Hexacoordinate oxy-globin models Fe(Por)(NH3)(O2) react with NO to form only the nitrato analogs Fe(Por)(NH3) (η1-ONO2), even at ～100 K

Article abstract of DOI:10.1039/c0cc02665d

The oxy-globin models Fe(Por)(NH₃)(O₂), prepared by sequential reactions of O₂ (¹⁸O₂) and NH ₃ with thin porous layers of Fe^II(Por), react with NO (¹⁵NO) at 80-100 K t

Full text of DOI:10.1039/c0cc02665d

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Hexacoordinate oxy-globin models Fe(Por)(NH3)(O2) react with NO to
form only the nitrato analogs Fe(Por)(NH3)(g1-ONO2), even at B100 Kw
Tigran S. Kurtikyan*a and Peter C. Ford*b
Received 19th July 2010, Accepted 1st October 2010
DOI: 10.1039/c0cc02665d
The oxy-globin models Fe(Por)(NH3)(O2), prepared by sequential
reactions of O2 (18O2) and NH3 with thin porous layers of
FeII(Por), react with NO (15NO) at 80–100 K to form only the
low-spin nitrato complexes Fe(Por)(NH3)(g1-ONO2), thus
implying that peroxynitrite intermediates, if formed, must
undergo very facile isomerization to the nitrato analog.
peroxynitrite intermediates;3 Goldstein et al.5 reported a
species with the same spectral properties upon reacting ferryl
myoglobin with NO2 and concluded that it was the
nitrato analog FeIII(ONO2) (II) and have argued that the
MbFeIII(OONO) species should be very short-lived (at ambient
temperature).5b Subsequent resonance Raman spectral (RRS)
studies of the ms intermediate formed by reaction of NO with
MbO2 in 3 1C alkaline solution and trapped by RFQ identified
this as a nitrato, not a peroxynitrito, complex.6 In model studies,
Karlin et al.7 used optical spectral changes to demonstrate that
reaction of NO with the five-coordinate dioxygen complex
FeII(F8P)(O2) (F8P = tetrakis(2,6-difluorophenyl)porphyrinato
dianion) in a THF solution at ꢀ80 1C gives the nitrato complex
FeIII(F8P)(Z1-ONO2) without an observable peroxynitrito inter-
mediate. However, when the tyrosine mimic 2-4-di-tert-butyl-
phenol (DTBP) was present, the analogous reaction led to a high
yield of DTBP nitration. This was interpreted as evidence that
the caged FeIV(O):NO2 pair is formed by homolytic cleavage of
the O–O bond of the putative FeIII(OONO) transient and that
this caged pair serves to nitrate DTBP (giving FeIII(OH) as the
other product) faster than recombining to give the nitrato
complex.7 Along similar lines, Su and Groves8 demonstrated
that reaction of horse heart met-Mb with OONOꢀ in pH 7.4,
25 1C aqueous solutions gives mostly nitrate but also gives the
ferryl form of Mb plus NO2. The latter results in measurable
nitration of the tyrosine-103 residues of this protein. In contrast,
Herold et al.3 reported quantitative nitrate formation from
NO reactions with HbO2 and MbO2 with no physiologically
significant nitration of the globins.
The principal sink for the bioregulatory molecule nitric oxide
in the cardiovascular system is the rapid reaction with oxy
hemoglobin HbO2 leading to the formation of nitrate ion and
the ferric protein, metHb.1,2 It is generally thought that this
occurs via the formation of a ferric peroxynitrite intermediate
FeIII(OONO) (I) (Scheme 1). However, ambiguity remains
regarding the lifetime of such intermediates and whether
isomerization to nitrate is initiated by homolytic O–O cleavage
(a) or concerted (c). The present low temperature spectro-
scopic study was initiated to probe the nature and reactivity of
intermediates in reactions of NO with the six-coordinate
complexes Fe(Por)(NH3)(O2) which are simple models of
oxy-heme proteins. Surprisingly, the putative intermediate I
was too short lived to be observed even at 100 K.
Kinetics studies by Herold et al. using rapid scan UV-vis
spectroscopy at 20 1C followed the reactions of NO with
HbO2 and with oxy-myoglobin (MbO2) and demonstrated the
formation of transients with ms lifetimes.3 Subsequently,
Olson et al.4 used EPR to probe rapid freeze-quenched
(RFQ) samples from the NO reaction with HbO2 and reported
the formation of a high-spin Fe(III) species on the same time
scale. Although these were initially assigned as ferri-heme
Computational treatments have also been contradictory.
