Engineering nitric oxide synthase chimeras to function as NO dioxygenases

Article abstract of DOI:10.1016/j.jinorgbio.2016.03.002

Nitric oxide synthases (NOSs) catalyze a two-step oxidation of l-arginine to form nitric oxide (NO) and l-citrulline. NOS contains a N-terminal oxygenase domain (NOSoxy) that is the site of NO synthesis, and a C-terminal reductase domain (NOSred) that binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin mononucleotide (FMN) and provides electrons to the NOSoxy heme during catalysis. The three NOS isoforms in mammals inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) share high structural similarity but differ in NO release rates and catalytic properties due to differences in enzyme kinetic parameters. These parameters must be balanced for NOS enzymes to release NO, rather than consume it in a competing, inherent NO dioxygenase reaction. To improve understanding, we drew on a global catalytic model and previous findings to design three NOS chimeras that may predominantly function as NO dioxygenases: iNOSoxy/nNOSred (Wild type (WT) chimera), V346I iNOSoxy/nNOSred (V346I chimera) and iNOSoxy/S1412D nNOSred (S1412D chimera). The WT and S1412D chimeras had higher NO release than the parent iNOS, while the V346I chimera exhibited much lower NO release, consistent with expectations. Measurements indicated that a greater NO dioxygenase activity was achieved, particularly in the V346I chimera, which dioxygenated an estimated two to four NO per NO that it released, while the other chimeras had nearly equivalent NO dioxygenase and NO release activities. Computer simulations of the global catalytic model using the measured kinetic parameters produced results that mimicked the measured outcomes, and this provided further insights on the catalytic behaviors of the chimeras and basis of their increased NO dioxygenase activities.

Full text of DOI:10.1016/j.jinorgbio.2016.03.002

JIB-09955; No of Pages 9
Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Engineering nitric oxide synthase chimeras to function as NO dioxygenases
a, ,1
b,1
b
b
Zhi-Qiang Wang ⁎ , Mohammad Mahfuzul Haque , Katherine Binder , Manisha Sharma ,
c
b,
Chin-Chuan Wei , Dennis J. Stuehr ⁎⁎
a
Department of Chemistry and Biochemistry, Kent State University Geauga, Burton, OH 44021, United States
Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, United States
Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL 62026, United States
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Nitric oxide synthases (NOSs) catalyze a two-step oxidation of L-arginine to form nitric oxide (NO) and L-citrul-
line. NOS contains a N-terminal oxygenase domain (NOSoxy) that is the site of NO synthesis, and a C-terminal
reductase domain (NOSred) that binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine
dinucleotide (FAD), and flavin mononucleotide (FMN) and provides electrons to the NOSoxy heme during catal-
ysis. The three NOS isoforms in mammals inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS
Received 12 November 2015
Received in revised form 10 March 2016
Accepted 12 March 2016
Available online xxxx
(
eNOS) share high structural similarity but differ in NO release rates and catalytic properties due to differences
Keywords:
Chimera
Electron transfer
Kox
Heme reduction
Catalysis
Stopped-flow
in enzyme kinetic parameters. These parameters must be balanced for NOS enzymes to release NO, rather than
consume it in a competing, inherent NO dioxygenase reaction. To improve understanding, we drew on a global
catalytic model and previous findings to design three NOS chimeras that may predominantly function as NO
dioxygenases: iNOSoxy/nNOSred (Wild type (WT) chimera), V346I iNOSoxy/nNOSred (V346I chimera) and
iNOSoxy/S1412D nNOSred (S1412D chimera). The WT and S1412D chimeras had higher NO release than the
parent iNOS, while the V346I chimera exhibited much lower NO release, consistent with expectations.
Measurements indicated that a greater NO dioxygenase activity was achieved, particularly in the V346I chimera,
which dioxygenated an estimated two to four NO per NO that it released, while the other chimeras had nearly
equivalent NO dioxygenase and NO release activities. Computer simulations of the global catalytic model
using the measured kinetic parameters produced results that mimicked the measured outcomes, and this provid-
ed further insights on the catalytic behaviors of the chimeras and basis of their increased NO dioxygenase
activities.
©
2016 Elsevier Inc. All rights reserved.
1. Introduction
Nitric oxide synthases (NOSs) are flavo-heme enzymes that catalyze
a stepwise oxidation of L-arginine (Arg) to form nitric oxide (NO) and L-
Abbreviation: Arg, L-arginine; DTT, dithiothreitol; NO, nitric oxide; EPPS, 4-(2-
ω
hydroxyethyl)-1-piperazinepropanesulfonic acid; NOHA, N -hydroxy-L-arginine; H
4
B,
(
6R)-5, 6, 7, 8-tetrahydro-L-biopterin; FMN, flavin mononucleotide; FAD, flavin adenine
citrulline [1–6]. The overall biosynthetic reaction consumes 1.5
nicotinamide adenine dinucleotide phosphate (NADPH) and 2 O and
2
dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; NED, N-1-
naphthylethylenediamine dihydrochloride; LDH, lactate dehydrogenase; SEITU, S-
ethylisothiourea; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase;
nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase; NOSoxy,
the oxygenase domain of nitric oxide synthase; NOSred, the reductase domain of nitric
involves two steps: the first being Arg hydroxylation to form
N-hydroxy-L-Arg (NOHA), and the second being NOHA oxidation to
form citrulline and NO (Scheme 1). NOSs are homodimeric enzymes
[5,7,8], with each monomer containing an N-terminal oxygenase
domain that binds Fe-protoporphyrin IX (heme), the substrate Arg,
II
oxide synthase; iNOS fl, the full length of inducible nitric oxide synthase; Fe , ferrous
III
II
III
heme species; Fe , ferric heme species; Fe O
2
, ferrous oxy species; Fe NO, ferric NO spe-
, ferric heme reduction rate; kox, ferrous heme-NO oxi-
, ferric heme-NO dissociation rate; kon, NO association to the ferric enzyme;
cat1, catalytic rate constant of Arg hydroxylation; kcat2, catalytic rate constant of NOHA ox-
idation; BSA, bovine serum albumin; SOD, superoxide dismutase.
Corresponding author.
