Mechanism of Inactivation of Neuronal Nitric Oxide Synthase by (S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic Acid

Article abstract of DOI:10.1021/jacs.5b01202

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and the second messenger nitric oxide. Three mechanistic pathways are proposed for the inactivation of neuronal NOS (nNOS) by (S)-2-amino-5-(2-(methylthio)acetimidamido)pentanoic acid (1): sulfide oxidation, oxidative dethiolation, and oxidative demethylation. Four possible intermediates were synthesized. All compounds were assayed with nNOS, their IC₅₀, K_I, and k_inact values were obtained, and their crystal structures were determined. The identification and characterization of the products formed during inactivation provide evidence for the details of the inactivation mechanism. On the basis of these studies, the most probable mechanism for the inactivation of nNOS involves oxidative demethylation with the resulting thiol coordinating to the cofactor heme iron. Although nNOS is a heme-containing enzyme, this is the first example of a NOS that catalyzes an S-demethylation reaction; the novel mechanism of inactivation described here could be applied to the design of inactivators of other heme-dependent enzymes.

Full text of DOI:10.1021/jacs.5b01202

Article
pubs.acs.org/JACS
Mechanism of Inactivation of Neuronal Nitric Oxide Synthase by
(S)‑2-Amino-5-(2-(methylthio)acetimidamido)pentanoic Acid
Wei Tang,† Huiying Li,‡ Emma H. Doud,§ Yunqiu Chen,§ Stephanie Choing,† Carla Plaza,‡
Neil L. Kelleher,*,§ Thomas L. Poulos,*,‡ and Richard B. Silverman*,†
†Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, and Center for Molecular
Innovation and Drug Discovery, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
‡Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine,
California 92697-3900, United States
§Department of Chemistry, Department of Molecular Biosciences, Chemistry of Life Processes Institute, and Proteomics Center of
Excellence, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States
S
* Supporting Information
ABSTRACT: Nitric oxide synthase (NOS) catalyzes the
conversion of L-arginine to L-citrulline and the second messenger
nitric oxide. Three mechanistic pathways are proposed for the
inactivation of neuronal NOS (nNOS) by (S)-2-amino-5-(2-
(methylthio)acetimidamido)pentanoic acid (1): sulfide oxidation,
oxidative dethiolation, and oxidative demethylation. Four possible
intermediates were synthesized. All compounds were assayed with
nNOS, their IC50, KI, and kinact values were obtained, and their
crystal structures were determined. The identification and
characterization of the products formed during inactivation
provide evidence for the details of the inactivation mechanism.
On the basis of these studies, the most probable mechanism for
the inactivation of nNOS involves oxidative demethylation with
the resulting thiol coordinating to the cofactor heme iron.
Although nNOS is a heme-containing enzyme, this is the first example of a NOS that catalyzes an S-demethylation reaction; the
novel mechanism of inactivation described here could be applied to the design of inactivators of other heme-dependent enzymes.
Alzheimer’s,11 Huntington’s,12 and Parkinson’s,13 diseases.14−16
INTRODUCTION
■
The NOS structure can be divided into three domains: a heme-
Nitric oxide (NO) is an important cell-signaling molecule,1,2
and tetrahydrobiopterin-containing oxygenase domain and a
which is synthesized in vivo by nitric oxide synthase (NOS, EC
1.14.13.39).3 In the presence of molecular oxygen, NOS
FAD-, FMN-, and NADPH-containing reductase domain
connected by a calcium−calmodulin domain.17 Because of the
converts L-arginine (L-Arg) to L-citrulline (L-Cit) and NO, with
essential roles of NO, nNOS inhibition to diminish excessive
the reducing equivalents derived from oxidation of NADPH to
NADP+ (Scheme 1). During this two-step reaction, 2 and 1.5
neuronal NO must be selective over iNOS and eNOS
inhibition to prevent detrimental side effects.
