Mechanistic studies of the inactivation of inducible nitric oxide synthase by N5-(1-iminoethyl)-L-ornithine (L-NIO)

Article abstract of DOI:10.1021/ja982318l

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L- citrulline and nitric oxide. N⁵-(1-iminoethyl)-L-ornithine (L-NIO, 5) is a natural product known to inactivate NOS, but the mechanism of inactivation is unknown. Upon incubation of iNOS with L-NIO a type I binding difference spectrum is observed, indicating that binding at the substrate binding site occurs. L-NIO is shown to be a time-dependent, concentration-dependent, and NADPH-dependent irreversible inhibitor of iNOS with K(I) and k(inact) values of 13.7 ± 1.6 μM and 0.073 ± 0.003 min^-1, respectively. During inactivation the heme chromophore is partially lost (Figure 1); HPLC shows that the loss corresponds to about 50% of the heine. Inclusion of catalase during incubation does not prevent heine loss. N⁵-(1-Imino-2-[¹⁴C]ethyl)- L-ornithine (11) inactivates iNOS, but upon dialysis or gel filtration, no radioactivity remains bound to the protein or to a cofactor. The only radioactive product detected after enzyme inactivation is N(ω)-hydroxy-L- NIO (12); no C(ω)-hydroxy-L-NIO (13) or N(δ)-acetyl-L-ornithine (14) is observed (Figure 2). The amount of 12 produced during the inactivation process is 7.7 ± 0.2 equiv per inactivation event. Incubations of 12 with iNOS show time-, concentration-, and NADPH-dependent inactivation that is not reversible upon dilution into the assay solution. Incubations that include an excess of L-arginine or with substitution of NADP⁺ for NADPH result in no significant loss of enzyme activity. The K(I) and k(inact) values for 12 are 830 ± 160 μM and 0.0073 ± 0.0007 min^-1, respectively. The magnitude of these kinetic constants (compared with those of 5) suggest that 12 is not an intermediate of L-NIO inactivation of iNOS. Compound 12 also is a substrate for iNOS, exhibiting saturation kinetics with K(m) and k(cat) values of 800 ± 85 μM and 2.22 min^-1, respectively; the product is shown to be N(δ)- acetyl-L-ornithine (14) (Figure 3). The k(cat) and k(inact) values for 12 can be compared directly to give a partition ratio (k(cat)/k(inact)) for inactivation of 304; i.e., there are 304 turnovers to give NO per inactivation event. This high partition ratio further supports the notion that 12 is not involved in L-NIO inactivation of iNOS. C(ω)-Hydroxy-L-NIO (13) is not an inactivator of iNOS. These results suggest that L-NIO inactivation occurs after an oxidation step (NADPH is required for inactivation) but prior to a hydroxylation step (12 and 13 are not involved). Inactivation of iNOS by N⁵-(1-imino-2-[²H₃]-ethyl)-L-ornithine (15) exhibits 3 kinetic isotope effect (H)k(inact)/(D)k(inact) of 1.35 ± 0.08 and on (H)(k(inact)/k(I)/(D)-(k(inact)/K(I) of 1.51 ± 0.3, suggesting that the methyl C-H bond is cleaved in a partially rate-determining step prior to hydroxylation, and that leads to inactivation. A new NADPH-dependent 400 nm peak in the HPLC of L-NIO-inactivated iNOS is produced (Figure 4). LC- electrospray mass spectrometry (Figure 5) demonstrates the m/z of the new metabolite to be 583, which is shown to correspond to biliverdin (23) (Figures 6 and 7). Two possible mechanisms for the formation of biliverdin during inactivation are proposed (Schemes 10 and 11). When 14 is incubated with iNOS, time-, concentration-, and NADPH-dependent loss of enzyme activity is observed (K(I) and k(inact) values are 490 mM and 0.24 min^-1, respectively); iNOS inactivation by 14 can be prevented by inclusion of L- arginine, but not D-arginine, in the inactivation mixtures, suggesting that the inactivator acts at the arginine binding site. However, 14 is not produced from L-NIO (Figure 2) and, therefore, is not involved in L-NIO inactivation.

Full text of DOI:10.1021/ja982318l

J. Am. Chem. Soc. 1999, 121, 903-916
903
Mechanistic Studies of the Inactivation of Inducible Nitric Oxide
Synthase by N5-(1-Iminoethyl)-L-ornithine (L-NIO)
Walter Fast,†,‡ Dejan Nikolic,§ Richard B. Van Breemen,§ and Richard B. Silverman*,†,|
Contribution from the Department of Chemistry and Department of Biochemistry, Molecular Biology, and
Cell Biology, Northwestern UniVersity, EVanston, Illinois 60208-3113, and the Department of Medicinal
Chemistry, UniVersity of Illinois at Chicago, Chicago, Illinois 60612-7231
ReceiVed July 2, 1998
Abstract: Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and nitric oxide.
N5-(1-Iminoethyl)-L-ornithine (L-NIO, 5) is a natural product known to inactivate NOS, but the mechanism of
inactivation is unknown. Upon incubation of iNOS with L-NIO a type I binding difference spectrum is observed,
indicating that binding at the substrate binding site occurs. L-NIO is shown to be a time-dependent, concentration-
dependent, and NADPH-dependent irreversible inhibitor of iNOS with KI and kinact values of 13.7 ( 1.6 µM
and 0.073 ( 0.003 min-1, respectively. During inactivation the heme chromophore is partially lost (Figure 1);
HPLC shows that the loss corresponds to about 50% of the heme. Inclusion of catalase during incubation does
not prevent heme loss. N5-(1-Imino-2-[14C]ethyl)-L-ornithine (11) inactivates iNOS, but upon dialysis or gel
filtration, no radioactivity remains bound to the protein or to a cofactor. The only radioactive product detected
after enzyme inactivation is Nω-hydroxy-L-NIO (12); no Cω-hydroxy-L-NIO (13) or Nδ-acetyl-L-ornithine (14)
is observed (Figure 2). The amount of 12 produced during the inactivation process is 7.7 ( 0.2 equiv per
inactivation event. Incubations of 12 with iNOS show time-, concentration-, and NADPH-dependent inactivation
that is not reversible upon dilution into the assay solution. Incubations that include an excess of L-arginine or
with substitution of NADP+ for NADPH result in no significant loss of enzyme activity. The KI and kinact
values for 12 are 830 ( 160 µM and 0.0073 ( 0.0007 min-1, respectively. The magnitude of these kinetic
constants (compared with those of 5) suggest that 12 is not an intermediate of L-NIO inactivation of iNOS.
Compound 12 also is a substrate for iNOS, exhibiting saturation kinetics with Km and kcat values of 800 ( 85
µM and 2.22 min-1, respectively; the product is shown to be Nδ-acetyl-L-ornithine (14) (Figure 3). The kcat
and kinact values for 12 can be compared directly to give a partition ratio (kcat/kinact) for inactivation of 304; i.e.,
there are 304 turnovers to give NO per inactivation event. This high partition ratio further supports the notion
that 12 is not involved in L-NIO inactivation of iNOS. Cω-Hydroxy-L-NIO (13) is not an inactivator of iNOS.
These results suggest that L-NIO inactivation occurs after an oxidation step (NADPH is required for inactivation)
but prior to a hydroxylation step (12 and 13 are not involved). Inactivation of iNOS by N5-(1-imino-2-[2H3]-
H
ethyl)-L-ornithine (15) exhibits a kinetic isotope effect on kinact/Dkinact of 1.35 ( 0.08 and on H(kinact/KI)/D-
(kinact/KI) of 1.51 ( 0.3, suggesting that the methyl C-H bond is cleaved in a partially rate-determining step
prior to hydroxylation, and that leads to inactivation. A new NADPH-dependent 400 nm peak in the HPLC of
L-NIO-inactivated iNOS is produced (Figure 4). LC-electrospray mass spectrometry (Figure 5) demonstrates
the m/z of the new metabolite to be 583, which is shown to correspond to biliverdin (23) (Figures 6 and 7).
Two possible mechanisms for the formation of biliverdin during inactivation are proposed (Schemes 10 and
11). When 14 is incubated with iNOS, time-, concentration-, and NADPH-dependent loss of enzyme activity
is observed (KI and kinact values are 490 mM and 0.24 min-1, respectively); iNOS inactivation by 14 can be
prevented by inclusion of L-arginine, but not D-arginine, in the inactivation mixtures, suggesting that the
inactivator acts at the arginine binding site. However, 14 is not produced from L-NIO (Figure 2) and, therefore,
is not involved in L-NIO inactivation.
Introduction
messenger and is synthesized in vivo from oxygen and L-
arginine by the enzyme nitric oxide synthase (NOS, EC
1.14.13.39).1 The importance of understanding the chemistry
and biochemistry of NO has been highlighted in fields as diverse
as immunology and reproductive technology, and the implication
of NO overproduction in septic shock, rheumatoid arthritis,
strokes, and Parkinson’s disease (among numerous other disease
states) has fueled an increasing interest in the unique enzymol-
ogy of NOS, how its catalysis occurs, and how it can be
inhibited.1
The relatively recent interest in nitric oxide (NO) has arisen
from the surprising discovery that NO serves as a biological
* Address correspondence to this author at Department of Chemistry,
Northwestern University. Phone: (847) 491-5653. Fax: (847) 491-7713.
E-mail: Agman@chem.nwu.edu.
† Department of Biochemistry, Molecular Biology, and Cell Biology,
Northwestern University.
‡ Present address: Department of Chemistry, The Pennsylvania State
University, 152 Davey Laboratory, University Park, PA 16802.
§ University of Illinois at Chicago.
| Department of Chemistry, Northwestern University.
(1) Kerwin, J. F.; Heller, M. Med. Res. ReV. 1994, 14, 23-74.
10.1021/ja982318l CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/22/1999