⁎ Correspondence to: D. J. Stuehr, Department of Pathobiology NC-22, Lerner Research
II
cies; Fe NO, ferrous NO species; k
dation rate; k
r
4
and the cofactor 6R-tetrahydrobiopterin (H B) [9–11], and a
d
C-terminal flavoprotein domain that binds flavin adenine dinucleotide
(FAD), flavin mononucleotide (FMN), and NADPH [12–15], with the
two domains being linked by a central calmodulin (CaM) binding
motif [6,16,17]. Neuronal NOS (nNOS) and endothelial NOS (eNOS)
k
⁎
⁎
Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, United
States.
2+
are reversibly activated by binding CaM in the presence of Ca . Where-
as iNOS binds CaM regardless of the Ca2 concentration and is continu-
+
E-mail addresses: zwang3@kent.edu (Z.-Q. Wang), stuehrd@ccf.org (D.J. Stuehr).
Both authors contributed equally to this work.
1
ously active [1,2,16,18]. The enzyme heme is ligated to a cysteine
http://dx.doi.org/10.1016/j.jinorgbio.2016.03.002
0162-0134/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
http://dx.doi.org/10.1016/j.jinorgbio.2016.03.002

2
Z.-Q. Wang et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Scheme 1. The two steps of NO synthesis.
thiolate and acts in conjunction with H
4
B to catalyze a reductive activa-
a unique catalytic profile, possibly to aid their specific functions in
biology [29].
r r r
NO biosynthesis by NOS enzymes is rate-limited by k , k ’, k ”
tion of molecular oxygen in both steps of NO synthesis [16,19–21].
Three mammalian NOS isozymes have been characterized: neuronal
NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS) [1,2,18,
r
(i.e., k
limit the rate of Fe NO formation in Fig. 1). Because the electron
transfer from two-electron reduced (hydroquinone) FMN (FMNH ) to
the heme is rate-limiting, it likely nullifies the importance of any Fe /
III
2
2–30]. Each is distinguished by its tissue location, regulation, and func-
2
II
III
tion [31–37]. Rates of NO synthesis (release) also differ among the
mammalian NOS with the rank order being iNOS N nNOS ≫ eNOS. Nu-
merous results show that their catalytic differences are predominantly
due to the differences in three main kinetic parameters: The rates of fer-
III
Fe thermodynamic differences that may exist among the various Fe
species. But increasing k also makes the enzyme partition more into
the futile cycle (k ”’, Fig. 1), which diminishes the proportion of NO re-
r
r
ric heme reduction (k
rous heme–NO oxidation (kox) (Fig. 1) [23,24,38–41]. In both the Arg
r
, k
r
’, k
r
”) ferric heme-NO dissociation (k
d
), and fer-
leased and increases the extent of NO dioxygenase activity and nitrate
production (Fig. 1). In fact, the global kinetic model predicts that each
hydroxylation and NOHA oxidation reactions of NO synthesis, the NOS
r
NOS has an optimal k setting for its NO release rate, above which the
ferric heme first accepts an electron from the reductase domain (k
r
&
NO release will fall and the NO dioxygenase activity will reciprocally in-
crease [29].
II
k
r
” in Fig. 1) to form the heme-dioxy species (Fe O
unreactive toward Arg or NOHA. An electron is then provided by cofac-
tor H B to form the ferric peroxy intermediate, and this H B-mediated
electron transfer is rate limiting for the subsequent catalytic steps in
Arg hydroxylation and in NO production from NOHA (kcat1, kcat2
2
, Fig. 1), which is
Some of the protein features in NOS that help determine set points
4
4
for the k
24,39,43,46]. In native enzymes, the identity of the NOS reductase
domain appears to determine the k value [39,47], with rank order
(fast to slow) nNOS N iNOS N eNOS, while the NOS oxygenase domain
primarily determines the kox and k settings [29], with rank orders for
ox being iNOS N eNOS N nNOS, and for k being nNOS N eNOS N iNOS.
Our studies indicate that nNOS evolved a near optimal k setting for its
NO release, whereas eNOS and iNOS have sub-optimal k rates [23,24,
r d
, kcat, k , and kox kinetic parameters have been identified [23,
,
r
Fig. 1) [19,21,42–44]. Practically all of the newly-generated NO binds
III
to the NOS ferric heme to form Fe NO before exiting the enzyme. The
d
d
dissociation of this ferric heme–NO complex (k ) is part of a “productive
k
d
cycle” that releases NO and is essential for NOS bioactivity. Conversely,
the attached reductase domain (NOSred) can reduce the ferric heme–
r
r
NO complex (k
r
”’; k
r
”’ = k
r
) [41] to channel the enzyme into an NO
29,48,49]. Accordingly, a point mutation (S1412D) that mimics
Akt-dependent Ser phosphorylation in the C-terminal region of nNOS
dioxygenase “futile cycle” that ultimately generates nitrate in place of
NO. Together, the productive and futile cycles create a global kinetic
increased it's heme reduction rate k , but also caused a lower NO release
r
rate [50–53], consistent with it's k going beyond the optimal setting.
This relationship was later confirmed and expanded in a study with
r
model for NOS catalysis. The global catalytic model reveals how the k
, and kox parameters must be balanced in order for NOS enzymes
to release the NO that they make, and to minimize destruction of the NO
in the futile dioxygenase pathway. Interestingly, the set points for k
, kox vary among NOS enzymes [27,29,41,45] and give each NOS
r
,
kcat, k
d
several nNOS mutants [30].
r
,
d
Regarding the control of k , there is a naturally-occurring Ile present
kcat, k
d
in the heme pocket opening in bacterial NOS enzymes that restricts
Fig. 1. Global kinetic model for NOS enzymes. During steady state catalysis, NOS molecules engage in a productive cycle that releases NO and in a futile NO dioxygenase cycle that releases
nitrate. Reduction of ferric enzyme to ferrous (k ) enables the heme to bind O and initiate catalytic reactions, kcat1 and kcat2 in concert with donation of a second electron from H B cofactor,
species and generates products in both the Arg hydroxylation and NOHA oxidation reactions, respectively. After NO is made a ferric heme–NO complex (Fe \\ NO)
r
2
4
II
III
that converts the Fe O
forms, which can either release NO (k
active ferric enzyme by reacting with O
2
II
d
) or become reduced (k
in a dioxygenase reaction (kox).