equiv of O2 and NADPH, respectively, are consumed to
generate 1 equiv of NO.4,5 There are three mammalian NOS
To improve the binding selectivity of arginine mimetic
inhibitors with nNOS, a sulfur atom was incorporated into a
isoforms: neuronal nitric oxide synthase (nNOS), important to
series of arginine amidines with the expectation that the sulfur
neuronal signaling; inducible nitric oxide synthase (iNOS),
atom might form a favorable interaction with the iron atom of
which produces NO as a defense mechanism; and endothelial
the heme.18 All but one of the sulfur-containing compounds
nitric oxide synthase (eNOS), which uses NO to regulate blood
prepared had poor selectivity and inhibitory activity. According
pressure. Low levels of NO are important as a second
messenger and as a neurotransmitter.6 However, NO can
to these earlier studies from our laboratory, compound 1
rapidly react with superoxide anion (O2·−) to form
(Table S1, Supporting Information) is a selective, time-
dependent irreversible inhibitor of nNOS, 185-fold more
selective for nNOS inhibition than for eNOS inhibition,
although only 3-fold more selective over iNOS. The crystal
peroxynitrite (ONOO−).7 The high levels of NO, O2·−, and
ONOO− are implicated in neurodegenerative diseases. The
overproduction of NO in nNOS has been implicated in several
pathological conditions; therefore, the inhibition of excess NO
from nNOS has been an important approach for the design of
drugs for the treatment of septic shock,8 stroke,9 migraine,10
Received: February 3, 2015
Published: April 15, 2015
© 2015 American Chemical Society
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Scheme 1. Synthesis of NO from L-Arginine
structure of 1 bound to the nNOS active site (Figure 1) shows
that compound 1 is a type I ligand to the heme with its sulfur
enzyme recovery after inactivation, metabolite formation, and
modification of the heme by treatment of nNOS with 2−6 were
determined. Other potential heme-coordinating sulfur-contain-
ing inhibitors of nNOS have been reported to be only
inhibitors, not inactivators (8−11).15,18 These inhibitors either
have a longer linker length between the amidine moiety and the
sulfur atom or a larger tail size attached to the sulfur atom. Our
results support an inactivation mechanism that involves S-
demethylation followed by heme−thiol complexation (Figure 2,
pathway C).
Figure 1. Crystal structure of 1 (cyan) bound in the active site of
nNOS.19 Key distances are marked in angstroms. All structure figures
were prepared with PyMol (www.pymol.org).
atom and vicinal methylene carbon atom at a distance of 5.1
and 3.7 Å, respectively; on the basis of the reactivity of L-Arg
and the inactivator N5-(1-iminoethyl)-L-ornithine (L-NIO),20
this predicted distance suggested that the sulfur atom could be
oxidized by the heme as an initial step in an inactivation
mechanism. Oxidation could result in the formation of 2 and/
or 3, thereby producing potential electrophiles having good
leaving groups (methyl sulfenite or methyl sulfinite anions) and
that could be attacked by an active site nucleophile (Figure 2,
pathway A) or produce an enolate when an acidic proton
between the amidine and sulfoxide or sulfone moieties is
removed; enolate addition to a cofactor could lead to
inactivation. Sulfide oxidation to the corresponding sulfoxide
and sulfone metabolites is the most common heme-catalyzed
reactions for sulfide-containing molecules.21 Heme also can
metabolize sulfides via oxidative dethiolation to the correspond-
ing aldehyde products,22 leading to metabolite 4 (Figure 2,
pathway B), which might undergo Schiff base formation with a
lysine residue. If 1 can be oxidized to 4 by nNOS, then 5, which
might arise from a hydroxide reaction with 2 or 3, also could be
oxidized to 4. A third common metabolite reaction of sulfides is
S-demethylation, which would convert 1 to 6 and form-
aldehyde; the generated thiol might form a complex with the
heme iron atom (Figure 2, pathway C). All of these possible
pathways are initiated by one-electron oxidation of the sulfide
sulfur atom by a heme iron-oxo species, leading to the
corresponding sulfenium cation radical (7, Figure 2).23
RESULTS AND DISCUSSION
■
Kinetics of Inhibition of nNOS by 1. Inhibition
experiments were performed by measuring the velocity of
NO generation after using different concentrations of the
inhibitor and different concentrations of L-Arg (substrate). The
NO hemoglobin assay was performed on 1−6 (see syntheses in
the Supporting Information), and their IC50 and Ki values were
obtained with rat nNOS (Table 1). Compound 6 was
comparable in potency to 1, compound 5 was half as potent,
and the remainder displayed considerably weaker potency.
