Heme/O2/?NO nitric oxide dioxygenase (NOD) reactivity: Phenolic nitration via a putative heme-peroxynitrite intermediate

Article abstract of DOI:10.1021/ja904832j

(Chemical Equation Presented) An oxy-heme complex, the heme-superoxo species (tetrahydrofuran)(F₈)Fe^III-(O₂ ^?-) (2) (F₈ = an ortho-difluoro substituted tetraarylporphyrinate), reacts with nitrogen monoxide (?NO; nitric oxide) to produce a nitrato-iron(III) compound (F₈)Fe^III-(NO ₃^-) (3) (X-ray). The chemistry mimics the action of ?NO Dioxygenases (NODs), microbial and mammalian heme proteins which facilitate ?NO detoxification/homeostasis. A peroxynitrite intermediate complex is implicated; if 2,4-di-tert-butylphenol is added prior to ?NO reaction with 2, o-nitration occurs giving 2,4-di-tert-butyl-6-nitrophenol. The iron product is (F₈)Fe^III-(OH) (4). The results suggest that heme/O₂/?NO chemistry may lead to peroxynitrite leakage and/or exogenous substrate oxidative/nitrative reactivity.

Full text of DOI:10.1021/ja904832j

Published on Web 07/23/2009
Heme/O2/•NO Nitric Oxide Dioxygenase (NOD) Reactivity: Phenolic Nitration
via a Putative Heme-Peroxynitrite Intermediate
Mark P. Schopfer,# Biplab Mondal,#,† Dong-Heon Lee,‡ Amy A. N. Sarjeant,#,§ and Kenneth D. Karlin*,#
Department of Chemistry, The Johns Hopkins UniVersity, Baltimore, Maryland 21218, and Department of Chemistry
& Research Institute of Physics and Chemistry, Chonbuk National UniVersity, Jeonju, Korea
Received June 12, 2009; E-mail: karlin@jhu.edu
The reaction of nitric oxide (•NO; nitrogen monoxide) with
oxygenated heme proteins is of considerable biological interest. Nitric
oxide is generated in vivo by the oxidation of L-arginine to L-citrulline
mediated by the enzyme Nitric Oxide Synthase (NOS).1 Nitric oxide
itself plays an important role in a number of physiological processes
that include as a signaling agent leading to smooth muscle vasodilation,
platelet disaggregation, neurotransmission, and immune response to
bacterial infection.2
Overproduction of •NO can lead to toxicological processes that
include DNA damage, protein nitration leading to cell death, and
formation of peroxynitrite (-OONdO) with the latter’s further
chemistry leading to highly reactive free radicals.3 It effects oxidation/
nitration of biomolecules; most evident is the nitration of tyrosine.3c,d,4
To maintain proper •NO levels, microbial heme protein •NO Dioxy-
genases (NODs) catalyze the reaction of O2 and •NO to yield the
biologically benign nitrate anion (NO3-).5
too short-lived to detect. This is consistent with a very recent report
employing rapid-freeze quench resonance Raman spectroscopy using
Mb. This shows that the high-spin FeIII species detected was not the
proposed peroxynitrite intermediate, but rather an iron-bound nitrate
complex formed prior to its decay to metMb.9
We report here the chemistry of a synthetic oxy-heme which exhibits
NOD reactivity, where the intermediacy of a peroxynitrite species is
implicated.15 As previously described,16 (F8)FeII (1) {F8 ) tetrakis(2,6-
difluoro-phenyl)porphyrinate(2-)} {λmax ) 422, 542 nm} reacts revers-
ibly with dioxygen to give a diamagnetic iron(III)-superoxo species
(S)(F8)FeIII-(O2•-) (2) {λmax ) 416, 535 nm}, which is stable in solution
below -40 °C in coordinating solvents (S) such as tetrahydrofuran
(THF), acetone, or propionitrile (Figure 1).16 Subsequent addition of
1 equiv of •NO (at -80 °C) produces the five-coordinate nitrato heme
complex (F8)FeIII-(NO3-) (3) {λmax ) 393, 505, 572, 634 nm (Figures
1, 2)} in near quantitative yield;17,18 its structure (X-ray) is shown in
Figure 2. In these benchtop experiments, where 10-20 s are required
to add •NO(g) in THF by syringe and record a spectrum, 3 has already
fully formed. No hint of an intermediate is observed, even if the
chemistry is carried out in 2-methyl-THF at -120 °C.18
•NO + O2 + e- NOD8 NO3-
Hemoglobin (Hb) and myoglobin (Mb) are also major targets/sinks
of •NO in mammals, and they exhibit NOD activity.6 High fidelity
incorporation of both 18O-labeled oxygen atoms into the nitrate (NO3-)
product occurs when •NO reacts with red blood cell oxyHb [Hb(FeIII-
18O2•-)], sperm whale oxyMb [Mb(FeIII-18O2•-)], and E. coli oxyfla-
vohemoglobin [flavoHb(FeIII-18O2•-)].7 The generally stated mechanism
of •NO dioxygenase (Scheme 1) involves direct reaction of the FeIII-
(O2•-) oxy complex with •NO, giving a peroxynitrite intermediate.