r
”’) to generate a ferrous heme–NO complex (Fe \\ NO), which dissociates extremely slowly and instead regenerates the
2
Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
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Z.-Q. Wang et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
3
newly-made NO from exiting the distal heme pocket, and also restricts
small molecule (i.e., O and NO) entrance into the heme pocket, as
compared to the animal NOS enzymes, which have the smaller Val in
place of Ile at this position. Thus, incorporating the V346I substitution
nNOS. Mutations (bold) and their corresponding oligonucleotides
were as follows: S1412D (sense) 5′-CTT AGA TCT GAG GAC ATC GCC
TTC ATC GAA-3′; S1412D (antisense) 5′-TTC GAT GAA GGC GAT GTC
CTC AGA TCT AAG-3′; V346I (sense) 5′-TAT GCA CTG CCT GCC ATT
GCC AAC ATG CTA-3′; V346I (antisense) 5′-TAG CAT GTT GGC AAT
GGC AGG CAG TGC ATA-3′. Restriction digestions, cloning, and bacterial
growth were performed using standard procedures. Transformations
were done using a TransformAid bacterial transformation kit
(ThermoFisher Scientific). Oligonucleotides used to construct site-
directed mutants in nNOS were obtained from Integrated DNA Technol-
ogies (Coralville, IA). Site-directed mutagenesis was done using a
QuikChange XL site-directed mutagenesis kit (Agilent Technologies).
The hybrid constructs and mutants were confirmed by DNA sequencing
at the Cleveland Clinic Genomics Core.
2
d
into iNOS decreased its k about 3-fold, decreased its affinity toward
binding external NO [49,54], and also increased the extent of it's
geminate or near-geminate heme-NO rebinding, as demonstrated in a
laser flash photolysis study of the enzyme heme–NO complex [55].
We took advantage of this knowledge described above to create
three NOS chimeras that were specifically engineered to be better NO
dioxygenases: iNOSoxy/nNOSred (WT chimera), iNOSoxy/S1412D
nNOSred (S1412D chimera), and V346I iNOSoxy/nNOSred (V346I
chimera). The rationale for using nNOSred is that it has the fastest k
r
among the three NOS, and so should cause the greatest fraction of
enzyme to partition into the futile NO dioxygenase cycle, which
depends on k
r
”’ (k
r
”’ was measured to be equivalent in rate to k
r
) [41]
2.3. Protein expression and purification
(
see Fig. 1). The S1412D mutation was added in an attempt to further
speed heme reduction and thus increases enzyme partitioning into the
Three chimeras, along with wild type iNOS with a six-histidine tag
futile cycle. The rationale for using iNOSoxy is that its kox parameter is
were each overexpressed in E. coli BL21(DE3) strain using pCWori vec-
tor and purified as reported previously by sequential Ni2
+
-
the fastest among the three NOS, and is faster than k
r
, which should
allow the enzyme to pass quickly through the futile cycle without accu-
mulating as the ferrous heme-NO species, which otherwise would
nitrilotriacetate (NTA) chromatography and CaM-sepharose affinity
column [18,23,45,49,58–60]. NOS concentrations were determined
from the 444 nm absorbance of the ferrous–CO complex, using an ex-
tinction coefficient 74 mM cm [61]. Buffer used for all experiments
was 40 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid
(EPPS, pH 7.6) containing 250 mM NaCl and 10% glycerol.
lower the overall catalytic cycling. The V346I mutation was added to
III
−1
−1
slow k
d
and prolong the lifetime of the Fe NO product species, and
thus give it more chance to become reduced and partition into the futile
cycle (the productive/futile partitioning ratio is determined by k ”’/k
r d
see Fig. 1).
We characterized the chimeras regarding their steady-state catalytic
activities, heme reduction rates, and NO versus nitrate production, and
then performed computer simulations using our global catalytic
model [18,27,29,41,45,56] and both measured and reported kinetic
values, to understand the behaviors of the three chimeras. The results
indicate that they all gained in NO dioxygenase activity, and that the
V346I chimera is a predominant NO dioxygenase, all generally
consistent with the computer modeling. Our study provides a better
understanding of NOS catalysis and it's protein structure–function rela-
tionships, regarding how NOS kinetic parameters are regulated and help
balance coincident NO release and NO dioxygenase activities.
2.4. UV–Vis spectra measurement
4
Spectra of chimeras in the presence of 10 mM Arg and 20 μM H B
were recorded at room temperature on Shimadzu UV-2410PC spectro-
photometer (Shimadzu Corp., Tokyo, Japan). After addition of small
amount of dithionite to the samples and bubbling with CO, the ferrous
CO complex was measured. Spectra were also taken after adding
31 mM imidazole to the purified proteins.
2.5. NO synthesis and NADPH oxidation rates
Steady-state rates of NO release were determined by the
spectrophotometric oxyhemoglobin assay using a difference extinction
coefficient of 38 mM cm for the oxyhemoglobin to methemoglobin
transition at 401 nm [26,45,58,60]. Sample solutions contained 0.1 μM
2
. Materials and methods
−
1
−1
2
.1. Materials
4
NOS enzyme, 4 μM H B, 2 mM Arg, 0.3 mM DTT, 0.1 mg/ml bovine
All reagents and materials were obtained from Aldrich, Alexis,
serum albumin (BSA), 10 U/ml superoxide dismutase (SOD), 346 U/ml
Sigma-Aldrich, or sources described previously [45,49,57].
catalase, 10 μM oxyhemoglobin, 4 μM FMN and FAD, 0.2 mM EDTA,
0
.7 mM CaCl
NADPH (100 μM final concentration), and the absorbance change at
01 nm was recorded at room temperature or at 10 °C. For the NADPH
oxidation rate measurements we used an extinction coefficient of
2
and 12.5 μg/ml CaM. Reactions were initiated by adding
2
.2. Constructing NOS chimeras
4
The oxygenase domain of nNOS was replaced with the oxygenase
−
1
−1
domain of iNOS using NdeI restriction site at 5′-end and KpnI site
generated by site directed mutagenesis) at 3′-end (Fig. 2). Chimeras
contain a 6-His tag at N-terminal and the CaM binding site from
6.22 mM
cm
at 340 nm. In some cases 250 μM NOHA or 2 mM
(
S-ethylisothiourea (SEITU) were added to the reaction solution in
place of Arg.