904 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Fast et al.
NO has been implicated in numerous biological functions;2
however, most systems can be grouped into one of three
paradigms for NO function which correlate with three known
isoforms of NOS: the immune response (inducible NOS or
iNOS), smooth muscle relaxation (endothelial NOS or eNOS),
and neuronal signaling (neuronal NOS or nNOS).
Scheme 1
NOS has been shown to bind a number of cofactors including
one FAD, one FMN, one NADPH, one protoporphyrin IX, and
one tetrahydrobioperin (BH4) per monomer. Each isoform is
also calmodulin dependent, although iNOS has been shown to
bind calmodulin tightly regardless of Ca2+ concentration
fluctuations.3 NOS is active only as a disulfide-linked ho-
modimer,4 with each monomer consisting of two subdomains.
Inspection of the NOS sequence and the crystal structure of
iNOS5 allows the identification of a C-terminal and N-terminal
domain. The C-terminal domain of NOS shows a high sequence
similarity to cytochrome P450 reductase and contains sequences
for FAD, FMN, and NADPH binding sites.6 This domain serves
to shuttle electrons from the obligate two electron donor,
NADPH, through the one or two electron acceptor/donors, FAD
and FMN, to the N-terminal domain of the other monomer.7 In
the N-terminal domain, which binds the heme and tetrahydro-
biopterin,8 the tetrahydrobiopterin is suspected to be the initial
electron acceptor, which subsequently transfers electrons to the
heme where substrate oxidation occurs.9 In the resting state, an
air-stable flavin semiquinone (presumably FMNH•) has been
detected by EPR.10 Unlike the C-terminal domain, the N-
terminal domain does not show any sequence similarity to
known cytochrome P450 related proteins. In the resting state,
NOS contains a ferric, five coordinate heme with a cysteine
thiolate as the proximal axial ligand. The N-terminal and
C-terminal domains are joined by a short sequence encoding a
of L-arginine14 and from the corresponding nitrogen in Nω-
hydroxy-L-arginine.12,15 One NADPH is used in producing Nω-
hydroxy-L-arginine (2), and 0.5 NADPH is used in conversion
of this intermediate to citrulline and NO.
Because of its similarity to cytochrome P450s, various heme-
based mechanisms have been proposed for the catalysis of
L-arginine to NO and L-citrulline. Like cytochrome P450, carbon
monoxide can inhibit both steps of the reaction (although at
different levels16), implicating a role for the heme in each
oxidation step.17 Following precedence for N-hydroxylation of
amidines by cytochrome P450s,18 a classical P450 type mech-
anism involving a perferryl Fe-heme complex ([FeO]3+ 19
) can
be proposed for the first step in the reaction (Scheme 2).
Formation of the perferryl Fe-heme complex usually requires
a general acid derived from the enzyme.20 No suitable residues
are found in the hydrophobic distal heme pocket; however,
substrate-assisted catalysis has been suggested.5,21 As with the
cytochrome P450 enzymes, uncoupled production of hydrogen
peroxide22 and superoxide23 can be observed under certain
conditions. Even though a similar perferryl Fe-heme type
mechanism can be proposed for the second oxidation step, White
and Marletta24 suggest that, following hydrogen atom abstaction
of 2, the second oxidation may be more like that proposed for
aromatase, also a multistep oxidative catalyst.25 In the third step
of aromatase, the electrophilicity of a substrate acyl-carbon
calmodulin binding site which serves as a sort of switch. Ca2+
/
calmodulin are required to allow electron transfer between the
C-terminal and N-terminal domains.11
NOS converts L-arginine (1, Scheme 1) and O2 to citrulline
(3) and NO with the concomitant oxidation of NADPH. Nω-
Hydroxy-L-arginine (2) has been shown to be a catalytically
competent intermediate;12 the hydroxyl group (and the ureido
oxygen of citrulline) is derived from molecular oxygen (not bulk
water).13 Nitric oxide is derived from the guanidino nitrogen
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InactiVation of Inducible Nitric Oxide Synthase
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 905
Scheme 2
functionality is thought to trap a nucleophilic ferric peroxide
arginine-like and Nω-hydroxyarginine-like ligands, yet dissimilar
to those caused by Nω-methylarginine-like or Nω-nitroarginine-
like ligands.32
(FeIIIOOH) intermediate (4), which can then fragment to yield
L-NIO is prototypical of a class of time-dependent carbami-
dine-containing NOS inhibitors. Other members of this class
include N5-(1-iminoethyl)-L-lysine (L-NIL, 6; homo-L-NIO)33
products. Chemical model studies for Nω-hydroxyguanidines
also support the role of a nucleophilic hydroperoxy species in
the catalytic mechanism for NOS.26
The arginine analogue N5-(1-iminoethyl)-L-ornithine (L-NIO)
(5) was originally isolated as a natural product from an
unclassified Streptomycete.27 The structure and pKa values of
L-NIO are very similar to those of L-arginine.27,28 In fact, L-NIO
can even serve as a substrate for the enzyme arginase, forming
ornithine as a product, and it is served by the same cationic
transport system (y+).29 Despite its structural similarities to
L-arginine, L-NIO also inhibits some arginine-dependent en-
zymes; L-NIO inhibited a crude preparation of NOS.30 Revers-
ible inhibition of NOS by L-NIO has reported IC50 values of
7.7, 5.8, and 10 µM for recombinant human inducible,
endothelial, and neuronal NOS, respectively,31 and, therefore,
is not an isoform-selective inhibitor. The binding of L-NIO to
the active site of nNOS, as characterized by EPR spectroscopy,
shows spectral changes intermediate between those caused by
and N-(3-(aminomethyl)benzyl)acetamidine (1400W, 7),34 which
contain the same pharmacophore and also demonstrate time-
dependent inhibition as well as many of the characteristics of a
mechanism-based inactivator. Probably the most thoroughly
studied amidine-containing inactivator is 6. Inactivation of iNOS
by 6 is time dependent, irreversible by dilution, and NADPH-
and O2-dependent.35 Reports of heme loss due to inactivation
differ,35,36 but inactivation seems to cause a destabilization of
the iNOS homodimer into inactive monomers.36 Use of radio-
labeled 6 failed to indicate covalent protein modification or
substrate oxidation.35,36 Likewise, use of [14C-amidine]-labeled
7 did not show any turnover products, but the low specific
activity of the compound prevented the detection of any E-I
(26) (a) Pufahl, R. A.; Wishnok, J. S.; Marletta, M. A. Biochemistry
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Feldman, P. L.; Bova, M. P.; Wong, P. J. Med. Chem. 1993, 36, 2666-
2670.
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Co.: Cleveland, OH, 1968; pp J-148-J189.
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T. P.; Currie, M. G. J. Med. Chem. 1994, 37, 3886-3888.
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4963.
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S. K.; Kozarich, J. W. Biochemistry 1998, 37, 4174-4180.
(36) Bryk, R.; Wolff, D. J. Biochemistry 1998, 37, 4844-4852.
(29) Baydoun, A. R.; Mann, G. E. Biochem. Biophys. Res. Commun.
1994, 200, 726-731.
(30) McCall, T. B.; Feelisch, M.; Palmer, R. M. J.; Moncada, S. Br. J.
Pharmacol. 1991, 102, 234-238.
(31) Moore, W. M.; Webber, R. K.; Fok, K. F.; Jerome, G. M.;
Kornmeier, C. M.; Tjoeng, F. S.; Currie, M. G. Bioorg. Med. Chem. 1996,
4, 1559-1564.