Subsequent homolytic O-O bond cleavage produces an oxo-ferryl
(FeIVdO) species and the free radical nitrogen dioxide (•NO2); the latter
attacks the ferryl O-atom to produce a N-O bond giving nitrate.3d,8-12
Figure 1. UV-vis spectra (THF solvent) of (F8)FeII (1) (blue), (thf)(F8)FeIII-
(O2•-) (2) (red) generated from 1 + O2 (g), and (F8)FeIII-(NO3-) (3) (green)
Scheme 1
generated from 2 + •NO(g)
.
Thus, we sought chemical evidence which might suggest the
formation of a peroxynitrite species. When the reaction of •NO(g) with
(thf)(F8)FeIII-(O2•-) (2) is followed by addition of the tyrosine mimic
2,4-di-tert-butylphenol (DTBP), (F8)FeIII-(NO3-) (3) is nevertheless
formed and unreacted DTBP is recovered.18 However, we do observe
effective nitration chemistry when DTBP (g1 equiv) is added prior
to addition of 1 equiv of •NO(g) to 2 (Scheme 2).18 Workup of the
reaction solution18 reveals that the ferric hydroxo product (F8)FeIII-
OH (4) forms (∼85% yield) along with high yields (>82%) of 2,4-
di-tert-butyl-6-nitrophenol (NO2DTBP).
The following control experiments indicate that a new heme-NOx
intermediate forms and is able to effect a phenol nitration reaction
faster than its own isomerization to the nitrate complex 3: (i) Use of
excess •NO(g) bubbled into solution had no effect on the products or
their relative yields. (ii) Bubbling excess •NO(g) into a solution
There have been some reports on the detection of a peroxynitrite
intermediate: (i) Under alkaline conditions, Herold and co-workers
obtained UV-vis spectroscopic evidence for a high-spin FeIII inter-
mediate for both the Hb and Mb systems.13 (ii) Olson and co-workers
suggested EPR evidence for a high-spin FeIII species (g ) 6) using
rapid-freezing techniques.8 However, based on thermokinetic14 and
theoretical10b arguments, the peroxynitrite-iron intermediate should be
# The Johns Hopkins University.
† Current address: Dept. Chemistry, IIT Guwahati, North Guwahati, Assam, India.
‡ Chonbuk National University.
§ Current address: Dept. Chemistry, Northwestern University, Evanston, IL.
9
11304 J. AM. CHEM. SOC. 2009, 131, 11304–11305
10.1021/ja904832j CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
reduced metal ion centers with O2 followed by •NO(g), compared to
conditions involving addition of peroxynitrite reagent to oxidized metal
ion centers. Further studies which address these issues and the
mechanism of the reaction observed here are in progress.
Acknowledgment. This work was supported by NIH Grant GM
60353 (K.D.K.).
Figure 2. Reaction of (thf)(F8)FeIII-(O2•-) (2) (ArF ) 2,6-difluorophenyl)
+ •NO(g) produces the nitrato complex (F8)FeIII-(NO3-) (3) (X-ray).17
Supporting Information Available: Details concerning synthesis,
spectroscopy, reactivity and CIF file. This material is available free of
charge via the Internet at http://pubs.acs.org.
containing 1 at -80 °C in the presence of DTBP and subsequent
warming yielded less than 2% of the NO2DTBP and no other products
besides the starting DTBP and the iron-nitrosyl (F8)FeII-(NO) (5);
excess •NO(g) is not solely responsible for significant DTBP nitration.
(iii) Warming 2 from -80 °C to RT in the presence of DTBP yielded
the oxidatively coupled phenol 3,3′,5,5′-tetra-tert-butyl-(1,1′-biphenyl)-
2,2′-diol (∼10%), unreacted DTBP (∼90%), and 4; •NO(g) is needed
for DTBP nitration. 4 is formed from thermal decomposition of 2.16a
(iv) Warming a -80 °C solution of (F8)FeIII-(NO3-) (3) in the presence
of DTBP gives no reaction of any kind; 3 itself will not nitrate DTBP.19
References
(1) (a) Marletta, M. A.; Hurshman, A. R.; Rusche, K. R. Curr. Opin. Chem.