Compounds 2 and 3 are possible metabolites from inactivation
pathway A (Figure 2), but both were very weak inhibitors, and
neither showed time-dependent inactivation with nNOS. The
proposed metabolite in pathway B is 4; 5 could be oxidized to
4. Compounds 4 and 5 inhibited nNOS, but 4 is much less
potent than 1, and neither was a time-dependent inhibitor.
Only 6 is more potent than 1 and also is a time- and
concentration-dependent inactivator of nNOS; on the basis of
steady-state kinetics, 6 appears to be a kinetically competent
intermediate in the nNOS inactivation mechanism by 1.
To test the viability of these proposed products and
mechanisms, the inhibitory activity, inactivation kinetics,
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Figure 2. Three possible inactivation mechanisms for nNOS by 1.
Table 1. IC50 and Ki Values for 1−6 with nNOS
compound
IC50 (μM)
Ki (μM)
1
2
3
4
5
6
5.59 0.55
461 20
0.64 0.063
53.0 2.4
507 49
58.3 5.6
199 7.1
13.0 0.67
3.64 0.15
22.9 0.82
1.50 0.08
0.42 0.017
Experiments varying the concentrations of 1 and 6 with rat
nNOS at room temperature were performed to determine their
KI and kinact values (Figure 3). On the basis of their KI values, 6
binds about 20 times more tightly to nNOS than does 1, and
the kinact values show that 6 has about half the inactivation rate,
indicating that 6 is about 9 times more efficient than 1 (Table
2), again indicating that, on the basis of steady-state kinetics, 6
is a kinetically competent intermediate. To support the
hypothesized mechanism that generates 6 (Figure 2, pathway
C), the nNOS generation of both 6 and formaldehyde was
determined.
spectrometry (LC/MS) was performed to detect possible
metabolites from inactivation of nNOS by 1. Incubation
mixtures in replicate with neither inhibitor nor NADPH were
studied as controls, and the data consistently showed that a
small amount of 2 was generated after inactivation (Supporting
Information, Figure S4). The mass of o-phthalaldehyde (OPA)
and β-mercaptoethanol (BME)-derivatized 2 (compound 32)
had a LC retention time of 17.7 min, confirmed using a
synthetic standard (Supporting Information, Figure S1).
However, sulfur oxidation of 1 was found to occur during the
LC/MS process. When this occurred, the m/z values of the
oxidized compounds were observed at the same retention times
as the standard nonoxidized compounds. For example, in both
Identification of Amino Acid Metabolites during
Inactivation of nNOS by 1. Liquid chromatography/mass
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Figure 3. (A) Time- and concentration-dependent inactivation of nNOS by 6. Each point represents the mean of three independent determinations.
(B) Kitz−Wilson replot for the inactivation of nNOS by 6; the equation for the plot is y = 43.9x + 15.38, R2 = 0.95.
more oxidized the compound (33 > 32 > 31), the earlier it
elutes from reverse-phase high-performance liquid chromatog-
raphy (HPLC), if it exists independently in the solution and is
not created as an artifact of electrospray ionization (ESI) mass
spectrometry.
It was further confirmed that 32 can be generated from the
incubation of nNOS with 31. No OPA and BME-derivatized 3,
4, or 6 were detected under these conditions. Compounds 4
and 6 were not detected using ESI in either the positive or
negative mode, but 6 might undergo oxidative degradation.
The amount of 2 generated from the inactivation was
determined using a standard linear curve (Supporting
Information, Figure S4). Approximately 0.53 μM 2 is produced
from the inactivation of nNOS by 1, which converts to 2.9%
turnover. Although 2 is a metabolite of this inactivation, its low
concentration and poor inactivation rate constants indicate that
it is not a kinetically competent intermediate.
Table 2. KI and kinact Values ( Standard Error) for 1 and 6
with nNOS
1
6
kinact (min−1
KI (μM)
)
0.11 0.01
59.0 6.0
0.046 0.002
2.9 0.2
k
inact/KI (μM−1 min−1
)
0.0019 0.0001
0.016 0.001
the reaction and injection of standard 31, an m/z
corresponding to 32 appeared at the same retention time (tR
= 18.8 min, Figures 4 and Supporting Information, Figure S1).