906 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Fast et al.
adducts,34 and the authors did not propose a mechanism for
inactivation.
Structurally more similar to the natural substrate, L-NIO also
has demonstrated many of the characteristics of mechanism-
based inactivation. Moncada’s group reported time-dependent,
irreversible (to substrate addition), substrate protectable, and
enantiomerically specific inactivation of unpurified iNOS by
L-NIO in phagocytic cells.30 Assays of L-NIO and L-NIL with
purified NOS, which show time-dependent inhibition, have been
mentioned.33
Except for Nω-methyl-L-arginine37 and Nω-allyl-L-arginine,38
which appear to act by similar mechanisms, the chemical
mechanisms for NOS inactivation by any of the other time-
dependent NOS inactivators remain obscure. While L-NIO has
physicochemical properties similar to those of L-arginine, the
same mechanisms for NOS inactivation by Nω-alkyl-L-arginine
analogues cannot be applied, and no mechanisms for the amidino
class of NOS inactivators have been proposed to date. Here we
report experiments that clarify the product of inactivation of
iNOS by L-NIO and suggest possible mechanisms consistent
with these results and those of similar amidine-containing
inactivators.
Figure 1. Loss of the heme chromophore before (t ) 0 h; s) and
after inactivation and gel filtration (t ) 2 h; - - -). See the Experimental
Section for details.
Scheme 3
Results
Spectral Changes during iNOS Inactivation by L-NIO.
L-NIO showed type I binding difference spectra (peak at 390
nm, trough at 430 nm) when used to titrate an iNOS‚imidazole
complex (see Supporting Information).
Kinetics of iNOS Inactivation by L-NIO. When tested for
time-dependent inactivation of iNOS, L-NIO showed time-,
concentration-, and NADPH-dependent inhibition that was not
reversed upon 60-fold dilution into the enzyme assay solution
which contained L-arginine in a 2600-fold excess (see Support-
ing Information). Control incubations do not lose significant
amounts of activity over time. Incubations that include an excess
of L-arginine do not exhibit time-dependent inactivation. No
inactivation by L-NIO was observed when NADP+ was sub-
stituted for NADPH. The rate of NOS inactivation was
concentration-dependent, saturatable, and had KI and kinact values
of 13.7 ( 1.6 µM and 0.073 ( 0.003 min-1, as determined by
the method of Kitz and Wilson,39 respectively (see Supporting
Information). Inactivation by L-NIO also was shown to be
irreversible. An inactivated sample was passed through a
Penefsky40 column to separate small molecules from the protein.
While a control solution maintained most of its activity, no
return of activity was seen with the inactivated sample.
Heme Loss during Inactivation. The heme cofactor appears
to be involved in the inactivation event. After inactivation, iNOS
was passed through a size exclusion column, and a loss in the
heme chromophore was observed (Figure 1). The loss of the
chromophore also corresponded with the loss of enzyme activity
(see Supporting Information). When inactivated NOS samples
were subjected to HPLC directly (without prior gel filtration),
the area of the peak due to the heme cofactor significantly
decreased. Inclusion of catalase in the inactivation mixtures
decreased the amount of heme lost in the control solutions but
did not eliminate significant loss of heme (47%) in the solutions
with L-NIO (see Supporting Information).
N5-(1-Imino-2-[14C]ethyl)-L-ornithine (11). N5-(1-Imino-2-
[14C]ethyl)-L-ornithine (11, Scheme 3; [14Cω]-L-NIO), was
synthesized from 2′-[14C]acetonitrile via ethyl acetimidate
hydrochloride (10) as shown in Scheme 3.
Radioactivity from 11 Bound to iNOS Protein. To deter-
mine if any radioactivity is irreversibly bound to the protein,
incubation mixtures were allowed to react and then were
subjected to dialysis. No significant radioactivity remained
bound to the protein either in incubations containing (42 dpm)
or omitting (25 dpm, control) NADPH, indicating that less than
0.01 equiv of inactivator remained bound to the protein and
that the remaining radiolabel was released with other small
molecules. To rule out the formation of a tight noncovalent
association of L-NIO (or a metabolite) with iNOS, an inactivated
sample was subjected to Sephadex G-50 chromatography.
Protein was observed to elute separately from the radioactivity.
Radioactivity from 11 Bound to Heme. To determine if
the inactivator is bound to the heme, 11-inactivated iNOS
mixtures were directly chromatographed by HPLC and the
elution monitored both by absorbance at 400 nm and by
radioactivity. As noted before, significant loss of heme was
measured (see Supporting Information). The radiotracer failed
to reveal NADPH-dependent formation of a new [14C]-labeled
peak (see Supporting Information).
Product Formation during Inactivation of iNOS by 11.
Turnover-dependent production of a [14C]-labeled peak with the
same retention time as Nω-hydroxy-L-NIO (12; o-fluoraldehyde-
derivatized) was observed, but no Nδ-acetyl-L-ornithine (14) (the
small peak at 17 min does not correspond to 14) or Cω-hydroxy-
(37) Olken, N. M.; Osawa, Y.; Marletta, M. A. Biochemistry 1994, 33,
14784-14791.
(38) Zhang, H. Q.; Dixon, R. P.; Marletta, M. A.; Nikolic, D.; Van
Breemen, R.; Silverman, R. B. J. Am. Chem. Soc. 1997, 119, 10888-10902.
(39) Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245-3249.
(40) Penefsky, H. Methods Enzymol. 1979, LVI, 527.

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J. Am. Chem. Soc., Vol. 121, No. 5, 1999 907
Figure 2. Turnover-dependent production of Nω-hydroxy-L-NIO (12)
from 11. Elution times for L-NIO (5), Cω-hydroxy-L-NIO (13), and
Nδ-acetyl-L-ornithine (14) standards are also indicated.
Figure 3. Time- and NADPH-dependent production of Nδ-acetyl-L-
ornithine (14) from Nω-hydroxy-L-NIO (12). (A) The top trace shows
retention times for 14 and 12 standards. The middle trace shows an
incubation mixture containing 12, iNOS, and NADPH. The bottom trace
shows an identical incubation mixture except without the NADPH. (B)
Traces at 1, 5, and 15 h time points of incubations including 12, iNOS,
and NADPH. Due to inactivation, fresh enzyme was added after 5 h
and the chromatogram adjusted for the dilution. To shorten run times,
the gradient for elution was changed from that in Figure 2, resulting in
shorter retention times for 12 and 14 (see the Experimental Section
for details).
Table 1. Km and kcat Values for L-Arginine and
Nω-Hydroxy-L-NIO (12) with iNOS
L-NIO (13) (Figure 2). When the dpm values from incubations
containing NADPH (29 534 dpm) and NADP+ (6396 dpm) are
subtracted, the amount of Nω-hydroxy-L-NIO (12) produced
during the inactivation process was calculated to be 7.7 ( 0.2
equiv per inactivation event.
L-arginine
12
K
k
k
m (µM)
cat (min-1
cat/Km (µM-1 min-1
5.9 ( 0.5
84.6
14.3
800 ( 85
2.22
)
)
2.8 × 10-3
Inhibition of iNOS by Nω-Hydroxy-N5-(1-iminoethyl)-L-
ornithine (12). Incubations of 12 with iNOS demonstrated time-
and concentration-dependent inactivation that was not reversible
upon dilution into the assay solution (see Supporting Informa-
tion). Incubations that included an excess of L-arginine or with
substitution of NADP+ for NADPH resulted in no significant
loss of activity. The KI and kinact values for 12 are 830 ( 160
µM and 0.0073 ( 0.0007 min-1, respectively.
dependent loss of enzyme activity was observed with 14 (see
Supporting Information). KI and kinact values for 14 inactivation
of iNOS were determined to be 490 mM and 0.24 min-1
respectively. iNOS inactivation by 14 can be prevented by
including L-arginine, but not D-arginine, in the inactivation
mixtures, suggesting that the inactivator acts at the arginine
binding site.
Inhibition of iNOS by N5-(1-Imino-2-hydroxyethyl)-L-
ornithine (13). Cω-Hydroxy-L-NIO (13) failed to cause time-
dependent inactivation of iNOS.
,
Substrate Activity of Nω-Hydroxy-N5-(1-iminoethyl)-L-
ornithine (12). Using the oxyhemoglobin assay for detection
of NO production (see the Experimental Section), 12 was
determined to be a substrate for iNOS and showed saturation
kinetics with Km and kcat values of 800 ( 85 µM and 2.22
min-1, respectively. However, 12 was only a poor substrate
relative to L-arginine, showing kcat/Km values 4 orders of
magnitude lower than those of L-arginine (Table 1). The kcat
and kinact values for 12 can be compared directly to give a
partition ratio (kcat/kinact ) for inactivation of 304; i.e., there are
304 turnovers to give NO per inactivation event.
Kinetic Isotope Effect on Inactivation of iNOS by N5-(1-
Imino-2-[C2H3]ethyl)-L-ornithine (15). Inactivation of iNOS
by 15 shows a kinetic isotope effect on Hkinact/Dkinact of 1.35 (
0.08 (0.073 ( 0.003/0.054 ( 0.001) and on H(kinact/KI)/D(kinact
/
KI) of 1.51 ( 0.3 (5.3 ( 0.8 × 10-3/3.5 ( 0.2 × 10-3) (see
Supporting Information).
Formation of Nδ-Acetyl-L-ornithine (14). Incubations of 12
with iNOS showed NADPH-dependent and time-dependent
conversion to 14 (Figure 3).
Characterization of Modified Heme. An enlargement of the
HPLC trace after inactivation of iNOS by 5 reveals a new
NADPH-dependent peak with absorbance at 400 nm at 47 min
(Figure 4). LC-electrospray mass spectrometry was used to
Inactivation of iNOS by Nδ-Acetyl-L-ornithine (14). For
incubations with iNOS, time-, concentration-, and NADPH-

908 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Fast et al.
Figure 4. HPLC of the NADPH-dependent formation of a new peak
(47 min) after inactivation of iNOS by L-NIO.
Figure 6. (A) MS/MS of the m/z 583 peak in Figure 5B. (B) MS/MS
of a biliverdin standard.
Scheme 4
Discussion
Recently, two reports of the inactivation of NOS by the
homologue of L-NIO, namely, L-NIL (6), were published.35,36
Some of the results published are consistent with the results
we report here; however, no cause for inactivation nor mech-
anisms of inactivation were reported. Considering the multistep
catalysis by NOS (Scheme 2), there are numerous possibilities
for inactivation of NOS by L-NIO at various stages along the
catalytic pathway. Inactivation could occur before any oxidation
occurs, after a one-electron oxidation, after hydroxylation to
yield either 12 or 13, or at an oxidation state beyond hydroxy-
lation. These possibilities will first be presented, and then
experiments designed to differentiate these possibilities will be
described and conclusions will be drawn on the basis of these
results. Each of these proposed mechanisms has a distinguishing
feature that can be determined experimentally. Elucidation of
the chemical mechanism of NOS inactivation by L-NIO should
provide a paradigm for understanding inactivation by not only
L-NIO but also by other amidines.
Figure 5. Mass spectra of the peaks at 52 min (A) and 47 min (B) in
Figure 4.
compare the control (no NADPH) and inactivated (NADPH)
mixtures (see Supporting Information). The mass spectra of the
peaks in the HPLC trace at 49 min (A) and at 42 min (B) from
the inactivated mixture are shown in Figure 5. The (m/z) values
for the peaks at 49 and 42 min are 616 (heme) and 583,
respectively. MS/MS of the 583 peak gave a spectrum (Figure
6A) that was the same as MS/MS of a biliverdin standard
(Figure 6B).
If inactivation occurs prior to any oxidation (Scheme 4),
ornithine would be produced and the enzyme would be modified
by imidoylation (16); this pathway would not require catalytic
turnover by NOS. If inactivation occurs prior to the first
hydroxylation step but after initial oxidation, possibly by a high-