Biol. 1998, 2, 656–663. (b) Crane, B. R. Biochem. Soc. Trans. 2008, 36,
1149–1154.
(2) (a) Ignarro, J. L.E. Nitric Oxide, Biology and Pathobiology; Academic Press:
San Diego, CA, 2000. (b) Moncada, S.; Palmer, R. M. J.; Higgs, E. A.
Pharmacol. ReV. 1991, 43, 109–142.
(3) (a) Denicola, A.; Radi, R. Toxicology 2005, 208, 273–288. (b) Beckman,
J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.; Freeman, B. A. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 1620–1624. (c) Groves, J. T. Curr. Opin.
Chem. Biol. 1999, 3, 226–235. (d) Pacher, P.; Beckman, J. S.; Liaudet, L.
Physiol. ReV. 2007, 87, 315–424.
(4) (a) Radi, R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4003–4008. (b) Szabo´,
C.; Ischiropoulos, H.; Radi, R. Nature 2007, 6, 662–680. (c) Gunaydin,
H.; Houk, K. N. Chem. Res. Toxicol. 2009, 22, 894–898. (d) Beckman,
J. S. Chem. Res. Toxicol. 1996, 9, 836–844.
(5) Gardner, P. R.; Gardner, A. M.; Martin, L. A.; Salzman, A. L. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 10378–10383.
Scheme 2
(6) (a) Ouellet, H.; Ouellet, Y.; Richard, C.; Labarre, M.; Wittenberg, B.;
Wittenberg, J.; Guertin, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5902–
5907. (b) Gardner, P. R. J. Inorg. Biochem. 2005, 99, 247–266.
(7) Gardner, P. R.; Gardner, A. M.; Brashear, W. T.; Suzuki, T.; Hvitved, A. N.;
Setchell, K. D. R.; Olson, J. S. J. Inorg. Biochem. 2006, 100, 542–550.
(8) Olson, J. S.; Foley, E. W.; Rogge, C.; Tsai, A.-H.; Doyle, M. P.; Lemon,
D. D. Free Radical Biol. Med. 2004, 36, 685–697.
(9) Yukl, E. T.; de Vries, S.; Moënne-Loccoz, P. J. Am. Chem. Soc. 2009,
131, 7234–7235.
(10) (a) Herold, S.; Koppenol, W. H. Coord. Chem. ReV. 2005, 249, 499–506.
(b) Blomberg, L. M.; Blomberg, M. R. A.; Siegbahn, P. E. M. J. Biol.
Inorg. Chem. 2004, 9, 923–935. (c) Ford, P. C.; Lorkovic, I. M. Chem.
ReV. 2002, 102, 993–1017.
(11) Computational chemistry indicates that peroxynitrous acid (HOONdO)
decomposition to nitrate involves the “caged radical pair” (HO•) (•NO2).
See: Gunaydin, H.; Houk, K. N. J. Am. Chem. Soc. 2008, 130, 10036.
(12) Protein heme-NO species also react with O2, leading to nitrate. The
mechanism is said to require NO dissociation, ligation of O2 to form the
oxy species, and its subsequent reaction with NO as in Scheme 1. See:
Herold, S.; Röck, G. Biochemistry 2005, 44, 6223–6231, and refs 10a,c.
From such a reaction involving Mb(NO), a visible spectrum of a putative
peroxynitrite intermediate was extracted; see: Møller, J. K. S.; Skibsted,
L. H. Chem. Eur. J. 2004, 10, 2291–2300.
As previously mentioned, these studies implicate a heme-peroxy-
nitrite intermediate (F8)FeIII-(-OONdO) (2a), formed from (thf)(F8)FeIII-
(O2•-) (2) (no excess O2 present) plus 1 equiv of •NO(g). Following
the literature suggestions, we can hypothesize that 2a undergoes
homolysis to give a ferryl + •NO2 (2b) (caged?), which however can
be captured by a phenol which is already present in solution (Scheme
2). The ferryl would oxidize the phenol to a phenoxyl radical which
will react with •NO2 to give the nitrophenol. The very stable heme-
FeIII-hydroxo complex 4 is formed by the wayside, as we observe.
