, 58). Indeed, certain pathologic
states might promote formation of ONOO
such as
ischemia/reperfusion injury (67). Formation of HO·, either
through metal ion-catalyzed H2O2 decomposition
(68) or from decomposition of ONOO
(69-71), at sensitive
cellular sites may also contribute to cytotoxicity. Finally, it is
worth noting that sequential formation of NO· and O
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Koustubh Panda for assistance and discussions.
![]() |
FOOTNOTES |
---|
* This minireview will be reprinted in the 2001 Minireview Compendium, which will be available in December, 2001. This work was supported in part by National Institutes of Health Grants RR-12257 and CA-69538 (to G. M. R.) and CA-53914 and GM-51491 (to D. S.).
To whom correspondence should be addressed: Dept. of
Pharmaceutical Sciences, University of Maryland School of Pharmacy, 725 W. Lombard St., Baltimore, MD 21201. Tel.: 410-706-0514; Fax: 410-706-8184; E-mail: grosen@umaryland.edu.
Published, JBC Papers in Press, March 9, 2001, DOI 10.1074/jbc.R100011200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: NOS, nitric-oxide synthase(s); H4B, (6R)-tetrahydrobiopterin.
![]() |
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