2.6. Rates of heme reduction
The kinetics of ferric heme reduction was determined at 10 °C as
described previously [18,26,30,58,59,62]. Reactions were carried out in
a Hi-Tech Scientific KinetAsyst SF-61 DX2 stopped-flow apparatus
equipped for anaerobic work and coupled to a diode array detector
(
Hi-Tech Scientific, Salisbury, UK). Reactions were initiated by rapidly
mixing anaerobic ferric enzyme solutions containing ~10 μM NOS,
1
00 μM H
4
B, 6 mM Arg, 0.4 mM Dithiothreitol (DTT), 1 mM EDTA,
and 12.5 μg/ml CaM with an anaerobic CO-saturated buffer
10 mM CaCl
2
solution containing 100 μM NADPH. Heme reduction was determined
by the absorbance increase at 444 nm due to formation of the ferrous–
CO complex, which occurs following an initial absorbance decrease at
Fig. 2. Cartoon showing the chimeric proteins used in this study. All chimeric proteins are
comprised of oxygenase domain from iNOS (iNOSoxy) and reductase domain from nNOS
(nNOSred) that also contains the nNOS calmodulin (CaM) binding domain.
Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
http://dx.doi.org/10.1016/j.jinorgbio.2016.03.002

4
Z.-Q. Wang et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
microplate reader, after adding Griess reagent (0.1% NED (N-1-
napthylethylenediamine dihydrochloride) solution and 1% sulfanil-
amide in 4.25% H PO ) to each well. Nitrite productions were then
3
4
quantitated based on nitrite solutions standard curve [29,49,63,64].
Total amount of nitrite plus nitrate was determined using a similar
method except adding nitrate reductase (0.1 U/ml) and incubating the
samples for extra 2 h at 37 °C before the addition of LDH and sodium
pyruvate [49]. Nitrate production was calculated by subtracting the
nitrite amount from the total amount.
2.8. Simulations of product formation and enzyme distribution during NO
synthesis
Computer simulations were based on the global kinetic model de-
scribed in Fig. 1 and were run in Gepasi 3.30 as described earlier [24].
The simulations assume constant values for [O ] = 180 μM and
NADPH] = 40 μM. Values of ferric heme reduction (k ), ferrous
heme-NO oxidation (kox), heme NO dissociation (k ), catalytic rate
2
[
r
d
constants (kcat1, kcat2) used for simulation are listed in tables.
Experimentally observed oxidation rates (kox) for ferrous heme–NO
complexes, which we derived at half air-saturated condition, were
multiplied by a factor of 1.5 to get a full air-saturated condition, based
2
on our previous study [65] where we determined the [O ] dependence
of kox for iNOS to determine the 2nd order rate constant. In simulation,
II
we also multiplied the observed oxygen binding rates to Fe intermedi-
ates by a factor of 2 to get a full air-saturated condition, assuming that
the rate is proportional to [O ]. The NADPH and chosen O concentra-
2 2
tions were kept constant during each simulation, to avoid secondary
effects due to their exhaustion.
3. Results and discussion
3.1. Protein properties
Fig. 3. Spectral properties of chimeric NOS enzymes. Spectra were recorded in the
Fig. 2 shows a cartoon of the chimeras we generated and used in our
4
presence of 10 mM Arg and 20 μM H B for the ferric enzymes (black lines), the ferric-
imidazole species (green lines), and the ferrous–CO complexes (red lines).
study. Purification yields were 38, 24, and 17 mg per liter of culture for
WT chimera, V346I chimera, and S1412D chimera, respectively, these
are all within the typical range seen when expressing mammalian
NOS full-length enzymes. All three chimeras displayed Soret absorbance
4
44 nm due to flavin reduction by NADPH. Rates were determined after
fitting the absorbance change to a bi-exponential equation.
peaks at 398 nm in the presence of H
contained a five-coordinate high-spin heme iron as occurs in wild type
iNOS when it has H B and Arg bound under similar conditions [18,34,42,
9,60,66]. The chimeras bound imidazole to form a normal Soret peak
4
B and Arg (Fig. 3), indicating they
2
.7. Nitrite and nitrate production in NADPH-driven reactions
4
4
9
0 μl 6–15 nM enzyme solutions containing 20 μM H
4
B, 3 mM Arg,
with maxima at 428 nm, although imidazole binding may be sterically
hindered in the V346I chimera. Addition of dithionite and CO to the
chimeras generated ferrous–CO complexes with Soret absorbance at
444 nm (Fig. 3), again similar to wild-type NOS. These properties dem-
onstrate that swapping the reductase domains, and introduction of the
point mutations, did not significantly perturb substrate/cofactor bind-
ing, CaM response, or the electronic properties of the NOS heme in the
chimeras.
0
.3 mM DTT, 0.1 mg/ml BSA, 10 U/ml SOD, 346 U/ml catalase, 4 μM
FMN and FAD and 12.5 μg/ml CaM were added into 96-well microplate
and incubated on ice for 30 min. The reaction was initiated by adding
0 μl 10 mM NADPH. After having reacted for 30 min in 37 °C incubator,
0 μl 15 mM SEITU was added to each well to stop the reaction. Excess
NADPH was consumed by the addition of 10 U/ml lactate dehydroge-
nase (LDH) and 10 mM sodium pyruvate. Nitrite was detected by
measuring the absorbance difference at 550 and 650 nm using a
1
1
Table 1
NO release and NADPH oxidation activities of the enzymes. Assays were run at 25 °C. Turnover number is expressed as moles of product formed per mole of NOS heme per min. Data are the
mean and S.D. of three to four determinations. N.D., not determined; W.T., wild-type.
Enzymes
H
4
B + Arg
H
4
B + NOHA
H
4
B + SEITU
Refs.