InactiVation of Inducible Nitric Oxide Synthase
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 909
Scheme 5
Scheme 8
Scheme 9
Scheme 6
irreversible enzyme inhibition.41 Alternatively, the nitroso
compound may also be attacked by a cofactor or enzyme based
nucleophile, resulting in methylation of the attacking group and
release of the amidoxime (20). Both of these pathways should
not require turnover by NOS after the initial Nω-hydroxylation.
An Amadori rearrangement of C-hydroxylated L-NIO (13;
Scheme 7) would give an aldehyde (21) that could form a Schiff
base with a lysine residue and lead to enzyme inactivation.
It also is possible that 12 may serve as an alternative substrate
for NOS, producing NO and 14 (Scheme 8). Production of nitric
oxide would require O2 and NADPH, even after the initial
formation of 12. Inactivation pathways also can be proposed
that require further oxidation of 14 (Scheme 9). Hydrogen atom
abstraction of the methyl group or of the enol gives an R-radical
(22) that could combine with either a cofactor or protein-based
radical or somehow uncouple peroxide formation, just like the
corresponding L-NIO radical (Scheme 5). This pathway should
require enzymatic turnover after the initial Nω-hydroxylation
and turnover after formation of 14. The potential mechanisms
in Schemes 4-9 were tested as described below.
Scheme 7
L-NIO shows type I binding difference spectra when used to
titrate an iNOS‚imidazole complex (data not shown). These
spectra are very similar to those exhibited by titrations of rat
nNOS with L-arginine, Nω-methyl-L-arginine, or Nω-hydroxy-
L-arginine42 and dissimilar to the type II difference spectra seen
with imidazole and L-thiocitrulline.43 The observation of type I
difference spectra indicates that L-NIO binds in a manner similar
to that of arginine and is capable of inducing similar changes
in the heme environment (a shift toward a high-spin pentaco-
ordinate ferric species), supporting the possibility that L-NIO
binds, and may be oxidized, in a manner similar to that of the
normal substrate.
energy iron-oxo or iron-peroxo species (Scheme 5), a radical
may be formed at the terminal carbon atom (17) by hydrogen
atom abstraction directly from the methyl group or from the
terminal amidino nitrogen followed by deprotonation; alterna-
tively, 17 could arise from a single electron transfer followed
by proton abstraction. The resulting radical could recombine
with a cofactor- or enzyme-based radical and result in covalent
modification and inactivation (18) or may somehow uncouple
peroxide formation. If the inactivation event occurs after the
initial hydroxylation, as it does for both Nω-methyl-L-arginine37
and Nω-allyl-L-arginine,38 either the terminal amidino nitrogen
(Scheme 6) or carbon (Scheme 7) positions may be hydroxy-
lated. Oxidation of amidino nitrogens18a and unactivated car-
bons19 have both been shown to be catalyzed by cytochrome
P450 enzymes. Isomerization of 12 to nitroso 19 (Scheme 6)
may inhibit the enzyme by coordination to the heme; nitroso-
(FeII) heme complexes are very stable and can result in pseudo-
(41) Mansuy, D.; Gans, P.; Chottard, J.; Bartoli, J. Eur. J. Biochem. 1988,
76, 607-615.
(42) McMillan, K.; Masters, B. S. S. Biochemistry 1993, 32, 9875-
9880.
(43) Frey, C.; Narayanan, K.; McMillan, K.; Spak, L.; Gross, S. S.;
Masters, B. S.; Griffith, O. W. J. Biol. Chem. 1994, 269, 26083-26091.

910 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Fast et al.
When tested for time-dependent inactivation of iNOS, L-NIO
shows time-, concentration-, and NADPH-dependent inhibition
that is not reversed by dilution into the enzyme assay solution
containing L-arginine in a 2600-fold excess or by passing
through a Penefsky40 column to separate small molecules from
the protein (data not shown). This type of saturation kinetics is
consistent with a requirement for enzymatic processing of a
latent inactivator, as proposed in most of the mechanisms above.
L-Arginine blocks the inactivation by L-NIO, reinforcing the
proposal that both L-NIO and L-arginine compete for the same
binding site. However, no inactivation by L-NIO was observed
when NADP+ was substituted for NADPH, indicating that an
oxidation is required for inactivation to occur. The O2 depen-
dence for inactivation NOS by L-NIL was noted earlier.35
Because NADPH is required for loss of activity, a simple
transamidination inactivation mechanism, such as shown in
Scheme 4, is not reasonable. This is in agreement with the
crystal structure of iNOS,5 which indicates that reactive nu-
cleophiles are not present near the guanidinium binding site.
The heme cofactor appears to be involved in the inactivation
event, as evidenced by the change in both the visible spectrum
(Figure 1) and by HPLC (data not shown). Loss of about 50%
of the heme chromophore following inactivation and gel
filtration indicates that the heme has either dissociated intact
from the enzyme or that it is modified or destroyed during
inactivation. The loss of the heme chromophore corresponds
with the loss of enzyme activity (data not shown). These
experiments suggest that cofactor loss is involved in the
inactivation process but do not distinguish between heme
modification or destruction and heme release without modifica-
tion (possibly due to disruption of the active iNOS homodimer
into inactive monomers). When inactivated NOS samples were
subjected to HPLC directly (without prior gel filtration), the
area of the peak due to the heme cofactor significantly decreased,
indicating that heme is either modified or destroyed during
inactivation and not just dissociated intact from the active
homodimer. Inclusion of catalase in the inactivation mixtures
decreased the amount of heme lost in the control solutions but
did not eliminate significant loss of heme (47%) in the solutions
with L-NIO. This rules out the possibility that heme loss is due
solely to uncoupled peroxide production44 and reinforces the
proposal of inactivator-mediated heme destruction. Modification
of only about half of the heme cofactor probably results from
either half sites reactivity or from disruption of the active iNOS
homodimer into inactive monomers; heme has been shown to
be essential for iNOS dimer formation.45 Inactivation of iNOS
by L-NIL also gives incomplete loss of heme35,36 with a
concomitant shift of the dimer/monomer population to predomi-
nantly monomer.36
by HPLC and the elution was monitored both by absorbance at
400 nm and by radioactivity. About half of the heme (t ) 52
min) was lost, and no radioactivity was observed in the
chromatogram at or near the heme peak. This indicates that the
inactivator does not covalently bond to released molecules (e.g.,
cofactors) or to the protein.
Possibly L-NIO is metabolized by iNOS to a product that
is responsible for inactivation. NADPH-dependent production
of a [14C]-labeled peak with the same retention time as Nω-
hydroxy-L-NIO (12; o-phthalaldehyde-derivatized) was ob-
served, indicating that the terminal amidino nitrogen can be
hydroxylated by iNOS (Figure 2); the amount produced corre-
sponds to 7.7 ( 0.2 equiv per inactivation event, a rather low
partition ratio. Neither Nδ-acetyl-L-ornithine (14; o-phthalalde-
hyde-derivatized) nor Cω-hydroxy-L-NIO (13) was detected as
a product.
Incubation of 12 with iNOS produced time-, concentration-,
and NADPH-dependent inactivation that was not reversed upon
dilution into the assay solution. Incubations that included an
excess of L-arginine or with substitution of NADP+ for NADPH
resulted in no significant loss of activity, demonstrating
substrate-protectable and turnover-dependent inactivation, re-
spectively, similar to that seen earlier with L-NIO. These results
suggest that isomerization of 12 to the nitroso species 19
(Scheme 6) or direct reaction of 19 with the enzyme is unlikely
because inactivation by 12 requires additional reducing equiva-
lents from NADPH for loss of activity.
It might, at first, be tempting to conclude that NOS inactiva-
tion by L-NIO is caused by Nω-hydroxylation to 12, followed
by enzyme inactivation; however, the KI and kinact values for
12, 830 µM and 0.0073 min-1, respectively, indicate that any
inactivation by L-NIO which proceeds via 12 can only account
for less than 10% of the total inactivation because the KI value
for L-NIO (13.7 ( 1.6 µM) indicates that L-NIO binds about
60 times more tightly to NOS than does 12, and the kinact value
for L-NIO (0.073 ( 0.003 min-1) is 10 times greater than that
for 12 (the kinact/KI for L-NIO is 3 orders of magnitude greater
than that for 12). Therefore, 12 is not a kinetically competent
intermediate. Although 12 is, at most, a minor contributor to
inactivation by L-NIO, at high concentrations it can serve as a
substrate for iNOS, exhibiting saturation kinetics with Km and
k
cat values of 800 ( 85 µM and 2.22 min-1, respectively (Table
1). As predicted by Scheme 8, concomitant production of 14 is
also observed in an NADPH- and time-dependent manner
(Figure 3). Since the Km and KI values for 12 are the same, the
kcat and kinact values can be compared directly to give a partition
ratio for inactivation (kcat/kinact) of 304. This provides further
evidence that during inactivation by L-NIO, 12 is not an
intermediate and is released into solution without further
processing. If even 10% of the inactivation proceeded through
12, then a significant amount of 14 would have been produced;
however, none was detected (Figure 2). Because of its high Km
value compared with that of L-NIO, it is unlikely that any 12
released into solution could compete with L-NIO for rebinding
and further turnover by iNOS.
Considering that inactivation does not proceed from 12, a
mechanism involving Cω-hydroxylation was considered. Cω-
Hydroxy-L-NIO (13) failed to cause time-dependent inactivation
of iNOS, which rules out the inactivation pathways proposed
that occur after Cω-hydroxylation (e.g., see Scheme 7). NOS
does not appear to be restrictive against binding hydroxylated
catalytic intermediates. For example, Nω-hydroxyarginine,15 Nω-
hydroxy-Nω-methylarginine,15 and Nω-hydroxy-Nω-allylargin-
ine38 are all examples of hydroxylated intermediates that are
accepted and processed in a kinetically competent manner by
NOS.
N5-(1-Imino-2-[14C]ethyl)-L-ornithine (11, Scheme 3) was
synthesized to determine if and where the inactivator becomes
attached to the enzyme. After inactivation, no significant
radioactivity remained bound to the protein either in the presence
or absence of NADPH. This is consistent with similar studies
using radiolabeled L-NIL.35,36 To rule out the formation of a
tight noncovalent association of L-NIO (or a metabolite) with
iNOS, a sample inactivated with 11 was subjected to gel
filtration. Protein was observed to elute separately from the
radioactivity, indicating that inactivator does not bind to the
protein. To determine if the inactivator is bound to the heme,
11-inactivated iNOS mixtures were directly chromatographed
(44) Heinzel, B.; John, M.; Klatt, P.; Bohme, E.; Mayer, B. Biochem. J.
1992, 281, 627-630.
(45) Baek, K. J.; Thiel, B. A.; Lucas, S.; Stuehr, D. J. J. Biol. Chem.
1993, 268, 21120-21129.

