Mechanism of nitric oxide synthase. Evidence that direct hydrogen atom abstraction from the O-H bond of NG-hydroxyarginine is not relevant to the mechanism

Full text of DOI:10.1021/ja005900u

2674
J. Am. Chem. Soc. 2001, 123, 2674-2676
Scheme 1
Mechanism of Nitric Oxide Synthase. Evidence that
Direct Hydrogen Atom Abstraction from the O-H
Bond of NG-Hydroxyarginine Is Not Relevant to the
Mechanism
Hui Huang, Jung-Mi Hah, and Richard B. Silverman*
Department of Chemistry and Department of
Biochemistry, Molecular Biology, and Cell Biology
Northwestern UniVersity, EVanston, Illinois 60208-3113
Scheme 2
ReceiVed December 19, 2000
Mammalian nitric oxide synthases (NOS, E.C. 1.14.13.39)
comprise a family of enzymes that catalyzes the conversion of
L-arginine (1) to L-citrulline (3) and the second messenger
molecule nitric oxide (4, Scheme 1).1 There are two constitutive
isozymes of NOS, neuronal nitric oxide synthase (nNOS), which
is believed to generate NO in the brain2 and is involved in
neurotransmission and long-term potentiation,3 and endothelial
nitric oxide synthase (eNOS), which is important as it is involved
in the regulation of smooth muscle relaxation and vascular tone.4
A third, inducible, isoform in macrophage (iNOS) is important
in the immune system defense against microorganisms and tumor
cells.5
Scheme 3
NOS is a complex enzyme that requires five cofactors for
activity. The N-terminus is the oxygenase domain, to which heme,
tetrahydrobiopterin, and the substrate bind. The C-terminus
reductase domain binds molecules of FMN, FAD, and NADPH;
the two domains are connected by a calmodulin-binding domain.6
The active form of the enzyme exists as a homodimer in which
the interaction occurs primarily between two oxygenase domains,7
but forms an extended dimer interface.8
The mechanism of action of this family of enzymes is not yet
clear, although it is known that the reaction proceeds from
L-arginine to L-NG-hydroxyarginine (2) and then to L-citrulline
and NO.9 Nω-Hydroxy-L-arginine was shown to be a kinetically
competent substrate for macrophage NO synthase that gives
stoichiometric amounts of L-citrulline and NO.10 [15N]N-Hydroxy-
L-arginine gives 15NO, indicating that the hydroxylamine nitrogen
becomes the N in NO.10b The first half-reaction consumes two
electrons from NADPH and incorporates one atom of oxygen from
molecular oxygen to give 2. The second half-reaction oxidizes
2, with the consumption of half an equivalent of NADPH and a
molecule of molecular oxygen to give the final products. The
simplest way to rationalize the conversion of 1 to 2 is via a
standard heme-dependent hydroxylation mechanism; however,
there is evidence that the tetrahydrobiopterin also may be involved
in the electron-transfer process of this reaction.11 The second half-
reaction presents an even greater challenge to the mechanistic
enzymologist, having no direct analogy in other systems. Con-
sequently, a variety of mechanistic possibilities have been
proposed over the years for this step. The early proposals, that 2
undergoes hydrolysis to 3 and NO,12 cannot be correct because
the oxygen atom in 3 was shown to come from O2, not from
H2O.
Schemes 2-8 show some of the different mechanistic pos-
sibilities previously proposed for the conversion of 2 to 3 and 4
which use molecular oxygen as the oxygen source for both half
reactions. The principal difference in these mechanisms involves
the order of bond cleavage. In all cases the O-H bond of 2 must
be cleaved, but in Schemes 2,13 3,14 4,15 5,16 and 617 the O-H
(1) (a) Griffith, O. W.; Stuehr, D. J. Annu. ReV. Physiol. 1995, 57, 707-
736. (b) Kerwin, J. F. J.; Lancaster, J. R. J.; Feldman, P. L. J. Med. Chem.
1995, 38, 4342-4362.
(2) Schmidt, H. H. H. W.; Murad, F. Biochem. Biophys. Res. Commun.
1991, 181, 1372-1377.
(3) Schmidt, H. H. H. W.; Walter, U. Cell 1994, 78, 919-925.
(4) (a) Fo¨rstermann, U.; Pollock, J. S.; Schmidt, H. H. H. W.; Heller, M.;
Murad, F. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1788-1792. (b) Palmer, R.
M. J.; Ferrige, A. G.; Moncada, S. Nature 1987, 327, 524-526.
(5) MacMicking, J.; Xie, Q. W.; Nathan, C. Annu. ReV. Immunol. 1997,
15, 323-350.