NO release
NADPH oxidation
NADPH/NO
NO release
NADPH oxidation
NADPH/NO
NADPH oxidation
−
1
−1
−1
−1
−1
(
min
)
(min
)
(min
)
(min
)
(min
)
WT-iNOS
WT-Chimera
V346I iNOS
V346I-Chimera
S1412D-Chimera
93 ± 1
119 ± 5
15 ± 1
11 ± 0.3
107 ± 6
160 ± 1
222 ± 3
59 ± 2
91 ± 2
222 ± 3
1.7
1.9
3.9
8.1
2.1
122 ± 2
157 ± 9
24 ± 1
20 ± 1
168 ± 4
117 ± 7
168 ± 5
36 ± 2
116 ± 3
236 ± 1
1.0
1.1
1.5
5.7
1.4
23 ± 1
39 ± 4
N.D.
35 ± 3
59 ± 2
This paper
This paper
[49]
This paper
This paper
Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
http://dx.doi.org/10.1016/j.jinorgbio.2016.03.002

Z.-Q. Wang et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
5
3.2. NO synthesis and NADPH oxidation
V346I iNOS (15 min−1) [49]. This is in accord with the V346I chimera
having a k /k ratio of 2.3 (see Table 2), which means that the increase
r
d
We measured steady-state NO synthesis activity (the NO release
in its heme reduction rate that is afforded by its nNOSred component
should mainly increase shunting of the enzyme Fe NO species into
the futile cycle (see Fig. 1), and thus should have a negative overall
effect on its NO release rate during the steady state.
III
activity) and the associated NADPH oxidation at 25 °C, using Arg or
the reaction intermediate NOHA as substrates. The results are given in
Table 1. Compared to wild-type iNOS, the NO release activities of the
WT and S1412D chimeras were 28% and 15% higher with Arg as
substrate, and were 29% and 38% higher with NOHA as substrate, re-
spectively. These increases are consistent with the predicted behavior
of these two chimeras: Their nNOSred component should support a
Although the k
r
values of the chimeras were all somewhat faster
that
under the same
than in parent iNOS, they were still 40% to 60% slower than the k
is seen for nNOS, which ranges from 3 to 6 s
r
−
1
experimental conditions [24,27,29,41,69] (Table 2). This difference
was unexpected, because our previous work with an eNOSoxy/nNOSred
chimera showed that the attached nNOSred component in that case
supported the same heme reduction rate in eNOSoxy as is seen in
faster heme reduction (k
component in native iNOS, and this effect on k
r
) to iNOSoxy as compared to the iNOSred
should enable these
r
two chimeras to display a greater steady-state NO release rate (see
Fig. 1). Indeed, the NADPH oxidation rates that accompanied NO
synthesis by the WT and S1412D chimeras were increased compared
to wild-type iNOS (Table 1). The ratio of NADPH oxidized per NO
formed was similar between wild-type iNOS and the WT chimera, but
was higher for the S1412D chimera (Table 1). Its increased ratio may re-
flect some increase in uncoupled NADPH oxidation that appears to be
inherent in the S1412 nNOSred, as judged from the S1412D chimera
having a higher NADPH oxidation rate when it was measured in the
presence of S-ethyl-isothiourea (SEITU, Table 1), which is a substrate-
competitive inhibitor that blocks iNOS heme reduction [67].
r
nNOS [39]. The slower k we observed in our nNOSred/iNOSoxy chi-
meras is not likely due to a hindrance in CO binding in iNOSoxy, because
CO binding to ferrous iNOSoxy is faster than the measured heme reduc-
tion rates [72]. We speculate that nNOSred may reduce the heme in the
attached iNOSoxy more slowly because the iNOS heme has a lower mid-
point potential than in nNOS [73], and/or because the iNOSoxy may
have a docking site for the reductase domain that contains comparative-
ly less charged residues available for docking when compared to the
eNOSoxy or nNOSoxy surfaces [30]. These possibilities deserve further
r
investigation. In any case, the slower than expected k that we achieved
In comparison, the V346I chimera displayed 88% and 94% lower NO
release activities with Arg or NOHA as substrates, respectively, relative
to native iNOS (Table 1). A decrease was expected, because our previous
work had shown that incorporating the V346I substitution alone into
iNOS lowered its NO release activity by 79% [49] (Table 1). The V346I
mutational effect on NO release activity was not related to poor
substrate binding because the V346I substitution does not alter the
substrate binding affinity [49]. NADPH oxidation by the V346I chimera
when catalyzing Arg oxidation was only 45% slower than the rate seen
in parent iNOS, and it's rate was similar to parent iNOS when it was
catalyzing NOHA oxidation (Table 1). Thus, the V346I chimera had
ratios of 8.1 or 5.7 NADPH oxidized per NO released with Arg or
NOHA as substrates, respectively, which are considerably higher than
ratios recorded for parent iNOS or for V346I iNOS (Table 1). The higher
NADPH consumption of the V346I chimera, compared to its NO release,
is consistent with the V346I chimera having an increased NO
dioxygenase activity.
in our nNOSred/iNOSoxy chimeras likely compromises their capacities
to act as NO dioxygenases, because the extent of enzyme partitioning
into the futile (NO dioxygenase) cycle is directly related to the magni-
r
tude of k ”’ (see Fig. 1).
3.4. Nitrate and nitrite production
NO that is released into air-saturated water primarily oxidizes to ni-
trite, whereas the NO dioxygenase reaction converts NO to nitrate [49,
55]. Thus, the nitrite to nitrate ratio can approximate what proportions
of a NOS enzyme partition through the productive versus futile cycles,
respectively, during catalysis. We measured the nitrate and nitrite
formed in the reactions catalyzed by each chimera and wild-type
iNOS. The WT and S1412D chimeras had nitrate:nitrite ratios similar
to the ratio we obtained with parent iNOS, suggesting that they had a
similar NO dioxygenase activity (Table 3). This is consistent with their
r
having a less than expected increase in their k values (see Table 2), as
3
.3. Kinetics of heme reduction
discussed above, and with their measured ratios of the NADPH oxidized
to NO released activities being only marginally greater than for wild-
type iNOS (see Table 1). In comparison, the nitrate:nitrite product
ratio of the V346I chimera was much higher and suggested it oxidized
on average 4.5 NO for every NO it released, assuming that all the re-
leased NO went on to form nitrite. Because this ratio is higher than the
2.8 nitrate:nitrite released ratio that we observed for V346I iNOS in a
similar study [49] (Table 3), it suggests that the 2.6 times faster heme re-
duction rate in the V346I chimera relative to V346I iNOS (Table 2)
makes a significant contribution toward increasing the V346I chimera's
We utilized stopped-flow spectroscopy to determine the heme
r
reduction rate (k ) of the chimeras. Ferric enzymes containing Arg,
H B, CaM and Ca were rapidly mixed with excess NADPH in the pres-
4
ence of CO-saturated buffer under anaerobic conditions [26,30,59,68],
and rate of heme reduction was determined as the rate of heme Fe CO
complex formation, as indicated by an absorbance increase at 444 nm.