Whereas when a standard of 32 was injected, the m/z
corresponding to both 32 and 33 appeared at tR = 17.7 min,
indicating the proclivity of the sulfur atom to oxidize under
typical electrospray ionization conditions. The OPA and BME-
derivatized 1 (Supporting Information, Figure S1) and 2
(Supporting Information, Figure S2) were used as standards.
The data were confirmed by spiking with standards 31 and 32
(Supporting Information, Figure S3). This confirms that the
Figure 4. (a) Total ion chromatogram of LC/MS of inactivation of nNOS by 1. (b) Extracted ion chromatogram of m/z corresponding to 31. (c)
Extracted ion chromatogram of m/z corresponding to 32.
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Figure 5. LC-MS of the NADPH-dependent formation of formaldehyde after inactivation of nNOS by 1. Traces from top to bottom: LC/UV (360
nm) of control with no 1 (pink), LC/UV (360 nm) of control with no NADPH (orange), LC/UV (360 nm) of 1-inactivated sample (blue);
extracted ion chromatogram (LC/MS) of DNPH-derivatized formaldehyde; extracted ion chromatogram (LC/MS) of DNPH.
Identification of Formaldehyde as a Metabolite.
Formaldehyde should be produced if 1 inactivates nNOS by
mechanistic pathway C. The amount of formaldehyde was
determined using 2,4-dinitrophenyl hydrazine (DNPH) deriva-
tization. LC with both UV and MS detection was performed,
and the generation of formaldehyde was identified (Figure 5).
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Figure 6. (a) Titration of rat nNOS−H4B with 6. (b) Titration of dithionite-reduced nNOS−H4B with 6. The plot was normalized at 430 and 435
nm, respectively. Note also, the trough positions are 400 and 410 nm in (a) and (b), respectively.
By using a standard curve for DNPH-derivatized formaldehyde
(Supporting Information, Figure S5), there is about 38%
conversion from 1.
Ferric and Ferrous nNOS Difference Spectra with 6.
For a heme thiolate enzyme such as NOS, the four heme
pyrrole nitrogen atoms and the proximal heme ligand from a
Cys residue form a pentacoordinated iron center. The NOS
substrate L-Arg binds to the NOS active site through its
extensive hydrogen-bonding interactions with the amino acid
side chains lining the active site but without making ligation
with heme. Therefore, the L-Arg is a type I, high-spin ligand
generating a Soret peak at 395 nm typical of high-spin NOS
and P450. However, if a heme type II, low-spin ligand is
available at the distal side, such as imidazole or a water
molecule, the heme becomes hexacoordinated, thereby showing
a red shift of its Soret peak (427 nm for imidazole).24 For NOS,
the binding of the H4B cofactor alone in the absence of L-Arg
can still form a high-spin heme with a Soret peak at 400 nm;
therefore, this can be used as the starting sample for titrations
with a type II ligand.
Compound 6 was found to be a type II ligand as its binding
to nNOS resulting in a bisthiolate species, having one native
Fe−thiolate bond from the proximal Cys415 residue and a
distal Fe−thiolate bond from 6. Figure S6 (Supporting
Information) shows the features of split Soret peaks character-
istic of the bisthiolate. The apparent binding constant (Ks) of 6
can be determined from the difference spectra between a peak
at 455 nm and a trough at 400 nm with rat nNOS, as shown in
Figure 6a. This is in contrast to parent compound 1, which is
known to be a type I ligand from its difference spectra18 and its
crystal structure (Figure 1). We have also checked the binding
of 6 to the dithionite-reduced nNOS−H4B complex. The
difference spectra exhibited a peak at 455 nm and a trough at
410 nm (Figure 6b). Again, 6 is still a type II ligand and may
ligate to the ferrous heme iron as well. This provides support
for pathway C as the inactivation mechanism. The apparent
binding constants of 6, Ks, to the ferric and ferrous nNOS are
1.8 0.1 and 1.6 0.3 μM, respectively; therefore, there is no
significant difference.