Blomberg et al.9 on the basis of density functional calculations
supported stepwise peroxo bond cleavage followed by
recombination of the ferryl:NO2 caged pair. However, a recent
adiabatic reactive molecular dynamics (ARMD) treatment
of a peroxynitrite–trHbN complex (trHbN = truncated
hemoglobin) concluded that FeO–ONO bond homolysis
should be much slower than the concerted rearrangement to
FeIII(ONO2).10
Scheme 1 Pathway(s) for NO di-oxygenation by oxy-heme proteins.
In our studies,11,12 porous layers are prepared by vacuum
sublimation of ferrous porphyrins FeII(Por) (Por = meso-
tetraphenyl- or meso-tetra-p-tolylporphyrinato dianion) onto
a CaF2 or KBr substrate cooled by liquid nitrogen (B80 K).
Volatile reactants are added sequentially at carefully
controlled temperatures and reactions with the heme models
studied spectroscopically. After each addition, the system is
evacuated to remove volatiles not coordinated to the metal
(see ESIw for a more detailed discussion of the experimental
procedures).
a Molecule Structure Research Centre (MSRC) of the Scientific and
Technological Centre of Organic and Pharmaceutical Chemistry,
National Academy of Sciences, 375014, Yerevan, Armenia.
E-mail: kurto@netsys.am; Fax: +374 10 282267;
Tel: +374 10 287323
b Department of Chemistry and Biochemistry, University of California,
Santa Barbara, Santa Barbara, CA 93106-9510, USA.
E-mail: ford@chem.ucsb.edu; Fax: +1 805-8934120;
Tel: +1 805-8932443
w Electronic supplementary information (ESI) available: More detailed
experimental procedures. Fig. S1: UV–Vis spectra of FeTPP,
FeTPP(O2), Fe(TPP)(NH3)(O2) and Fe(TPP)(NH3)(Z1-ONO2).
Comments on the potential rate of reaction in the 100 K layered
solids. See DOI: 10.1039/c0cc02665d
In the present case, dioxygen complexes Fe(Por)(O2) were
obtained by low-T interaction of O2 with layered FeII(Por)
c
8570 Chem. Commun., 2010, 46, 8570–8572
This journal is The Royal Society of Chemistry 2010

View Article Online
Scheme 2 Preparation of six-coordinate O2 complexes in layered
solids.
solids. Gaseous NH3 was then introduced to give
Fe(Por)(NH3)(O2) (Scheme 2). With the substrate at B80 K,
NO was then supplied, and FTIR and visible spectra of the
resulting material were recorded periodically while slowly
warming.
The reaction of Fe(Por) with O2 leads to the appearance of a
new medium intensity band at 1184 cmꢀ1 for Fe(TPP) and
1177 cmꢀ1 for Fe(TTP) that we assign to the dioxygen n(O2)
stretch of the five-coordinate complexes Fe(Por)(O2).11c In the
experiments with 18O2, the new band shifts to 1113 cmꢀ1 and
1116 cmꢀ1, respectively, confirming this assignment. Changes
in the visible absorption spectra are shown in Fig. S1 (ESIw).
When NH3 (B1 mm equiv.) was then supplied and the layers
slowly warmed to 140 K, the n(O2) bands moved to lower
frequencies, 1163 cmꢀ1 (1095 cmꢀ1) for Fe(TPP) and 1160 cmꢀ1
(1096 cmꢀ1) for Fe(TTP) (values in parenthesis for 18O2).
These shifts indicated the formation of the 6-coordinate
Fe(Por)(NH3)(O2) complexes and were accompanied by changes
in the visible spectra (Fig. S1, ESIw).
The cryostat was then evacuated at B150 K to remove
excess NH3, the substrate cooled to B80 K, and the IR
spectrum recorded to confirm that the Fe(Por)(NH3)(O2)
complexes remain intact.z A measured quantity of NO was
then supplied, and the FTIR spectra were repeatedly recorded
in the course of slow warming to 100 K (Fig. 1). During this
period, the intensity of the n(O2) band decreased, and new
bands characteristic of Z1-coordinated nitrate12 grew at
1496m, 1268s and 936w cmꢀ1 for Por = TTP (1499m, 1268s
and 938w cmꢀ1 for Por = TPP). For analogous experiments
with 18O2 and 15NO, these bands were shifted to lower
frequencies as expected (see Fig. 2 and Table 1).
Fig. 2 FTIR spectra of Fe(TTP)(NH3)(O2) after low-T (80 K–100 K)
reaction with NO (solid line) or with 15NO (dashed line).