Another reaction mechanism that should be considered is that
(F8)FeIII-(-OONdO) (2a) undergoes heterolytic OsO bond cleavage,
producing nitronium (NO2+) ion which is an effective phenol nitrating
agent.20 Generation of NO2+ in reactions of copper-zinc superoxide
dismutase (CuZnSOD) with peroxynitrite was in fact suggested by
Beckman,21 and other literature reports suggest this chemistry may
be preferred when metal ions are present.4c,20 However, for metal-
loporphyrins, OsO heterolytic cleavage to give NO2+ seems to have
been ruled out or not considered.22
In summary, we have here described a heme complex that acts as
a nitrogen monoxide dioxygenase, facilitating the reaction of O2 and
•NO to yield the nitrate anion NO3-. Generation of a heme-
peroxynitrite species is implicated; it can be trapped by a phenolic
substrate, leading to o-nitration. The results lead to the suggestion that
in heme proteins peroxynitrite may leak and effect nitration of nearby
residues or exogenous substrates. While Herold observed essentially
no peroxynitrite leakage in oxyMb reactions with •NO,13 other studies
show that addition of peroxynitrite to various metal complexes and
metalloproteins leads to protein tyrosine (including for Mb) or
exogenous phenol nitration.23 Other examples include MnSOD and
CuZnSOD.3d,24 Perhaps there are subtle differences in reactions of
(13) Herold, S.; Exner, M.; Nauser, T. Biochemistry 2001, 40, 3385–3395.
(14) Goldstein, S.; Merenyi, G.; Samuni, A. J. Am. Chem. Soc. 2004, 126,
15694–15701.
(15) Heme-NO complexes may react with O2 to give (heme)FeIII-nitrates; for exam-
ple, see:Munro, O. Q.; Scheidt, W. R. Inorg. Chem. 1998, 37, 2308–2316.
(16) (a) Ghiladi, R. A.; Kretzer, R. M.; Guzei, I.; Rheingold, A. L.; Neuhold,
Y.-M.; Hatwell, K. R.; Zuberbühler, A. D.; Karlin, K. D. Inorg. Chem.
2001, 40, 5754–5767. (b) Kim, E.; Helton, M. E.; Wasser, I. M.; Karlin,
K. D.; Lu, S.; Huang, H.-W.; Moënne-Loccoz, P.; Incarvito, C. D.;
Rheingold, A. L.; Honecker, M.; Kaderli, S.; Zuberbühler, A. D. Proc.
Natl. Acad. Sci. U.S.A. 2003, 100, 3623–3628.
(17) For comprison of properties, a separately synthesized nitrate complex (3)
was generated via the reaction, (F8)FeIII-(Cl) + AgNO3 f (3).18
(18) See Supporting Information.
(19) DTBP in a metal-complex free solution bubbled with excess O2(g) and •NO(g)
yielded a 50/50 mixture of NO2DTBP and a dinitrated product, 2-tert-
butyl-4,6-dinitrophenol. Free •NO2 which might derive from 2 + •NO(g)
chemistry is cabable of effecting phenol nitration, but since production of
only one •NO2 per heme complex is possible, the yield of NO2DTBP would
then be less than 50%, not as observed.
(20) (a) Reynolds, M. R.; Berry, R. W.; Binder, L. I. Biochemistry 2007, 46,
7325–7336. (b) van der Vliet, A.; Eiserich, J. P.; O’Neill, C. A.; Halliwell,
B.; Cross, C. E. Arch. Biochem. Biophys. 1995, 319, 341–349.
(21) Ischiropoulos, H.; Zhu, L.; Chen, J.; Tsai, M.; Martin, J. C.; Smith, C. D.;
Beckman, J. S. Arch. Biochem. Biophys. 1992, 298, 431–437.
(22) Lee, J.; Hunt, J. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 6053–6061.
(23) (a) Bourassa, J. L.; Ives, E. P.; Marqueling, A. L.; Shimanovich, R.; Groves,
J. T. J. Am. Chem. Soc. 2001, 123, 5142–5143. (b) Crow, J. P. Arch.
Biochem. Biophys. 1999, 371, 41–52.
(24) Demicheli, V.; Quijano, C.; Alvarez, B.; Radi, R. Free Radical Biol. Med.
2007, 42, 1359–1368. (b) Quint, P.; Reutzel, R.; Mikulski, R.; McKenna,
R.; Silverman, D. N. Free Radical Biol. Med. 2006, 40, 453–458.
JA904832J
9
J. AM. CHEM. SOC. VOL. 131, NO. 32, 2009 11305