Figure 7. Crystal structures of 2 (a, green) and 3 (b, magenta) bound
to the rat nNOS active site. Two alternate conformations were
observed for the >SO moiety of 2, where the directions of methyl
(green) and SO (red) vary but the sulfur atom (yellow) remains in
the same position. Key distances are marked in angstroms.
Although once the heme iron is reduced and molecular oxygen
is bound, the ligand conformation might undergo some changes
that are not reflected in the crystal structures, S-oxidation is not
likely the major turnover pathway considering the far Fe−S
distance observed in the structures. On the other hand, S-
demethylation of 1 to give 6 should be easier than S-oxidation,
since the S−C bond is more accessible to the heme−oxo
species, leading to pathway C for nNOS inactivation (Figure 2).
The distance between the vicinal methylene carbon atom and
the heme iron is 3.7 Å in 1 (Figure 1), which is a reasonable
distance for carbon oxidation, and potential intermediate 4
might be generated after oxidation. The crystal structure for 4
bound to the nNOS active site (Figure 8a) shows a binding
mode similar to that of 1 (Figure 1). A crystal structure was also
obtained for 5 bound to the nNOS active site (Figure 8b). Its
binding mode is similar to that of the substrate Nω-
hydroxyarginine. The main difference between 4 and 5 is that
the CO group in 4 bends significantly away from the plane
defined by the Ne, Cz, Ch, and Nh atoms, while in 5 the
hydroxyl group is almost coplanar. As a result, binding of 5 to
nNOS is stronger than 4 (Table 1) because of an extra H bond
from the OH to the backbone amide of Gly586, while the C
O group in 4 makes no significant contact with protein.
Crystal Structures of 1−6. The crystal structures of 1
(Figure 1) as well as 2 and 3 (Figure 7) show a distance of
about 5.1, 4.7, and 4.8 Å, respectively, between the S atom and
the Fe atom. In addition, the lone-pair electrons of the sulfur
atom on 1 are not properly oriented to interact with the iron−
oxo bond required for S-oxidation in pathway A (Figure 2).
Although the difference spectra indicated that 6 was a type II
ligand (Figure 6), the Fe−S bond was not observed initially in
the crystal structures for 6 bound to the nNOS active site.
While the first structure obtained at pH 6.0 (pH of the
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CONCLUSIONS
■
Compound 1 is an inactivator of nNOS; three possible
mechanistic pathways were proposed. The corresponding
metabolites were synthesized, and their enzymatic kinetic data
were determined. Compounds 2, 3, and 4 showed much larger
IC50 values than 1, suggesting that they are not relevant to the
inactivation mechanism. Compound 6 gave a slightly smaller Ki
value compared to 1, indicating that they have comparable
binding energies. Furthermore, 6 turns over about 10 times
faster than 1 with nNOS, which suggests, on the basis of steady-
state kinetics, that 6 is a kinetically competent intermediate for
inactivation of nNOS by 1. The production of formaldehyde
occurred in 38% of 1 turnovers to products, although LC/MS
did not reveal the presence of 6 after inactivation. The
generation of 2 in about 3% of turnovers was confirmed, but
this could be an irrelevant side reaction. The crystal structure of
6 bound to nNOS confirmed the existence of an Fe−S bond.
These experiments support an inactivation mechanism that
involves heme oxo S-demethylation and coordination of the
thiolate produced with the heme iron (Figure 2, pathway C).
Although nNOS is a heme-containing enzyme, this is the first
example of an S-demethylation reaction catalyzed by nNOS,
and it also is a novel inactivation mechanism, which could be
applied to other heme-dependent enzymes.
Figure 8. Crystal structures of 4 (a, orange) or 5 (b, yellow) bound to
the rat nNOS active site. Key distances are marked in angstroms.
crystallization buffer) showed a type I ligand binding mode for
6 with its S atom pointing away from the heme (data not
shown), the second structure obtained by transferring crystals
to the cryo-soaking solutions at pH 7.5 revealed two alternate
conformations (Figure 9a). The major conformation (70%) still
EXPERIMENTAL SECTION
■
General Methods. All chemicals were purchased from Sigma−
Aldrich and were used without further purification. LC/MS studies of
amino acids were carried out on an Agilent 1200 LC and a Thermo
Exactive (ESI) mass spectrometer using a Phenomenex (Torrance,
CA, USA) Luna C18 100A (5 μm, 150 cm × 2 cm) column.