Table 1 IR frequencies (in cmꢀ1) of coordinated nitrato group in the
six-coordinate nitrato complexes Fe(Por)(L)(nitrate)
nas(NO2)
(m)
ns(NO2)
(s)
n(O–N)
(w)
Compound
Fe(TPP)(NH3)(Z1-ONO2)a
Fe(TPP)(NH3)(Z1-18ON(18O)O)a
Fe(TPP)(NH3)(Z1-O15NO2)a
Fe(TTP)(NH3)(Z1-ONO2)a
Fe(TTP)(NH3)(Z1-18ON(18O)O)a
Fe(TTP)(NH3)(Z1-O15NO2)a
Fe(TPP)(NO)(Z1-ONO2)b
1499
1481
1472
1496
1482
1469
1505
1472
1491
1457
1486
1454
1268
1252
1249
1268
1253
1249
1265
1246
1280
1258
1277
1258
938
913
925
936
914
925
969
954
B997
986
B995
984
Fe(TPP)(NO)(Z1-O15NO2)b
Fe(TPP)(THF)(Z1-ONO2)c
Fe(TPP)(THF)(Z1-O15NO2)c
Fe(TTP)(THF)(Z1-ONO2)c
Fe(TTP)(THF)(Z1-O15NO2)c
a
b
c
This work. Ref. 12a. Ref. 12b.
The identities of the Fe(Por)(NH3)(Z1-ONO2) complexes
were confirmed by preparing the same nitrato complexes via
the low-T reaction of NH3 with porous layers of the five-
coordinate Z2-nitrato analogs FeIII(Por)(Z2-O2NO)13 similar
to the previous preparation of Fe(TPP)(THF)-(Z1-ONO2)12b
(see ESIw). This reaction is accompanied by nitrate isomerization
to the Z1-isomers (eqn (1)), as seen for reactions with other
ligands.12a,b
ð1Þ
Detailed analysis of the FTIR spectra following the reaction
of NO with Fe(Por)(NH3)(O2) showed no bands indicating
other species, specifically, the expected peroxynitrito inter-
mediate(s). Even at 80–100 K, the only spectrally detected
product is the six-coordinate complex Fe(Por)(NH3)(Z1-ONO2).
Thus, if the peroxynitrito complex is formed initially, its
Fig. 1 FTIR spectral changes as NO (0.1 mm equiv.) is supplied into
the cryostat containing layer of Fe(TTP)(NH3)(O2) on a KBr substrate
at 80 K followed by slow warming to 100 K.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8570–8572 8571

View Article Online
isomerization to the nitrato product must have a very small
barrier (rB7 kcal molꢀ1, see ESIw) given the low-T conditions
of these experiments. A similar conclusion can be drawn for
Karlin’s five-coordinate Fe(F8P)(OONO) model discussed
above, although the temperature in that case was significantly
higher, thus the upper limit for the barrier to isomerization was
correspondingly higher.7
temperatures, hence the barrier for such isomerization must be
quite low, regardless of the mechanism.
ð2Þ
The surprising observation of only the nitrate product does
not resolve whether isomerization of I to II is stepwise or
concerted, since even the faster concerted pathway predicted in
the ARMD computations would still be expected to demonstrate
a measurable barrier.10 One possibility is that the reaction of
the FeII(O2) moiety with NO is so exergonic that the immediate
species formed is sufficiently activated to undergo O–O bond
cleavage or concerted isomerization prior to vibrational cooling,
but this suggestion is not consistent with DFT calculations of the
relevant energies.9
PCF acknowledges support from the US National Science
Foundation (CHE-0749524).
Notes and references
z Some residual NH3 remains, since complete removal of this could
not be effected without some disruption of the Fe(Por)(NH3)(O2)
complex.
1 (a) P. R. Gardner, A. M. Gardner, W. T. Brashear, T. Suzuki,
A. N. Hvitved, K. D. Setchell and J. S. Olson, J. Inorg. Biochem.,
2006, 100, 542–550; (b) L. Ignarro, Nitric Oxide Biology
and Pathobiology, ed. L. J. Ignarro, Elsevier Inc., Burlington,
MA, 2nd edn, 2010.
2 P. C. Ford and I. M. Lorkovic, Chem. Rev., 2002, 102, 993.
3 (a) S. Herold, FEBS Lett., 1998, 439, 85; (b) S. Herold, M. Exner
and T. Nauser, Biochemistry, 2001, 40, 3385.