(6) (a) Mayer, B.; Hemmens, B. Trends Biochem. Sci. 1997, 22, 477-
481. (b) Masters, B. S. S.; McMillan, K.; Sheta, E. A.; Nishimura, J. S.; Roman,
L. J.; Martasek, P. FASEB J. 1996, 10, 552-558.
(7) Stuehr, D. J. Annu. ReV. Pharmacol. Toxicol. 1997, 37, 339-359.
(8) (a) Li, H.; Raman, C. S.; Glaser, C. B.; Blasko, E.; Young, T. A.;
Parkinson, J. F.; Whitlow, M.; Poulos, T. L. J. Biol. Chem. 1999, 274, 21276-
21284. (b) Fischmann, T. O.; Hruza, A.; Niu, X. D.; Fossetta, J. D.; Lunn, C.
A.; Dolphin, E.; Prongay, A. J.; Reichert, P.; Lundell, D. J.; Narula, S. K.;
Weber, P. C. Nat. Struct. Biol. 1999, 5, 602-611. (c) Crane, B. R.; Arvai, A.
S.; Ghosh, D. K.; Wu, C.; Getzoff, E. D.; Stuehr, D. J.; Tainer, J. A. Science
1998, 279, 2121-2126.
(9) Marletta, M. A.; Hurshman, A. R.; Rusche, K. M. Curr. Opin. Chem.
Biol. 1998, 2, 656-663.
(10) (a) Stuehr, D. J.; Kwon, N. S.; Nathan, C. F.; Griffith, O. W.; Feldman,
P. L.; Wiseman, J. J. Biol. Chem. 1991, 266, 6259-63. (b) Pufahl, R. A.;
Nanjappan, P. G.; Woodard, R. W.; Marletta, M. A Biochemistry 1992, 31,
6822-8.
(11) (a) Hurshman, A R.; Krebs, C.; Edmondson, D. E.; Huynh, B. H.;
Marletta, M. A. Biochemistry 1999, 38, 15689-15696. (b) Gorren, A. C. F.;
Bec, N.; Schrammel, A.; Werner, E. R.; Lange, R.; Mayer, B. Biochemistry
2000, 39, 11763-70. (c) Raman, C. S.; Li, H.; Martasek, P.; Kral, V.; Masters,
B. S.; Poulos, T. L. Cell 1998, 95, 939-950.
(12) (a) Marletta, M. A.; Yoon, P. S.; Iyengar, R.; Leaf, C. D.; Wishnok,
J. S. Biochemistry 1988, 27, 8706-11. (b) Tayeh, M. A.; Marletta, M. A. J.
Biol. Chem. 1989, 264, 19654-8. (c) Hibbs, J. B., Jr.; Taintor, R. R.; Vavrin,
Z. Science 1987, 235, 473-6. (d) De Master, E. G.; Raij, L.; Archer, S. L.;
Weir, E. K Biochem. Biophys. Res. Commun. 1989, 163, 527-33.
(13) Marletta, M. A. J. Biol. Chem. 1993, 268, 12231-4.
(14) Korth, H.-G.; Sustmann, R.; Thater, C. C.; Butler, A. R.; Ingold, K.
U. J. Biol. Chem. 1994, 269, 17776-9.
10.1021/ja005900u CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/27/2001

Communications to the Editor
J. Am. Chem. Soc., Vol. 123, No. 11, 2001 2675
Scheme 4
Scheme 8
Scheme 5
lose a proton to give the oxime radical much faster than
nucleophilic addition of the ferric hydroperoxide could occur, and
therefore the mechanism in Scheme 8 was not reasonable.
Recently, ENDOR spectroscopy of 2 bound to nNOS indicated
that the N-H hydrogen of 2 was juxtaposed closer to the heme
iron than was the O-H hydrogen.20 From the crystal structure of
2 bound to the oxygenase domain of iNOS, Crane et al.21
concluded that the hydroxyl hydrogen was not oriented correctly
for hydrogen atom abstraction, but the NG N-H was properly
positioned. Density functional theory calculations of the structures
and relative energies of various model forms of 2 showed that
the most likely form of L-NG-hydroxyarginine bound to NOS is
the protonated form (5), and to rationalize the ENDOR results, it
was found that a reasonable alternative to O-radical formation
from 5 would be N-radical formation (6) during conversion of 2
to 3 and 4.22
Scheme 6
Scheme 7
In this communication we describe experiments with analogues
of L-NG-hydroxyarginine (2) to test whether initial O-H or N-H
bond cleavage occurs in the conversion of 2 to 3 and 4. The results
of these experiments suggest that initial O-H bond cleavage is
not relevant to the reaction, providing chemical support for the
ENDOR spectroscopic,20 crystallographic,21 and theoretical22
proposals for how NOS catalyzes the degradation of 2.