Determination of formaldehyde and analytical reverse-phase HPLC/
MS were performed on an Agilent 1200 series system (Agilent
Technologies, Santa Clara, CA, USA) using a Phenomenex Gemini-
NX C18 column (4.6 cm × 50 cm, 5 μm). This system was equipped
with an Agilent G1315C DAD detector and an Agilent 6130
quadrupole MS detector. Preparative runs were performed with a
Phenomenex Gemini-NX C18 column (21.20 cm × 150 cm, 5 μm).
Chemical Syntheses. See details in the Supporting Information.
(S)-2-Amino-5-(2-(methylthio)acetimidamido)pentanoic Acid (1).
This compound was prepared according to the method of Litzinger et
al.18
Figure 9. (a) Crystal structure of 6 (pink) bound to the rat nNOS
active site obtained at pH 7.5; two alternate conformations were found
for the terminal sulfur atom. (b) The ferric nNOS−6 structure
obtained with the composite data collection procedure at pH 7.5; a
ligation bond at 2.7 Å was observed.
had the S atom pointing away from the heme, similar to what
was seen at pH 6.0; the other conformation (30%) showed the
S atom pointing toward the heme with an Fe−S distance of 3.0
Å. The observed Fe−S distance is longer than that for an Fe−S
ligation bond, but reduction of the heme iron in the X-ray beam
very likely alters ligand affinity.
In previous work,18 we developed a composite data
collection protocol that ensures the iron remains in the ferric
oxidation state. This involves merging data obtained from
several crystals, each of which is exposed to X-rays for only a
short time, thereby avoiding iron reduction. The structure of 6
bound to nNOS obtained using this approach shows a single
conformation of 6 with its S atom 2.7 Å from the heme iron
(Figure 9b). Although this is longer than the proximal Fe−
thiolate bond length with the Cys ligand, it is reasonable for a
ligation bond in an iron bisthiolate species (there is no known
bisthiolate heme in native proteins). This observed ligation
bond provides the structural basis for pathway C (Figure 2) as
the inactivation mechanism.
(2S)-2-Amino-5-(2-(methylsulfinyl)acetimidamido)pentanoic
Acid (2). This compound was prepared from 1 equiv oxidation of 1
with H2O2.
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Hemoglobin NO Generation Assay. Recombinant rat nNOS
was expressed and purified from Escherichia coli.28 The production of
NO was measured by the rapid oxidation of oxyHb to metHb by
NO.29 The inhibition assays contained 10 μM L-Arg, 104 μM
NADPH, 833 μM CaCl2, 3 U CaM, 80 μM H4B, 0.75 μg of oxyHb,
and different amounts of inhibitors. The final volume was adjusted to
600 μL with 100 mM HEPES buffer, pH 7.4. The enzymatic reaction
was initiated by addition of 10 μL of nNOS stock, and the rate of NO
production was monitored by the change in absorbance at 401 nm in
the initial 60 s on a spectrophotometer at 37 °C. For Ki
determinations, IC50 values were calculated using nonlinear regressions
(dose−response inhibition, four-parameter variable slope). Subsequent
Ki values were calculated using the Cheng−Prusoff relationship: Ki =
IC50/(1 + [S]/Km) (Km for murine nNOS is 6.9 μM).30
(S)-2-Amino-5-(2-(methylsulfonyl)acetimidamido)pentanoic Acid
(3). This compound was prepared following the synthetic route above.
LC/MS Study of 1. The inactivation mixture of 1 with nNOS was
derivatized with OPA and BME. Inactivation mixtures contained 56
μM 1, 0.0055 mg of catalase, 6% glycerol, 3.51 mM NADPH, 0.56 mM
H4B, 3 mM CaCl2, 0.018 U CaM, and 12 μM nNOS stock in a total
volume of 100 μL. HEPES buffer (100 mM, pH 7.4) was used as the
solvent. The mixture was incubated at 37 °C for 3 h until completion
of inactivation. Aliquots (10 μL) were removed and added to 20 μL of
OPA/BME (20:1, v/v) reagent. The sample was injected into the
HPLC chromatograph; the gradient was started with 2% B, held for 5
min, and then, 2−98% B over 30 min (A = H2O with 0.1% formic acid
and B = MeCN with 0.1% formic acid), followed by a wash of 98% B
for another 5 min. MS data were collected in positive ESI mode.