4 J. S. Olson, E. W. Folley, C. Rogge, A.-L. Tsai, M. L. Dohle and
D. D. Lemon, Free Radical Biol. Med., 2004, 36, 685.
5 (a) S. Goldstein, G. Merenyi and A. Samuni, J. Am. Chem. Soc.,
2004, 126, 15694; (b) S. Goldstein, J. Lind and G. Merenyi, Chem.
Rev., 2005, 105, 2457.
Certain ring vibration modes of Fe(TPP) and Fe(TTP)
complexes at B1350 cmꢀ1 (n(Ca–Cm) mixed with n(Cm–phenyl))
and at B450 cmꢀ1 (d(Pyr. rotation)) have been shown to be
sensitive indicators of the iron spin state.14,15 These appear at
lower frequencies for the high-spin complexes and shift higher
upon transition to a low-spin state. For both the five-coordinate
Fe(Por)(O2) and the six-coordinate Fe(Por)(NH3)(O2), these
bands are disposed in the frequency ranges indicating low-spin
complexes. Furthermore, these bands remain characteristic of
low-spin states upon reaction of Fe(Por)(NH3)(O2) with NO to
give Fe(Por)(NH3)(Z1-ONO2). The UV-visible spectral changes
also support this conclusion (Fig. S1, ESIw). Notably, a similar
analysis of the spin-sensitive RRS bands for the ms RFQ
samples from the Mb(O2) reaction with NO system concluded
that the intermediate observed is a high-spin nitrato complex6
consistent with the earlier EPR studies.4 We cannot readily
explain the difference except to note that the analogous
Fe(Por)(THF)(Z1-ONO2)12b complex is high-spin, so the proximal
ligand has a significant effect in this regard.
6 E. T. Yukl, S. de Vries and P. Moenne-Loccoz, J. Am. Chem. Soc.,
2009, 131, 7234.
7 M. P. Schopfer, B. Mondal, D.-H. Lee, A. A. N. Sarjeant and
K. D. Karlin, J. Am. Chem. Soc., 2009, 131, 11304.
8 J. Su and J. T. Groves, J. Am. Chem. Soc., 2009, 131, 12979.
9 L. M. Blomberg, M. R. A. Blomberg and P. E. M. Siegbahn, JBIC,
J. Biol. Inorg. Chem., 2004, 9, 923.
10 S. Mishra and M. Meuwly, J. Am. Chem. Soc., 2010, 132, 2968.
11 (a) T. S. Kurtikyan and P. C. Ford, Coord. Chem. Rev., 2008, 252,
1486; (b) T. S. Kurtikyan, G. G. Martirosyan, A. V. Gasparyan,
M. E. Akopyan and G. A. Zhamkochyan, J. Appl. Spectrosc.,
1990, 53, 67 (in Russian); (c) G. G. Martirosyan, V. H. Chinaryan,
A. M. Dalaloyan and T. S. Kurtikyan, Vib. Spectrosc., 2009, 51,
294.
12 (a) T. S. Kurtikyan, G. G. Martirosyan, M. E. Hakobyan and
P. C. Ford, Chem. Commun., 2003, 1706; (b) G. M. Gulyan,
T. S. Kurtikyan and P. C. Ford, Inorg. Chem., 2008, 47, 787.
13 M. A. Philippi, N. Baenziger and H. M. Goff, Inorg. Chem., 1981,
20, 3904.
In summary, the six-coordinate oxy-globin models
Fe(Por)(NH3)(O2) were constructed by sequential reactions of
O2 (18O2) and NH3 with thin layers of FeII(Por). The reactions of
these models with NO (15NO) at quite low temperature
(80–100 K) led exclusively in each case to the formation of
the low-spin nitrato complex Fe(Por)(NH3)(Z1-ONO2) as
characterized by IR and visible spectroscopy. Peroxynitrite
complexes were not observed, and this result implies that,
if formed, isomerization of this putative intermediate to the
nitrato product (eqn (2)) must be quite facile even at these low
14 (a) H. Oshio, T. Ama, T. Watanabe, J. Kincaid and K. Nakamoto,
Spectrochim. Acta, 1984, 40A, 863; (b) F. Paulat, V. K.
K. Praneeth, C. Nather and N. Lehnert, Inorg. Chem., 2006, 45,
2835.
¨
15 T. S. Kurtikyan, A. A. Hovhannisyan, G. M. Gulyan and
P. C. Ford, Inorg. Chem., 2007, 46, 7024.
c
8572 Chem. Commun., 2010, 46, 8570–8572
This journal is The Royal Society of Chemistry 2010