InactiVation of Inducible Nitric Oxide Synthase
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 911
abstraction (or electron transfer-proton transfer) of L-NIO is
involved in heme destruction without attachment to it or that,
if attachment does occur, it is only the first step in a process
that eventually releases this radical from the heme.
To determine if either of these proposals based in Scheme 5
is reasonable, an experiment was carried out to determine if
methyl C-H bond cleavage is involved in the inactivation
process. In fact, a kinetic isotope effect on inactivation of iNOS
by N5-(1-imino-2-[C2H3]ethyl)-L-ornithine (15, d3-L-NIO)
(Hkinact/Dkinact of 1.35 ( 0.08; H(kinact/KI)/D(kinact/KI) of 1.51 (
0.3) was observed. Although the isotope effects on kinact and
kinact/KI are small, they are significant and not uncommonly small
for primary isotope effects observed in other enzymatic reac-
tions.46 They are also similar in magnitude to isotope effects
observed on inactivation rates for deuterated mechanism-based
inactivators of other enzymes. For example, a keto-enol
tautomerism of an R-chloro ketone was proposed in an inactiva-
tion mechanism for UDP galactose-4-epimerase and was sup-
ported with an isotope effect on kinact of 1.4 (Hkinact/Dkinact).47
Also, an N-methyl mechanism-based inactivator of cytochrome
P450 demonstrated a kinact isotope effect of 2 when a trideute-
riomethyl group was substituted.48 The observation of a primary
kinetic isotope effect on NOS inactivation by L-NIO suggests
that C-H bond scission is a partially rate-determining step in
the inactivation mechanism, and this is consistent with the first
step in the inactivation mechanism proposed in Scheme 5.
The conundrum, based on the mechanism in Scheme 5, of
having no inactivator bound to either protein or to the heme is
discussed below, but first the question of what the heme is
converted into is considered. From Figure 4 it is apparent that
a new NADPH-dependent peak in the HPLC chromatogram at
about 47 min (400 nm) results from L-NIO inactivation of NOS.
LC-electrospray mass spectrometry was carried out to identify
the masses of the peaks at 47 and 52 min. The peak at 52 min
has a m/z value (Figure 5A) of 616 and corresponds to
unmodified heme. The peak at 47 min has a m/z value (Figure
5B) of 583, which is consistent with the mass of the heme
degradation product biliverdin IX (23 shows biliverdin IXR, that
Figure 7. Heme loss requiring both NADPH and L-NIO. (A) HPLC
chromatograms of iNOS and catalase incubation mixtures containing
(from top to bottom) L-NIO only, NADPH only, L-NIO and NADPH,
and a biliverdin standard. For easier viewing of heme loss (t ) 52
min), the retention times for all three experiments are offset by 2 min
from the previous experiment. (B) An expanded view of (A) between
30 and 80 min.
is, oxidative cleavage at the R-meso carbon). MS/MS of this
product (Figure 6A) was the same as that for a biliverdin
standard (Figure 6B). Formation of biliverdin requires oxidative
cleavage of the heme macrocycle to a linear tetrapyrrole and
loss of a meso carbon.
To rule out L-NIO independent heme destruction caused only
by the presence of NADPH (and subsequent enzymatic forma-
tion of an oxidant), incubations of iNOS and catalase containing
L-NIO only, NADPH only, or L-NIO and NADPH were
compared with a biliverdin standard (Figure 7). Only the
Since neither Nω-hydroxy-L-NIO (12) nor Cω-hydroxy-L-NIO
(13) is responsible for inactivation of iNOS by L-NIO, and
oxidation (NADPH) is required for inactivation, the inactivation
must precede the hydroxylation step but follow an oxidation
step. This precludes the mechanism shown in Scheme 9 (via
Scheme 8) from being relevant. The only mechanism presented
that is consistent with these requirements is the one in Scheme
5; however, it was shown that radioactive L-NIO does not
become bound to either the protein or to a cofactor, which would
be inconsistent with structure 18. If this mechanism is correct,
then it means that either the radical generated by hydrogen atom
(46) Klinman, J. P. AdV. Enzymol. 1978, 46, 415-494.
(47) Flentke, G. R.; Frey, P. A. Biochemistry 1990, 29, 2430-2436.
(48) Kunze, K. L.; Trager, W. F. Chem. Res. Toxicol. 1993, 6, 649-
656.

912 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Fast et al.
incubation containing both NADPH and L-NIO showed signifi-
cant loss of heme and formation of a biliverdin peak at 47 min.
Thus, the presence of L-NIO is mandatory for heme destruction.
The peak area for biliverdin (at 400 nm) varies with concentra-
tion in a linear manner over the concentrations detected in the
inactivation solutions. On the basis of these standards, the
average peak area of three separate inactivations indicates that
0.63 ( 0.1 equiv of biliverdin is produced per inactivation event,
which corresponds to the loss of about half of the heme from
the UV-vis and HPLC experiments described above.
Scheme 10
Simple peroxide formation also cannot be the cause for heme
degradation to biliverdin. Peroxide, produced both enzymatically
(NADPH-P450 reductase, NADPH, and O2) and nonenzymati-
cally, is known to degrade free heme and cytochrome P450
bound heme.49 Initially, it was reported that biliverdin is a very
small (3-14%) byproduct of heme degradation;50,51 however,
the major products of peroxidative heme cleavage were later
shown49 to consist of various propentdyopents, maleimides,
formic acid (the meso carbons), and hematinic acid, and
biliverdin was shown not to be an intermediate in this process.
Peroxynitrite is known to inactivate iNOS, possibly by an
oxidation of the heme thiolate ligand.52 However, peroxynitrite
cannot be responsible for inactivation by L-NIO because no nitric
oxide (which reacts with superoxide to give peroxynitrite) is
produced from L-NIO. Nitric oxide is produced from N-hydroxy-
L-NIO (12), but thiols have a protective effect,52 and the
inactivation studies were carried out in the presence of 10 mM
dithiothreitol and catalase (100-1000 units). Furthermore, it
was shown that only about 8 equiv of 12 are generated during
inactivation and are not further processed. Therefore, it is doubt-
ful that peroxynitrite is responsible for inactivation by L-NIO.
A summary of the data describing the inactivation of iNOS
by L-NIO is as follows: complete inactivation converts ap-
proximately half of the heme cofactor to biliverdin, does not
covalently modify the protein, does not proceed through Cω-
hydroxylation, releases a small amount of the Nω-hydroxylation
product as a turnover product, and exhibits a kinetic isotope
effect upon substitution of L-NIO with d3-L-NIO. On the basis
of these experimental results, at least two reasonable, but
speculative, mechanisms for the inactivation by L-NIO can be
proposed. In the first mechanism, L-NIO serves to disrupt the
normal redox cycle of NOS following one-electron oxidation,
possibly by shifting the heme spin state, increasing uncoupled
peroxide production, and altering the heme environment, thereby
resulting in oxidative cleavage of the heme macrocycle to
biliverdin IX. Our results indicate that catalase does not prevent
inactivation, but that result only supports the notion that excess
peroxide from solution is not responsible for inactivation. It does
not preclude a mechanism in which some of the hydrogen
peroxide generated at the active site causes heme destruction
prior to release into solution. It is known that biliverdin is
produced by the enzyme heme oxygenase,53 an enzyme that
cleaves the R-meso bridge of the heme regiospecifically,
proceeding through R-meso-hydroxyheme (24) and verdoheme
(25) as intermediates, liberating the meso carbon as carbon
monoxide, and leaving biliverdin IXR (23) as the product
(Scheme 10).53,54 The proposal that L-NIO causes uncoupled
peroxide production is consistent with the idea of substrate-
assisted catalysis5,21 in the first oxidation step in which the
proton of the terminal NH2 group of L-arginine provides the
catalysis for conversion of peroxoheme to the purported
FeVdO species. L-NIO, however, does not have the terminal
NH2 group to deliver a proton to peroxoheme, so that leaves
the heme-based oxidant in the peroxo state, possibly resulting
in the production of excess peroxide.
In a second proposed mechanism, L-NIO is oxidized initially
by one of the pathways in Scheme 5, where X is the heme. The
partitioning between turnover and inactivation occurs with C-H
bond cleavage. If L-NIO is bound in a manner similar to that of
aminoguanidine21 or L-arginine5 and orients the amidino nitrogen
toward the heme (for electron transfer), glutamate 371 would
be correctly positioned to abstract a proton from the terminal
methyl group of (one-electron oxidized) L-NIO, producing 17
(Scheme 5). Alternatively, if L-NIO was bound with the terminal
methyl group pointing toward the heme, it would be correctly
placed for heme peroxo abstraction of a hydrogen atom directly
from the carbon to give 17 (Scheme 5). Formation of L-NIO-
heme adduct 26 (equivalent to 18, X ) heme, in Scheme 5) is
proposed to initiate a rapid series of reactions leading to the
formation of biliverdin and regenerating L-NIO, as depicted in
Scheme 11 (R-meso-alkylation is arbitrary; it could alkylate at
any of the meso-positions). The conversion of alkylheme 26 to
biliverdin (23) and L-NIO shown in Scheme 11 is similar to
the mechanism proposed by Ortiz de Montellano and co-
workers55 for the heme oxygenase-dependent conversion of
R-meso-methylheme to biliverdin. According to this mechanism,
the R-meso-alkyl heme is first converted to the heme hydrop-
eroxide (27) followed by protonation and electrophilic substitu-
tion at the R-meso carbon to give cation 28. A two-electron
reduction of O2 leads to formation of peroxide 29, which breaks
down by homolytic cleavage of the O-O and C-C bonds to
give 30. In the case of R-meso-methylheme, the fate of the
methyl and R-meso carbon is not known although verdoheme
was detected as an intermediate.54 In our case, we also propose
the formation of verdoheme (25), possibly via 31 in addition to
Cω-carboxy-L-NIO (32), which should undergo rapid decar-
boxylation and release unmodified L-NIO. This would account
(49) Schaefer, W. H.; Harris, T. M.; Guengerich, F. P. Biochemistry 1985,
24, 3254-3263.
(50) Guengerich, F. P. Biochemistry 1978, 17, 3633-3639.
(51) Docherty, J. C.; Masters, B. S. S.; Firneisz, G. D.; Schacter, B. A.
Biochem. Biophys. Res. Commun. 1982, 105, 1005-1013.
(52) (a) Humer, A. F. R.; Nishida, C. R.; Ortiz de Montellano, P. R.;
Schoneich, C. Chem. Res. Toxicol. 1997, 10, 618-626. (b) Pasquet, J. P.
E. E.; Zou, M. H.; Ullrich, V. Biochimie 1996, 78, 785-791.
(53) (a) Maines, M. D. FASEB J. 1988, 2, 2557-2568. (b) Tenhunen,
R.; Marver, H. S.; Schmid, R. J. Biol. Chem. 1969, 244, 6388-6394.
(54) Torpey, J.; Ortiz de Montellano, P. R. J. Biol. Chem. 1996, 271,
26067-26073.
(55) Torpey, J.; Lee, D. A.; Smith, K. M.; Ortiz de Montellano, P. R. J.
Am. Chem. Soc. 1996, 118, 9172-9173.