Fig. 4 contains kinetic traces from representative reactions that were
run with each chimera and spectra are shown in the supplemental fig-
ure. The initial absorbance decrease at 444 nm that is seen in all cases
was due to flavin reduction that takes place before electrons can transfer
to the ferric heme [26,48,68–70]. All three chimeras exhibited a similar
extent of heme reduction, that was also similar to parent iNOS [29,48,
2
+
II
NO dioxygenase activity. As noted above, this makes sense because the
III
increase in k
r
places the Fe NO partition ratio (k
r d
”’/k ) of the V346I chi-
mera above unity, which then favors its entry into the futile cycle during
steady-state catalysis (see Fig. 1).
4
9,71], suggesting no large differences in the thermodynamics. The
The NO dioxygenase activity of iNOS can also involve its catalyzing
the dioxygenation of NO that has been released into solution, if the
NO concentration builds up sufficiently for it to bind to the iNOS heme
[74]. Interestingly, V346I iNOS actually displays a diminished
dioxygenase activity toward external NO relative to wild-type iNOS,
because the V346I substitution restricts NO entrance into the iNOS
heme pocket [49]. Thus, the greater NO dioxygenase activity that we
see for the V346I chimera is likely due to its catalyzing an increased
dioxygenation of the newly-formed NO within its heme pocket, and
does not likely involve any increase in its dioxygenase activity toward
external NO.
rates of absorbance change at 444 nm were fit to a biphasic equation,
which gave estimated heme reduction rates of 1.9 ± 0.08, 1.8 ± 0.15
and 2.2 ± 0.05 s , respectively, for the WT, V346I, and S1412D
chimeras (Table 2).
−
1
The nNOSred component that was part of each chimera supported
heme reduction rates (k
seen in the parent iNOS or in V346I iNOS (Table 2), thus achieving one
of our protein engineering goals for the chimeras. Despite the k of the
V346I chimera being 2.6 times faster than in V346I iNOS, the NO release
r
) that were somewhat faster than the rates
r
−
1
rate of the V346I chimera (11 min , Table 1) was 27% less than that for
Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
http://dx.doi.org/10.1016/j.jinorgbio.2016.03.002

6
Z.-Q. Wang et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
NOS catalysis as shown in Fig. 1 [18,24,29,45,49], in order to model the
NO release activity, NADPH oxidation, and product ratios, as well as any
changes in steady-state enzyme distribution, of the three chimeras rel-
ative to the parent iNOS. In addition, we measured the steady-state cat-
alytic activities of the enzymes at 10 °C (Table 5), to obtain experimental
measures that could be directly compared with the simulation results,
which utilize kinetic parameters that were measured at 10 °C (i.e., the
values in Table 5). The simulation results are valuable because they
help to test our ideas about how the altered kinetic parameters in the
chimeras may change their steady state activity outcomes (NO release
&
NADPH oxidation rates, NO to nitrate produced), particularly with
regard to their having increased NO dioxygenase activity.
In our study, the “outcomes” (steady state catalytic activities and
product ratios) showed a similar rank order among the enzymes
when measured at 10 and 25 °C. This implies that temperature differ-
ence is not a major factor in projecting how the enzymes may behave
at their physiologic temperature. Regarding a lack of large (5–10×)
differences in some of our individual rate or activity measures among
the chimeras, and the possibility of such smaller differences to influence
outcomes: It is important to note that the outcomes (NO release activity,
NADPH consumption, nitrate production) all rely on the blending of the
individual measured rate parameters within the global catalytic model,
and so relatively small differences among the rate parameters can in
some cases have compounded effects on outcome, depending on how
they blend in relation to one another. One good example is in how the
III
III
ratio of the rate of Fe NO reduction (k
ciation (k ) (i.e., k ”’/k ) determine the enzyme partitioning between
the productive versus futile cycles. A 30% change in k ”’ would not
have a big impact if the NOS k was 2 or 3 times faster than the original
”’. However, in a situation where the original k ”’ and k are of similar
speed, the same 30% increase in k ”’ would make it surpass the k , and
r
”’) and the rate of Fe NO disso-
d
r
d
r
d
k
r
r
d
r
d
thus would significantly shift the ratio for enzyme partitioning between
the two cycles. Thus, more modest (30 to 200% changes) in any one of
the rate parameters can still have significant impact on outcomes (and
do so in our study), and thus can be informative for understanding the
enzyme cycling and steady state behaviors in the chimeras. In fact,
there are several similar studies where meaningful comparisons
among NOS enzymes and mutants have been achieved, despite the
overall change for any one rate parameter being fairly small [30,56,59].
Fig. 5 depicts the measured and simulated rates we obtained for
steady-state NO release and NADPH consumption by iNOS and each of
the three chimeras. The measured and simulated outcomes distribute
in the same rank order: NO release rates were S1412D chimera, WT
chimera ≥ iNOS ≫ V346I chimera, and NADPH oxidation rates were
S1412D chimera, WT chimera N iNOS N V346I chimera. The magnitudes
of each simulated and measured rate were well within a factor of two of
one another. Thus, the simulations provided a reasonable match to the
measured outcomes. The bottom panel of Fig. 5 compares the NADPH
oxidized per NO released ratios of each enzyme, when derived from
either the measured or simulated data in the upper and middle panels
of Fig. 5. This analysis particularly distinguishes the V346I chimera for
its poor NO release activity relative to its NADPH consumption, which
is the expected behavior of a NOS enzyme that has become a predomi-
nant NO dioxygenase.