To test initial O-H bond cleavage of 2 by NOS, NG-tert-
butyloxy- (7)23 and NG-(3-methyl-2-butenyl)oxy-L-arginine (8)24
were synthesized by the same route used to prepare 2,10b,25
bond is broken during the one-electron oxidation step. In Schemes
718 and 819 the O-H bond is broken after an initial one-electron
transfer to an unspecified group (X) and O2 (Scheme 7) or to a
heme peroxy radical (Scheme 8). In both cases, the peroxide that
is formed then adds to the carbon-nitrogen double bond to give
a species that either decomposes spontaneously to 3 and proto-
nated NO (Scheme 7) or undergoes decomposition via a six-
membered transition state involving simultaneous deprotonation
and conversion to products (Scheme 8). On the basis of
electrochemical model studies and thermodynamics, Korth et al.14
suggested that the oxime radical cation in Schemes 7 and 8 would
(15) (a) Kerwin, J. F., Jr.; Lancaster, J. R., Jr.; Feldman, P. L. J. Med.
Chem. 1995, 38, 4342-62. (b) Babu, B. R.; Griffith, O. W. Curr. Opin. Chem.
Biol. 1998, 2, 491-500. (c) Griffith, O. W.; Stuehr, D. J. Annu. ReV. Physiol.
1995, 57, 707-736.
(20) Tierney, D. L.; Huang, H.; Martasek, P.; Masters, B. S. S.; Silverman,
R. B.; Hoffman, B. M. Biochemistry 1999, 38, 3704-3710.
(21) Crane, B. R.; Arvai, A. S.; Ghosh, S.; Getzoff, E. D.; Stuehr, D. J.;
Tainer, J. A. Biochemistry 2000, 39, 4608-21.
(16) Rosen, G. M.; Eccles, C. U.; Pou, S. The Neurologist 1995, 1, 311-
(22) Tantillo, D. J.; Fukuto, J. M.; Hoffman, B. M.; Silverman, R. B.; Houk,
K. N. J. Am. Chem. Soc. 2000, 122, 536-537.
325.
(17) Wang, C. C. Y.; Ho, D. M.; Groves, J. T. J. Am. Chem. Soc. 1999,
121, 12094-12103.
(23) 1H NMR (D2O) δ 4.07 (t, 1 H), 3.28 (t, 2 H), 1.95 (m, 2 H), 1.72 (m,
2 H), 1.23 (s, 9 H); HRMS (M + 1) calcd for C10H22O3N4 247.1715, found
247.1714.
(18) Feldman, P. L.; Griffith, O. W.; Hong, H.; Stuehr, D. J. J. Med. Chem.
1993, 36, 491-6.
(24) 1H NMR (D2O) δ 5.38 (t, 1 H), 4.24 (d, 2 H), 3.63 (t, 1 H), 3.05 (t,
2 H), 1.82 (m, 2 H), 1.74 (s, 3 H), 1.68 (s, 3 H), 1.59 (m, 2 H).
(25) Wagenaar, F. L.; Kerwin, J. F., Jr. J. Org. Chem. 1993, 58, 4331-8.
(19) Feldman, P. L.; Griffith, O. W.; Stuehr, D. J. Chem. Eng. News 1993,
71 (51), 26-38.

2676 J. Am. Chem. Soc., Vol. 123, No. 11, 2001
Communications to the Editor
Scheme 9
Figure 1. Michaelis-Menten plot of NO formation for 7 (A) and for 8
(B).
substituting either O-(tert-butyl)hydroxylamine (Aldrich) or O-(3-
methyl-2-butenyl)hydroxylamine26 for hydroxylamine. The tert-
butyl and 3-methyl-2-butenyl substituents would prevent a
mechanism that requires an initial hydrogen atom or proton
abstraction from the O-H bond. However, if initial N-H bond
cleavage or electron transfer from that nitrogen atom occurs, the
intermediate (9, Scheme 9) could readily decompose, with loss
of either tert-butyl cation or 3-methyl-2-butenyl cation to give
the same intermediate proposed after hydrogen atom abstraction
from 2 (10, Scheme 9), and 3 and 4 would be produced.