InactiVation of Inducible Nitric Oxide Synthase
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 913
Scheme 11
for why no radiolabeled adduct or modified amino acids (other
than 12) were detected. The verdoheme (25) produced could
then be converted to biliverdin corresponding to the reaction
catalyzed by heme oxygenase. Although heme oxygenase
catalyzes destruction of both heme and alkylated hemes, this
proposal suggests that, in the case of NOS, only after heme
alkylation does the enzyme initiate a reaction similar to that of
heme oxygenase. It is not known if the biliverdin resulting from
NOS inactivation by L-NIO consists of a single isomer or a
mixture of isomers. Because no L-NIO-biliverdin adducts were
isolated, heme cleavage must occur at the point of inactivator
attachment and not at an unsubstituted meso carbon, consistent
with the work of Ortiz de Montellano and co-workers.55
This second proposed inactivation mechanism is significant
for several reasons. The similarities between L-NIO and the usual
substrate, L-arginine, suggest that NOS may use the normal
catalytic mechanism to process both of these compounds. While
previously studied mechanism-based inactivators of NOS target
the second oxidation step, the oxidation of Nω-hydroxy-L-
arginine to citrulline and NO,38 L-NIO would be the first known
probe of the first step, the hydroxylation of L-arginine to Nω-
hydroxy-L-arginine. A standard heme [FeVdO] species is usually
invoked in mechanisms for the first oxidation step, but this
proposal has been based largely on similarities to P450 type
chemistry, X-ray crystal structures, and on partial CO inhibition
and lacks rigorous chemical evidence for a heme-based rather
than a tetrahydrobiopterin-based oxidant. Formation of an L-NIO
heme adduct, albeit unstable, would be indicative of a heme-
based radical providing more compelling evidence as to the
identity of the actual oxidant. It remains unclear as to why the
reported allyl-heme adduct38 is further reduced, whereas this
L-NIO-heme adduct is further oxidized; perhaps the N-hydroxy-
N-allylarginine intermediate blocks productive O2 binding to
the heme iron, so the electrons meant for the O2 are transferred
to the heme instead.
To complete the picture of the metabolism of L-NIO by iNOS,
we investigated the further reaction of 12, which, as noted above,
does not appear to be involved in inactivation of iNOS. Since
NO is produced from 12 (the assay for substrate activity of 12
was to monitor the conversion of oxyhemoglobin to methemo-
globin by NO), then as shown in Scheme 8, concomitant
production of Nδ-acetyl-L-ornithine (14) also should be observed.
Oxidation of 12 to NO and 14 is observed only at high
concentrations of 12. Incubations of iNOS with 12 led to a
NADPH- and time-dependent conversion to 14 (Figure 3).
Interestingly, when 14 was incubated with iNOS, time-,
concentration-, and NADPH-dependent loss of enzyme activity
also was observed; the KI and kinact values for 14 inactivation
of iNOS were determined to be 490 mM and 0.24 min-1
,
respectively. The KI value for 14 is quite high, probably
reflecting the loss of affinity due to the lack of a guanidinium
or amidinium ion. iNOS inactivation by 14 can be prevented
by inclusion of L-arginine, but not D-arginine, in the inactivation
mixtures, suggesting that the inactivator acts at the arginine
binding site. While the KI for this compound is very large (too
large to be relevant to the inactivation by L-NIO), 14 is still of
interest because it provides another example of a nonguanidino,
neutral side chain, amino acid inhibitor of NOS. This type of
compound will presumably be transported by a neutral trans-
porter (perhaps the same as citrulline29) and, therefore, should
be clinically interesting as a lead compound for use in
conjunction with arginine transporter inhibitors in combination
therapy, which could target both NO production by NOS and
arginine delivery by the y+ transporter.
Experimental Section
General Methods and Reagents. Optical spectra and enzyme assays
were recorded on a Perkin-Elmer Lambda 10 UV/vis spectrophotometer.
NMR spectra were recorded on a Varian Gemini-300 300 MHz