Irreversible Inhibition Kinetics. The same inactivation mixtures
were prepared as above. The reactions were initiated by the addition of
enzyme, and 10 μL aliquots were removed at specified time points and
tested via the initial velocity assay. Controls were performed by
replacing the inhibitor or NADPH volume with HEPES buffer. KI and
kinact values were determined by the method of Kitz and Wilson.31
DNPH Derivatization of Formaldehyde. DNPH-derivatization
reactions were prepared by mixing 20 μL of formaldehyde standard or
sample with 10 μL of 20 mM DNPH in 0.4 M H2SO4 in 10% H2O,
90% MeCN. The derivatization reactions were complete in 10 min.
The sample was injected onto a Phenomenex Gemini-NX C18 column
at 360 nm; mobile phase A is H2O with 1% formic acid, and B is
MeCN with 1% formic acid. A gradient from 10−90% B was run over
7 min at 1.0 mL/min. Then, B was increased to 100% in 0.1 min and
held until 10 min. Under these conditions, DNPH eluted at 3.2 min,
and DNPH-formaldehyde eluted at 4.3 min. A DNPH−formaldehyde
standard curve (R2 = 0.981) was linear from 10 to 120 μM.
(S)-2-Amino-5-(2-oxoacetimidamido)pentanoic Acid (4). This
compound was prepared following the synthetic route above.
Spectral Titration of Ferrous and Ferric nNOS with 6. These
experiments were similar to those reported earlier.18 The inhibitor−
ferric difference spectrometry assays were performed on a Cary 300
(Agilent) spectrophotometer. Full-length nNOS (5 μM) in 600 μL of
HEPES buffer (50 mM, pH 7.5, 100 mM NaCl) was titrated with 1 or
2 μL of 6, and spectral changes were monitored from 350 to 550 nm.
The total volume of the aliquots did not exceed 2% of the buffer
volume.
(S)-2-Amino-5-(2-hydroxyacetimidamido)pentanoic Acid (5).
This compound was prepared following the synthetic route above.25
For ferrous difference spectrometry assays, the spectral titrations of
inhibitor 6 binding to the ferrous nNOS were carried out under
anaerobic conditions with a Cary 4 (Varian) spectrophotometer. The
HEPES buffer (50 mM, pH 7.5, 100 mM NaCl) was sealed in serum
vials and degassed by purging the solution with ultrapure argon and
pulling vacuum in the head space of the vial for a few alternating cycles
within 2 h. All of the inhibitor stock solutions were made with
degassed buffer inside a glovebox (COY Laboratory Products, Inc.).
The full-length nNOS was reduced by adding grains of dithionite, and
then, the reduced sample was passed through a 10-DG desalting
column (Bio-Rad Laboratories) in the glovebox to remove excess
dithionite. An aliquot of reduced nNOS was used to take a spectral
scan, which showed a Soret peak at 410 nm, confirming the reduction
of heme. To both the sample and reference septum-sealed cuvettes
was added 600 μL of buffer containing ∼6.2 μM nNOS; then, the
baseline was set up with both cuvettes in place using the double beam
mode of the instrument. Each addition of 1−2 μL of 6 to the sample
cuvette, with stock concentrations of 1, 5, and 20 mM, was performed
inside the glovebox. The total volume of the aliquots was less than 2%
(S)-2-Amino-5-(2-mercaptoacetimidamido)pentanoic Acid (6).
This compound was prepared following the synthetic route above.26,27
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of the buffer volume (600 μL) in the sample cuvette. After each
addition, spectral scans were carried out in the range 350−600 nm and
repeated a few times until no further spectral change was observed.