Table 1. Kinetic Constants for 2, 7, and 8
compd
kcat/Km
(product assay)
K
m (mM)
kcat (min-1
)
(mM-1 min-1
)
7 (NO)
7 (citrulline)
8 (NO)
3.27 ( 0.56
2.96 ( 0.73
0.82 ( 0.09
0.67 ( 0.06
6.92 ( 0.36
6.82 ( 0.60
11.2 ( 0.5
8.92 ( 0.28
2.12
2.30
13.66
8 (citrulline)
13.31
L-NG-OHArg (2) 0.015 ( 0.002 21.2 ( 1.5
1.44 × 103
Both 7 and 8 were shown to be substrates for nNOS,27
producing both citrulline (data not shown) and NO (Figure 1, A
and B, respectively) with Michaelis-Menten kinetics (Table 1).
Both compounds were shown to be free of possible contaminating
L-arginine (1) and L-NG-hydroxyarginine (2) by treatment with
o-phthalaldehyde reagent28 and HPLC analysis. The limit of
detection for 1 or 2 was 1 µM. Because neither 1 nor 2 was
detectable in the incubation mixture, no citrulline produced from
them would be detectable either (citrulline formation is detected
by conversion to the o-phthalaldehyde derivative as well). The
rate constants for 7 and 8 are similar to those for substrates 1
and 2; the high Km values for 7 and 8 reflect the steric hindrance
of the tert-butyl and dimethylallyl groups, respectively, toward
binding. The fact that both act as substrates indicates that it is
highly unlikely that initial O-H bond cleavage in the first step
is relevant to the mechanism. This supports either initial N-H
bond cleavage (Scheme 9, R′ ) H) or electron transfer from the
nitrogen atom to which the hydroxyl group of 2 is attached
(Scheme 9 except electron transfer instead of hydrogen atom
abstraction).
These results further support the density functional theory
calculations22 indicating that the product of hydrogen atom
abstraction from the N-H of 5 is similar in energy to the product
of hydrogen atom abstraction from the O-H. The results also
are consistent with the crystal structure and mechanistic conclu-
sions of Crane et al.,21 who suggested that the peroxo-heme
initially removes the N-H hydrogen. The mechanism shown in
Scheme 9 would be consistent with all of these results, but the
mechanisms in Schemes 2-6 are inconsistent.
(26) 1-Chloro-3-methyl-2-butene (2 g, 20 mmol) was treated with anhydrous
K2CO3 (2.76 g, 20 mmol) and N-hydroxyphthalimide (3.3 g, 20 mmol) in
DMSO (30 mL) at room temperature. After being stirred for 24 h the mixture
was poured into cold water, and the white precipitate was collected;
recrystallization from ethanol gave white prisms (3 g, 66%): 1H NMR (CDCl3)
δ 7.85 (m, 2H), 7.75 (m, 2H), 5.53 (t, 1H), 4.72 (d, 2H, J ) 7.4), 1.77 (s,
3H), 1.74 (s, 3H). This product (3 g, 13 mmol) was treated with hydrazine
hydrate (3.6 mL, 20 mmol) in refluxing ethanol (60 mL) for 30 min. Following
cooling, the phthalazine was dissolved in 3% Na2CO3, and the product was
extracted with ether. After drying (MgSO4), HCl gas was bubbled into the
ether solution, and the white flakes (1.0 g, 76%) were collected: 1H NMR
(D2O) δ 5.36 (t, 1H), 4.53 (d, 2H), 1.79 (s, 3H), 1.73 (s, 3H).
Acknowledgment. The authors are grateful to Professor Michael A.
Marletta for the recombinant E. coli cells that express murine macrophage
iNOS and to the National Institutes of Health (GM49725) for financial
support of this research.
(27) A recombinant murine iNOS/calmodulin overexpression system in
Escherichia coli was obtained, and the enzyme was purified (Hevel, J. M.;
White, K. A.; Marletta, M. A. J. Biol. Chem. 1991, 266, 22789-22791). The
enzyme was quantified according to the methods of Stuehr and Ikeda-Saito
(Stuehr, D. J.; Ikeda-Saito, M. J. Biol. Chem. 1992, 267, 20547-20550) using
ꢀ397 ) 71 mM-1 cm-1 and a molecular mass of 150 kDa.
Supporting Information Available: Syntheses of 7 and 8, LC-MS
for the control reactions to show the absence of contaminating L-arginine
and L-NG-hydroxyarginine, and assay procedures (PDF). This material is
available free of charge via the Internet at http://pubs.acs.org.
(28) Roth, M. Anal. Chem. 1971, 43, 880-2.
JA005900U