914 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Fast et al.
instrument. Chemical shifts are reported as δ values in parts per million
downfield from Me4Si as the internal standard in CDCl3, unless stated
otherwise. Mass spectra were recorded on a VG Instruments VG70-
250SE high-resolution spectrometer. LC-electrospray ionization mass
spectra were obtained with a Hewlett-Packard series 1050 HPLC
coupled to a Micromass Quattro II triple quadrupole electrospray mass
spectrometer. Melting points were obtained with a Fisher-Johns melting
point apparatus and are uncorrected.
NR-Boc-L-ornithine was purchased from either Bachem Bioscience
Inc. (King of Prussia, PA) or ICN Pharmaceuticals Inc. (Costa Mesa,
CA). NR-Boc-Nδ-Cbz-L-ornithine, NADPH, HEPES, DTT, calmodulin,
and human ferrous hemoglobin were purchased from Sigma Chemical
Co. Tetrahydrobiopterin (BH4) was obtained from B. Schircks Labo-
ratories (Jona, Switzerland) or from Alexis Biochemicals (San Diego,
CA). tert-Butyl alcohol was purchased from Mallinckrodt. D2O was
purchased from Cambridge Isotope Laboratories, and CDCl3 was
purchased from Aldrich Chemical Co. Acids, bases, and conventional
organic solvents were purchased from Fisher. DCC, DMAP, 10% Pd/
C, ethanol-d, acetonitrile-d3, and acetyl chloride were purchased from
Aldrich. TLC plates (silica gel 60-F254, 250 µM), and silica gel 60
(230-400 mesh) were purchased from VWR Scientific.
solvents were removed by high-vacuum rotary evaporation to yield 12
1
(0.8 g, 3.1 mmol, 89%): [R]D ) +40°(6 N HCl); H NMR (D2O) δ
1.6-2.05 (m, 4 H), 2.17 (s, 3 H), 3.43 (t, 2 H), 4.02 (t, 1 H); 13C NMR
(D2O) δ 12.73, 24.65, 26.96, 42.05, 52.92, 161.37, 172.332; M‚H+
obs
) 190.1184, M‚H+calcd ) 190.1192. For elemental analysis, an analytical
sample of 12 was recrystallized as the monoflavinate salt. Calcd for
C17H21N5O11S: C, 40.55; H, 4.21; N, 13.92. Found: C, 40.78; H, 4.57;
N, 13.76.
Nδ-Acetyl-L-ornithine (14). NR-Boc-Nδ-Cbz-L-ornithine, tert-butyl
ester15 (245 mg, 0.58 mmol), was dissolved in 10 mL of methanol and
stirred with a catalytic amount of 10% Pd on activated carbon under 1
atm H2(g) for 15 h. The resulting solution was filtered through Celite.
Volatile solvents were removed by rotary evaporation to yield a white
powder, which was subsequently dissolved in CH2Cl2 and cooled on
ice. First acetyl chloride (50.2 mg, 0.64 mmol) and then triethylamine
(70.1 mg, 0.7 mmol in 5 mL of CH2Cl2) was added dropwise with
stirring, and the resulting solution was allowed to warm to room
temperature and to stir for 15 h. Volatile solvents were removed by
rotary evaporation, and the resulting compound was stirred in 3 mL of
trifluoroacetic acid for 1.5 h at room temperature. To this reaction
mixture, 30 mL of water was added and the solution was loaded onto
5 mL of AG 50W-X8 resin, washed with water until neutral, and eluted
with 1 M NH4OH. The ninhydrin positive fractions were pooled and
rotary evaporated to a white powder (0.049 g, 0.28 mmol, 48%).
Analytical characterization matched literature values for this com-
pound:56 1H NMR (D2O) δ 1.4-1.9 (m, 4 H), 1.93 (s, 3 H), 3.17 (t, 2
H), 3.69 (t, 1 H); 13C NMR (D2O) δ 21.84, 24.19, 27.77, 38.75, 54.34,
N5-(1-Iminoethyl)-L-ornithine (L-NIO, 5). This compound was
either purchased as the hydrochloride salt from Research Biochemicals
International (Natick, MA) or was synthesized as the dihydrochloride
salt by literature methods (0.81 mmol, 38%):33 1H NMR (D2O) δ 1.6-
2.1 (m, 4 H), 2.15 (s, 3 H), 3.25 (t, 2 H), 4.06 (t, 1 H); 13C NMR
(D2O) δ 22.47, 26.66, 30.98, 45.29, 56.40, 66.5 (dioxane), 168.82,
174.19, 174.41; M‚H+ ) 175.1064, M‚H+
) 175.1083.
175.60; M‚H+ ) 174.1267, M‚H+
) 174.1243.
obs
calcd
obs
calcd
N5-(1-Imino-2-hydroxyethyl)-L-ornithine (13). 2-Hydroxyacetim-
idic acid ethyl ester hydrochloride was synthesized by a procedure
similar to literature methods.57 To a solution of potassium cyanide (13
g, 200 mmol) in 40 mL of water at 0 °C, paraformaldehyde (6 g, 200
mmol) was added in a portionwise manner over 15 min. After an
additional 1 h of stirring the mixture was adjusted to pH 2 with 7.5 N
H2SO4 and pH 3-5 with Na2CO3 and then extracted with 3 × 15 mL
of cold Et2O. The organic layers were combined, dried with Na2SO4
and Drierite, and then stirred at 0 °C as dry HCl(g) was bubbled through
the mixture for 2 h. The reaction mixture was then concentrated by
rotary evaporation until a white product crystallized from solution
which, after filtration and drying, gave 2-hydroxyacetimidic acid ethyl
ester hydrochloride (7.03 g, 50.4 mmol, 25%): 1H NMR (DMSO) δ
1.34 (t, 3 H), 4.33 (s, 2 H), 4.45 (q, 2 H); 13C NMR (DMSO) δ 13.60,
58.59, 69.52, 180.13; EI HRMS Mobs ) 103.0620, Mcalcd ) 103.0633.
NR-Boc-L-ornithine (1.1 g, 4.7 mmol) was stirred in 22 mL of water
and adjusted to pH 10 with 2.5 N NaOH. 2-Hydroxyacetimidic acid
ethyl ester hydrochloride was added portionwise, maintaining pH 9
with concomitant addition of 2.5 N NaOH, until all of the NR-Boc-L-
ornithine was consumed, as shown by TLC. After an additional 45
min of stirring, the pH was adjusted to 7.5, and the reaction was stirred
overnight. The reaction mixture was loaded onto 30 mL of AG50W-
X8 resin, washed with water, and then eluted with 10% aqueous
pyridine. Volatile solvents were removed by high vacuum rotary
evaporation to give NR-Boc-N5-(1-imino-2-hydroxyethyl)-L-ornithine
(NR-Boc-Cω-hydroxy-L-NIO) as a pale yellowish oil (0.25 g, 0.87 mmol,
19%): 1H NMR (D2O) δ 1.65-2.1 (m, 4 H), 3.38 (t, 2 H), 4.12 (t, 1
H), 4.40 (s, 2 H); 13C NMR (D2O) δ 22.89, 27.00, 41.14, 52.46, 58.09,
N5-(1-Imino-2-[14C]ethyl)-L-ornithine ([14Cω]-L-NIO, 11). Three
aliquots of cold EtOH saturated with HCl(g) were used to wash 2.5
µL of 14CH3CN (21 mCi/mmol; Sigma) from an ampule cooled in a
CCl4/CO2(s) bath into a tared Wheaton vial. Unlabeled CH3CN (21
µL) was added, and the vial was capped and stirred on ice for 0.5 h
and then at room temperature for 15 h. The resulting ethyl acetimidate‚
HCl salt (10) was precipitated with addition of cold Et2O, the remaining
solution removed with a pipet, and the solid dried with a stream of
N2(g). The solid was washed three times with Et2O and then dried under
vacuum for 3 h (56.9 mg, 0.46 mmol). NR-Boc-L-ornithine (60 mg, 1.7
equiv) was added as a powder to the vial, and then NaOH (0.46 mmol,
1 equiv) was added along with 500 µL of water; the resulting solution
was stirred. The pH was adjusted to 10 (pH paper) with 5 µL aliquots
of 5 N NaOH, and the solution was stirred. After 3 h, it was applied
directly to a 1 mL column of AG 50W-X8 resin, washed with water,
eluted with 10% aqueous pyridine, and dried under vacuum attached
to a rotary evaporator. The protecting group was removed by stirring
with 4 N HCl in ethyl acetate for 0.5 h at room temperature resulting
in 11 (33.7 mg, 0.14 mmol, 30%). Specific radioactivity was determined
to be 2.5 mCi/mmol, and radiopurity to be 96% by TLC (10% NH4-
OH/MeOH or 10% HOAc/MeOH). Radiopurity was increased to 99%
by loading 11 onto 1 mL of AG50W-X8 resin and washing with water,
10% aqueous pyridine, water, and 1 N HCl. Compound 11 was eluted
with 4 N HCl and dried by vacuum rotary evaporation. The product
coeluted with authentic L-NIO (5) by TLC (10% NH4OH/MeOH or
10% HOAc/MeOH).
Nω-Hydroxy-N5-(1-iminoethyl)-L-ornithine (12). NR-Boc-L-orni-
thine (2.82 g; 12.16 mmol) was dissolved in 56 mL of methanol, and
then ethyl N-hydroxyacetimidate (10 g, 97 mmol) was added all at
once. The reaction mixture was adjusted to a basic pH with 2 N NaOH
and was allowed to react for 48 h at room temperature. The desired
compound was then purified from the reaction mixture by silica gel
chromatography (1:2 CHCl3/MeOH) followed by a silica gel plug
washed with 4:1 CHCl3/MeOH and eluted with MeOH. The resulting
compound had slight impurities which were removed by silica gel
chromatography (1:5 CHCl3/MeOH), leaving NR-Boc-Nω-hydroxy-N5-
(1-iminoethyl)-L-ornithine (1.18 g; 4.08 mmol; 34%): 1H NMR (D2O)
δ 1.39 (s, 9 H), 1.45-1.85 (m, 4 H), 1.92 (s, 3 H), 3.20 (t, 2 H), 3.86
(m, 1 H); 13C NMR (D2O) δ 12.96, 26.70, 27.74, 28.57, 41.51, 55.69,
166.94, 171.64; M‚H+ ) 290.184, M‚H+
) 290.172.
obs
calcd
NR-Boc-Cω-hydroxy-L-NIO (0.2 g, 0.7 mmol) was dissolved in 10
mL of 4 N HCl/ethyl acetate and stirred at room temperature for 1 h.
Volatile solvents were removed by high-vacuum rotary evaporation to
yield a white solid, which was then dissolved in methanol and
precipitated as a white solid upon addition of ethyl acetate. Filtration
and subsequent drying resulted in the desired compound as a very
hygroscopic white solid (0.17 g, 0.6 mmol, 92%): 1H NMR (D2O) δ
1.36 (s, 9 H), 1.5-1.8 (m, 4 H), 3.29 (t, 2 H), 3.84 (t, 1 H), 4.35 (s, 2
H); 13C NMR (D2O) δ 22.81, 26.95, 41.04, 52.38, 57.99, 166.91, 171.60;
FAB+ HRMS, C7H16N3O2: M‚H+
) 190.119 19, M‚H+
)
obs
calcd
81.01, 133.39, 157.68, 179.87; FAB+ HRMS, C12H24N3O5: M‚H+
obs
(56) Miller, S. P. F.; Thompson, J. J. Biol. Chem. 1987, 262, 16109-
16115.
(57) McCasland, G. E.; Tarbell, D. S. J. Am. Chem. Soc. 1946, 68, 2393-
2395.
) 290.171 45; M‚H+
) 290.171 60.
calcd
The above compound (1 g, 3.5 mmol) was dissolved in 7 mL of 4
N HCl dioxane and stirred at room temperature for 30 min. Volatile