Fig. 4. Kinetics of heme reduction in the chimeric enzymes. Anaerobic solutions containing
H B, Arg, CaCl and CaM and ~10 μM ferric NOS protein were rapidly mixed with an
4 2
anaerobic CO-saturated buffer solution containing 100 μM NADPH at 10 °C in a stopped
flow spectrophotometer with diode array detection. Scans were recorded and heme
reduction was followed by the formation of the ferrous–CO complex with Soret
maximum at 444 nm. The lines of best fit are black.
3
.5. Simulation of catalytic behaviors
We utilized the kinetic parameters listed in Table 4, which were de-
rived from our measures at 10 °C and from our previous publications as
noted in the Table, to run computer simulations of our global model for
Table 2
Kinetic parameters for NOS enzymes and chimeras. Values of the k
r
, kox, and k
d
parameters were determined at 10 °C unless otherwise noted. k
r
, rate of ferric heme reduction. k
d
, disso-
ciation rate of the ferric heme-NO complex. kox, rate of reaction between the ferrous heme-NO complex and approximately 140 μM O
2
(half air-saturated conditions). WT., wild-type.
(s−1)
FeIIINO dissociation k
(s−1
)
II
Fe NO oxidation kox (s−1
)
Refs.
NOS
heme reduction k
r
d
WT-Chimera
V346I-Chimera
S1412D-Chimera
V346I iNOS
WT- iNOS
WT-nNOS
1.9 ± 0.08
1.8 ± 0.15
2.2 ± 0.05
0.69 ± 0.02
0.9–1.5
2.3 ± 0.1
0.77 ± 0.03
2.3 ± 0.1
0.77 ± 0.03
2
5
5
3.1 ± 0.15
1.8 ± 0.01
3.1 ± 0.15
1.8 ± 0.01
3
This paper, [49]
This paper, [49]
This paper, [49]
[49]
[29,41,49,73,74]
[24,29,41,69]
[27,29,50]
3–4
5.4 ± 0.4
0.2
0.2
S1412D nNOS
Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
http://dx.doi.org/10.1016/j.jinorgbio.2016.03.002

Z.-Q. Wang et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
7
Table 3
Nitrite and nitrate production in NADPH-driven reaction at 37 °C. Reactions were run for 30 min prior to quenching as described under “Section 2.” The values are the mean ± S.D. of three
measurements.
−
−
−
−
NOS
NO
2
produced (mol/mol NOS)
NO
3
produced (mol/mol NOS)
NO
3
/NO
2
Refs.
WT-iNOS
WT-Chimera
V346I-iNOS
V346I-Chimera
S1412D-Chimera
1572 ± 18
1853 ± 43
2400
303 ± 11
1934 ± 32
1409 ± 16
1848 ± 49
6800
1351 ± 21
1973 ± 16
0.9
1.0
2.8
4.5
1.0
This paper
This paper
[49]
This paper
This paper
Table 4
Rates used for the computer simulation.
WT-iNOS
1.1
106
12.5
1.1
1.1
106
36.7
2.0
1.1
WT-Chimera
V346I-Chimera
S1412D-Chimera
III
II
k
k
k
k
k
k
k
k
k
k
k
k
k
1
Fe (a) ➔ Fe (a)
k
r
1.9
106
12.5
1.9
1.8
31
3.7
1.8
1.8
31
4.5
0.8
1.8
2.2
106
12.5
2.2
2.2
106
36.7
2.3
2.2
4.65
0.00006
11.0
0.023
II
II
2
Fe (a) (+ O
2
) ➔ Fe O
2
(a)
II
III
3
Fe O
2
(a) ➔ Fe (b*)
k
k
k
cat1
r’
Fe (b*) ➔ FeIII(b)
III
III
4
II
5
Fe (b) ➔ Fe (b)
r”
1.9
II
II
6
Fe (b) (+ O
2
) ➔ Fe O
2
(b)
106
36.7
2.3
II
III
7
Fe O
2
(b) ➔ Fe NO
k
k
k
k
cat2
d
III
III
8
Fe NO ➔ Fe (a) + NO
III
II
9
Fe NO ➔ Fe NO
r”‘
1.9
II
) ➔ FeIII(a) + NO
10
11
12
13
Fe NO (+ O
2
II
x
ox
4.5
0.00006
11.0
0.3
4.65
0.00006
11.0
0.3
2.7
0.00006
2.3
II
Fe NO ➔ Fe (a) + NO
II
III
Fe O
2
(b) ➔ Fe (b)
(a) ➔ Fe (a)
II
III
Fe O
2
0.023
III
(
a) Denotes L-Arg-bound enzyme; (b) denotes NOHA-bound enzyme; Fe (b*) indicates the ferric enzyme with NOHA and H
4
B radical bound, see scheme in Fig. 1 for details. All values are
, k10) the value used in the calculations is the product of the “actual” value and the concentration of oxygen, which is considered to remain constant
the values used are the experimental values multiplied by 2 (assumes the rate is proportional to [O ], experimental values are determined in half air-
−
1
in s . For bimolecular reactions (k
during the reaction. For k and k
2 6
, k
2
6
2
saturated conditions). For k10 the values used are the experimentally determined values ×1.5 (based on observed oxygen dependence seen in Tejero et al. [65]. k11 values are interpolated
from Salerno et al. paper [76]. k12 and k13 values are used from Wang et al. paper [49].
Fig. 6 informs on the measured and simulated NO dioxygenase
activities of each enzyme, as judged from their nitrate versus nitrite
productions in Table 3, and from their simulated nitrate to NO release
ratios in Table 5, respectively. In both analyses, the V346I chimera is
indicated to have ratios that indicate that its steady-state catalysis
mainly occurs through the futile cycle (nitrate) versus productive
partitioning into the futile cycle due to their faster k
to iNOS, and in the case of the V346I chimera, is also consistent with
r
values compared
II
d 2
its having a slower k . The predicted greater buildup of the Fe O species
in the V346I chimera can be justified from it's having a faster heme
II
reduction (k
r r 2
, k ’) than iNOS, which speeds formation of the Fe O
species, coupled with it's having slower kcat1 and kcat2 rates, which pro-
II
(
NO) pathway. It is unclear why the simulations indicate a lesser extent
longs the lifetime of the Fe O
2
species. Consequently, the V346I chimera
of futile cycling for the V346I chimera compared to the experimental
estimate we derived from the nitrate and nitrite production, but in
any case, they both indicate that this chimera has a predominant NO
dioxygenase activity.
should be more susceptible to uncoupling, due to increased partitioning
of the Fe O species to Fe and superoxide [49] (see Fig. 1). This process
2
II
III
in turn may contribute to the higher NADPH oxidation rate we see for
the V346I chimera, beyond the level that is needed to explain its
increased NO dioxygenase activity.