The absorbance differences from the peak (∼455 nm) to the trough
(400 nm for ferric and 410 nm for ferrous heme) positions were
extracted from titration curves and then plotted against the
micromolar concentration of inhibitor 6. The resulting plots showing
the saturation features that could not be fit properly with a hyperbolic
function in SigmaPlot (ΔA = Bmax [L]/(Ks + [L]); instead, a quadratic
function was used to derive the apparent Ks values from the plot:
ASSOCIATED CONTENT
■
S
* Supporting Information
Structures of compounds 1−6 and 31−33; HPLC of
metabolites of nNOS oxidation of 1; LC/MS of formaldehyde
formation from inactivation of nNOS by 1; crystallographic
data; chemical synthesis; and NMR spectra of final products.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.5b01202.
AUTHOR INFORMATION
■
ΔA = A0 + (Bmax/2[E])((Ks + [E] + [L])
Corresponding Authors
*n-kelleher@northwestern.edu
*poulos@uci.edu
*Agman@chem.northwestern.edu
Notes
− ((Ks + [E] + [L])2 − 4[E][L])1/2
)
where Bmax is the maximum absorbance change to infinite ligand
concentration, [E] is the total enzyme concentration, [L] is the ligand
concentration, and A0 is a constant.
The authors declare no competing financial interest.
Inhibitor Complex Crystal Preparation. The nNOS heme
domain proteins used for crystallographic studies were produced by
limited trypsin digest from the corresponding full-length enzymes and
further purified through a Superdex 200 gel filtration column (GE
Healthcare); the nNOS heme domain (at 9 mg/mL containing 20 mM
histidine) was used for the sitting drop vapor diffusion crystallization
setup under conditions as described previously.32 Fresh crystals (1−2
days old) were first passed stepwise through cryoprotectant solutions
and then soaked with 10 mM inhibitor for 4−6 h at 4 °C before being
flash cooled with liquid nitrogen.
X-ray Diffraction Data Collection, Data Processing, and
Structural Refinement. The cryogenic (100 K) X-ray diffraction
data were collected remotely at the Stanford Synchrotron Radiation
Lightsource or Advanced Light Source (ALS) through the data
collection control software Blu-Ice33 and a crystal mounting robot.
When a Q315r CCD detector was used, 90−100° of data were
typically collected with 0.5° per frame. If a Pilatus pixel array detector
was used, 140−150° of fine-sliced data were collected with 0.2° per
frame. Raw CCD data frames were indexed, integrated, and scaled
using HKL2000,34 but the pixel array data were processed with XDS35
and scaled with Scala (Aimless).36 The composite data of nNOS
bound with 6 were collected with about 20 crystals at ALS BL12.3.1.
For each crystal, a snapshot was taken, and the image was indexed to
get the orientation matrix. The Strategy routine in MOSFLM37 was
used to determine the starting scanning angle for data collection. Then
5° of data at 1 s exposure per frame (1°) were collected. Once the next
crystal was mounted and the snapshot image was indexed, the Strategy
in MOSFLM was used again to determine the best starting angle for
the current crystal by incorporating crystal orientation matrices from
the previous crystal(s). The calculation was necessary to avoid
collecting data in the reciprical space that had been covered by the data
from the previous crystals. Data frames from multiple crystals were
integrated with XDS and then merged and scales with Aimless. Data
frames from certain crystals which showed poor merging R values were
rejected in the final scaling.
The binding of inhibitors was detected by the initial difference
Fourier maps calculated with REFMAC.38 The inhibitor molecules
were then modeled in COOT39 and refined using REFMAC or
PHENIX.40 Water molecules were added in REFMAC or PHENIX
and checked by COOT. The TLS41 protocol was implemented in the
final stage of refinements with each subunit as one TLS group. The
omit Fo − Fc density maps were calculated by repeating the last round
of TLS refinement with the inhibitor coordinate removed from the
input PDB file to generate the map coefficients DELFWT and
SIGDELWT. The refined structures were validated in COOT before
deposition in the protein data bank. The crystallographic data
collection and structure refinement statistics are summarized in
Table S2 (Supporting Information) with the PDB accession codes
included.
ACKNOWLEDGMENTS
■
This work was supported by the National Institutes of Health
(GM049725 to R.B.S. and GM057353 to T.L.P.).
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