InactiVation of Inducible Nitric Oxide Synthase
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 915
190.119 17. For elemental analysis, an analytical sample of 13 was
recrystallized as the monoflavinate salt. Calcd for C17H21N5O11S: C,
40.56; H, 4.20; N, 13.91. Found: C, 40.17; H, 4.41; N, 13.45.
N5-(1-Imino-2-[2H3]ethyl)-L-ornithine (15, d3-L-NIO). 2-[C2H3]-
Acetimidic acid ethyl ester hydrochloride was synthesized by the
method of Burkholder et al.58 except that ethanol-d was substituted for
ethanol (56.3 mmol, 55%): 1H NMR (D2O) δ 1.41 (t, 3 H), 4.40 (q, 2
H); 13C NMR (D2O) δ 13.40, 70.12, 179.14. Longer aquisition times
also revealed a septuplet at 18.87. For determining HRMS, deuterated
samples were dissolved in H2O immediately before analysis. FAB+
Absorbance Spectral Changes during Inactivation. Spectra were
not recorded directly because of high concentrations of NADPH and
BH4 present in the incubation mixtures. Instead, at various time points,
100 µL aliquots of the inactivation mixture were taken and small
molecules were removed by centrifuged size exclusion chromatography
column according to the procedures of Penefsky,40 except that the
buffers used contained 11% glycerol and 100 mM Hepes. The
absorption spectra (350-550 nm) of the collected fractions were
recorded in 100 µL cuvettes against a 100 mM Hepes blank.
HPLC of the Heme Cofactor. Chromatograms of iNOS (60-250
µg) inactivated by L-NIO (5, 45-300 µM) were obtained using
gradient elution of an Econosil C18 HPLC column (Alltech, 10 µm,
250 mm × 4.6 mm) and detection at 400 nm. The elution gradient (1
mL/min) consisted of 5 min of 0% B, 60 min of 5-75% B, and then
2 min of 75-100% B followed by 100% B with buffer A consisting
of H2O and 0.1% TFA and buffer B of CH3CN and 0.1% TFA. The
peak at 16 min derived from the BH4 stock solutions and the peak at
64 min coeluted with heme standards. Chromatograms of iNOS
inactivated by [14C]-L-NIO (11) were obtained using gradient elution
(1 mL/min) of an Alltima C18 HPLC column (Alltech, 5 µm, 250 mm
× 4.6 mm) and detection at 400 nm. The elution gradient consisted of
5 min of 0% B and then 80 min of 0-100% B with buffer A consisting
of H2O and 0.1% TFA and buffer B of CH3CN and 0.1% TFA.
Fractions (1 mL) were collected, diluted with 15 mL of Ultima Gold
liquid scintilation cocktail (Packard), and counted for [14C] dpm values.
Gel Filtration of Inactivated iNOS. [14C]-L-NIO-inactivated iNOS
was subjected to gravity flow Sephadex G-50 (fine) column chroma-
tography according to the procedures of Furfine et al.63 except that
100 mM Hepes was used as the buffer. Fractions of 200 µL were
collected, 190 µL of which were counted for [14C] dpm and 10 µL of
each were used to estimate protein concentration with Coomassie Plus
staining reagent (Pierce).
Dialysis of [14C]-L-NIO-Inactivated iNOS. Samples of iNOS were
inactivated with 100 µM [14C]-L-NIO as described above (140 µL total
volume). After inactivation, these were injected into a Pierce Slide-
A-Lyser (10 000 MWCO) and subjected to dialysis against 800 mL of
5 mM Hepes, pH 7.5, at 4 °C. Buffer was changed at 4 or 8 h intervals
until no radioactivity was detected in the solvent. The contents of the
dialysis chamber was removed, mixed with 15 mL of Packard Ultima
Gold LSC cocktail, and analyzed for [14C] dpm. Control experiments
showed g70% protein recovery.
HPLC of Amino Acid Metabolites. Inactivation mixtures were
derivatized with o-fluoraldehyde reagent (Pierce) and injected onto an
Econosil C18 HPLC column (Alltech, 10 µm, 250 mm × 4.6 mm). A
Beckman System Gold 125P solvent module was used to control the
gradient elution as follows: For samples inactivated with 11, the
gradient consisted of 25-45% B over 10 min followed by 45-75% B
over 10 min where the % B was held until all metabolites eluted. For
the assay of NOS-mediated conversion of 12 to 14, the gradient
consisted of 35-55% B over 16 min where the % B was held until all
metabolites eluted. In each case solvent A consisted of 95% 100 mM
NaOAc at pH 7.2, 4.5% methanol, and 0.5% THF. Solvent B consisted
of 95% methanol and 5% 100 mM NaOAc at pH 7.2. Sample elution
was detected by either absorbance at 340 nm or a Spectra/glo filter
fluorimeter fitted with o-phthaldehyde fluorescence filters. For counting
the radioactivity elution profiles, 0.5 mL fractions were collected, diluted
with 5 mL of Packard Ultima Gold liquid scintilation cocktail, and
counted for 4 min each in a Packard Tri-Carb 2100TR liquid
scintillation analyzer. The [14C] dpm values from NIO were counted
from the HPLC peak eluting from 12 to 15 min and 12 from 20 to 23
min.
HRMS: calcd for C4H6NOD3, M‚H+
) 90.087 24, M‚H+
)
calcd
found
90.086 15.
N5-(1-Imino-2-[2H3]ethyl)-L-ornithine (15) was synthesized by the
same route to make L-NIO41 except that 2-[2H3]acetimidic acid ethyl
ester hydrochloride was used as the starting material and all reactions
were carried out in D2O (87 mg, 0.34 mmol): 1H NMR (D2O) δ 1.55-
2.051 (m, 4 H), 3.23(t, 2 H), 4.04 (t, 1 H); 13C NMR (D2O) δ 23.00,
27.17, 41.65, 52.51, 164.96, 171.66; FAB+ HRMS: calcd for
C7H13N3O2D3, M‚H+
) 177.143 08, M‚H+
) 177.141 04.
calcd
found
Expression and Purification of Recombinant iNOS. A recombi-
nant murine iNOS/calmodulin overexpression system in Escherichia
coli was obtained, and the enzyme was purified.59 The enzyme was
quantified according to the methods of Stuehr and Ikeda-Saito60 using
ꢀ397 ) 71 mM-1 cm-1 and a MW of 150 kDa.
Difference Spectra. Spectra were recorded according to the
procedure of McMillan and Masters.61 Briefly, a solution of 1.55 µM
iNOS in 10 µM BH4, 10% glycerol, and 100 mM Hepes at pH 7.5
was scanned for absorbance from 370 to 470 nm against a blank
containing 10 µM BH4, 10% glycerol, and 100 mM Hepes at pH 7.5
in a 100 µL quartz cuvette. Following addition of 1 mM imidazole,
another scan was measured over the same absorbance range (subtraction
of the first spectrum revealed a type II spectral shift). A 250 µM
solution of L-NIO was then added in 2 µL aliquots and scanned over
the same absorbance range. The iNOS‚imidazole spectrum was then
subtracted from each of these to obtain difference spectra. The total
volume of aliquots did not exceed 5% of the total volume.
Initial Velocity Measurements via the Hemoglobin Assay. The
generation of nitric oxide by iNOS was measured by the rapid oxidation
of oxyHb to metHb by nitric oxide.62 The assay mixture contained iNOS
(0.1 µM), L-arginine (2 mM), NADPH (0.1 mM), tetrahydrobiopterin
(10 µM), dithiothreitol (100 µM), and oxyhemoglobin (7.5 µM),
diluted to a total volume of 600 mL with Hepes buffer (100 mM, pH
7.5). The relative rate of nitric oxide synthesis was determined by
monitoring the NO-mediated conversion of oxyhemoglobin to meth-
emoglobin at 401 nm on a Perkin-Elmer Lambda 10 UV/vis spectro-
photometer. All assays were performed at 30 °C.
Irreversible Inhibition Kinetics. iNOS (5-10 µM) was incubated
at 37 °C in Hepes buffer (100 mM, pH 7.5), containing catalase (1000
units for kinetic measurements, 100 units for metabolite studies, and
none for the experiment in Figure 1), NADPH (9 mM), tetrahydro-
biopterin (1 mM), dithiothreitol (10 mM), glycerol (10% v/v), and
inactivator in a total volume of 70-550 µL. The reactions were initiated
by the addition of iNOS, and 10 µL aliquots were removed to assay
for enzyme activity at various times. Controls were performed by
omitting the inhibitors or by omitting or replacing NADPH with
NADP+.
Irreversibility of Inactivation. Aliquots (100 µL) of inactivation
mixtures were removed at various time points and were subjected to a
centrifuged Sephadex G-50 (fine) chromatography column according
to the method by Penefsky,40 except that 100 mM Hepes at pH 7.5
was used as the buffer. Activity was judged using the oxyhemoglobin
NO assay as described above.
LC-Electrospray Mass Spectra of Inactivation Mixtures. Samples
of iNOS (864 µg in 140 µL total volume) were incubated with L-NIO
(250 µM) for 2.5 h as described earlier either with or without (control)
NADPH. Either 25 µL (control) or 30 µL (inactivated) of these samples
was injected directly onto a Hypersil XDB C18 column (2.0 × 250
mm) and using an Applied Biosystems 140 solvent delivery system
was eluted at a rate of 190 µL/min with the following gradient:
(58) Burkholder, C. D.; Jones, W. E.; Wasson, J. S. J. Org. Chem. 1980,
45, 4515.
(59) Rusche, K. M.; Spiering, M. M.; Marletta, M. A. Manuscript
submitted for publication.
(60) Stuehr, D. J.; Ikeda-Saito, M. J. Biol. Chem. 1992, 267, 20547-
20550.
(61) McMillan, K.; Masters, B. S. S. Biochemistry 1993, 32, 9875-
9880.
(62) Hevel, J. M.; Marletta, M. A. Methods Enzymol. 1994, 233, 250.
(63) Furfine, E. S.; Harmon, M. F.; Paith, J. E.; Garvey, E. P.
Biochemistry 1993, 32, 8512-8517.

916 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Fast et al.
5-100% B over 80 min, with solvent A consisting of water and solvent
B of CH3CN. All solvents were 0.5% in formic acid. After 10 min, the
column outlet was attached to a Micromass Quattro II mass spectrometer
without splitting for monitoring column output using positive mode
ionization. Source parameters: capillary voltage 3.43 kV; counter
electrode 0.53 kV; cone voltage 84 V; temperature 130 °C; unit
resolution scan 550-820 at 2 s/scan. Cone voltage for the MS/MS
experiment was 60 V, the collision energy was 40 eV, and the argon
gas pressure (the collision gas) was 3 × 10-3 mbar.
supported by an instrument grant from the NIH (S10 RR10485)
to R.B.V.B. We thank Professor Michael A. Marletta (University
of Michigan) for a generous gift of the cloned E. coli cells with
an overexpression system for inducible nitric oxide synthase.
W.F. carried out all of the experiments except for the HPLC-
electrospray mass spectral work with the modified heme. D.N.
and R.B.V.B. carried out the HPLC-electrospray mass spectral
work with the modified heme.
Supporting Information Available: Ten figures of data (12
pages, print/PDF). See any current masthead page for ordering
and Web access instructions.
Acknowledgment. The authors are grateful to the National
Institutes of Health for financial support of this work to R.B.S.
(Grant R01 GM49725) and to W.F. (NIH Predoctoral Training
Grant T32 GM08382). The electrospray mass spectrometer is
JA982318L