The impact of replacing the natural iNOSred with nNOSred in the
V346I chimera can be further appreciated by comparing the simulated
enzyme distributions of the chimera versus V346I iNOS [49]. The rela-
The simulations can also predict how each enzyme distributes
among the five major enzyme species that form during steady-state
catalysis (Table 6 and Fig. 7). In all cases, the simulations predict that
the ferric form of each enzyme should remain the predominant species
during the steady state, but also indicate that its proportion will be
diminished in all three chimeras relative to native iNOS, and will be
diminished to the greatest extent in the V346I chimera. The loss of the
ferric form is counterbalanced by increased buildup of the other enzyme
species. In particular, all three chimeras should have increased buildup
r
tively faster k in the V346I chimera is expected to cause more accumu-
II
lation of the Fe NO species during steady-state NO synthesis, due to its
causing more enzyme to partition into the futile cycle. The simulations
support this notion, predicting 12.5% of the V346I chimera would accu-
II
mulate as its Fe NO complex (Table 6), versus only 4.5% predicted for
II
II
of their Fe NO species, which is consistent with their having increased
V346I iNOS [49]. Because the Fe NO complex is actually an inhibited
Table 5
Measured and simulated rates at 10 °C.
H4B + Arg + CaM
NO release NADPH oxidation
H4B + Arg -CaM
NADPH oxidation
Simulation data
Enzymes
NADPH Ox/NO
release
NO release
NADPH oxidation
NADPH Ox/NO
release
NOx/NO
−
1
−1
−1
−1
−1
(
min
)
(min
)
(min
)
(min
)
(min
)
WT- iNOS
27 ± 2
36 ± 3
2.5 ± 0.2
43 ± 2
42 ± 4
1.55
1.8
8.8
13 ± 1
18
22
5.2
21
46
64
34
58
2.5
2.9
6.5
2.8
0.68
0.93
2.17
0.96
WT-Chimera
V346I-Chimera
S1412D-Chimera
66 ± 3.5
22 ± 2.8
72 ± 7
12 ± 0.8
3.5 ± 0.4
10 ± 1
1.7
Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
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8
Z.-Q. Wang et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
Fig. 6. NO dioxygenase activities of different NOS enzymes. The ratio of NO deoxygenated
per NO released was determined as the ratio of nitrate to nitrite formed by each enzyme
−
−
(NO
3 2
/NO ) from reactions described in Table 3, and from the ratio of nitrate formed
per NO released (NOx/NO) from the simulation data in Table 5.
4. Conclusions
In our study we aimed to engineer iNOS proteins to function primar-
ily as NO dioxygenases, by altering structural features that were known
to impact two kinetic parameters (k , and k ) that help to determine the
r
d
extent of NOS enzyme cycling between its productive (NO-releasing)
and futile (NO dioxygenase) pathways. We succeeded in creating one
NOS chimera with predominant NO dioxygenase function relative to
its NO release. Our work provides a better understanding of how distinct
protein structures or structural features regulate key kinetic parameters
in NOS enzymes, and how interplay between these parameters can tune
and balance the inherent NO releasing and NO dioxygenase activities
that are present in all NOS enzymes. Besides adding to the general
fundamental understanding, our study provides a template for rational
design of NOS enzymes in order to regulate or change their functions in
various biological settings [75].
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2016.03.002.
Acknowledgments
This work was supported by the National Institutes of Health grants
GM51491 and CA53914 (D. J. S.), the American Heart Association
Beginning Grant-in-aid 0565297B (Z-Q. W.), KSU Farris Innovation
Award (Z-Q. W.), KSU Faculty Professional Improvement Leave Award
(
0
Z-Q. W.), the American Heart Association Beginning Grant-in-aid
365175B (C.C.W), and SIUE See Grants for Transitional and Exploratory
Projects (C.C. W).
Fig. 5. Comparison of experimental and simulated NO release and NADPH consumption
rates for the different NOS enzymes. Upper panel — Bar graph showing the measured NO
release rates and NADPH consumption rates for each enzyme at 10 °C. Middle panel —
Bar graph showing simulated NO release and corresponding NADPH oxidation rates for
each enzyme at 10 °C. Lower panel — The ratio of NADPH consumed per NO released for
each enzyme in the experimental and simulated conditions.
Table 6
Simulations of enzyme distribution during steady-state catalysis. Values were obtained by
computer simulation of the global kinetic model described in “Section 2” and Fig. 1. WT,
wild type.
WT-iNOS
WT-Chimera
V346I-
Chimera
S1412D-
Chimera
V346I-iNOS
(
or poisoned) form of NOS enzymes, its increased buildup will slow
FeIII%
82.3
3.1
0.8
3.6
10.2
76.5
7.0
1.0
4.9
10.6
59.7
10.1
2.6
15.5
12.1
73.9
8.4
1.1
5.3
11.3
75.1
5.6
1.3
7.7
10.3
II
overall enzyme cycling, decrease steady-state activity, and will increase
the importance of the kox parameter and its associated NO dioxygenase
reaction in determining the behavior of enzyme molecules during
catalysis.
Fe NO%
II
Fe %
II
Fe O
2
%
III
Fe NO%
Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
http://dx.doi.org/10.1016/j.jinorgbio.2016.03.002

Z.-Q. Wang et al. / Journal of Inorganic Biochemistry xxx (2016) xxx–xxx
9
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Please cite this article as: Z.-Q. Wang, et al., Engineering nitric oxide synthase chimeras to function as NO dioxygenases, J. Inorg. Biochem. (2016),
http://dx.doi.org/10.1016/j.jinorgbio.2016.03.002