The functional model complex [Fe2(BPMP)(OPr)(NO) 2](BPh4)2 provides insight into the mechanism of flavodiiron NO reductases

Article abstract of DOI:10.1021/ja309782m

Flavodiiron nitric oxide reductases (FNORs), found in many pathogenic bacteria, are able to detoxify NO by reducing it to N₂O. In this way, FNORs equip these pathogens with immunity to NO, which is a central immune defense agent in humans. Hence, FNORs are thought to promote infection of the human body, leading to chronic diseases. Despite this importance of FNORs for bacterial pathogenesis, the mechanism of NO reduction by these enzymes is not well understood. Here we present the synthesis and spectroscopic characterization of the diiron dinitrosyl model complex [Fe₂(BPMP) (OPr)(NO)₂](BPh₄)₂. The crystal structure of this complex shows two end-on-coordinated {FeNO}⁷ units that, based on spectroscopic and electrochemical results, are only weakly electronically coupled. Importantly, reduction of this complex by two electrons leads to the clean formation of N₂O in quantitative yield. This complex therefore represents the first example of a functional model system for FNORs. The results provide key mechanistic insight into the mechanism of FNORs and, in particular, represent strong support for the proposed super-reduced mechanism for these enzymes.

Full text of DOI:10.1021/ja309782m

Communication
pubs.acs.org/JACS
The Functional Model Complex [Fe (BPMP)(OPr)(NO) ](BPh )
4 2
2
2
Provides Insight into the Mechanism of Flavodiiron NO Reductases
†
†
Sheng Zheng, Timothy C. Berto, Eric W. Dahl, Melissa B. Hoffman, Amy L. Speelman,
and Nicolai Lehnert*
Department of Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-1055, United States
*
S Supporting Information
Scheme 1. Structural Comparison of the Desulfovibrio gigas
FNOR Active Site (Top) and Model Complexes
ABSTRACT: Flavodiiron nitric oxide reductases
FNORs), found in many pathogenic bacteria, are able
(
[
Fe (BPMP)(OPr)](BPh ) (1, Bottom Left) and
2 4 2
to detoxify NO by reducing it to N O. In this way, FNORs
R,X
2
[Fe(BMPA-PhO )](OTf) (Bottom Right)
equip these pathogens with immunity to NO, which is a
central immune defense agent in humans. Hence, FNORs
are thought to promote infection of the human body,
leading to chronic diseases. Despite this importance of
FNORs for bacterial pathogenesis, the mechanism of NO
reduction by these enzymes is not well understood. Here
we present the synthesis and spectroscopic character-
ization of the diiron dinitrosyl model complex [Fe2-
(
BPMP)(OPr)(NO) ](BPh ) . The crystal structure of
2 4 2
7
this complex shows two end-on-coordinated {FeNO}
units that, based on spectroscopic and electrochemical
results, are only weakly electronically coupled. Impor-
tantly, reduction of this complex by two electrons leads to
the clean formation of N O in quantitative yield. This
2
complex therefore represents the first example of a
functional model system for FNORs. The results provide
key mechanistic insight into the mechanism of FNORs
and, in particular, represent strong support for the
proposed “super-reduced” mechanism for these enzymes.
and water ligation (Scheme 1, top), which is typical for non-heme
iron enzymes. However, one key difference is the presence of the
redox-active FMN cofactor in very close proximity (∼4 Å) to the
diiron active site. Currently, the role of this cofactor for catalysis
is unknown. Importantly, previous studies have shown that the
ferrous non-heme iron centers in FNORs bind NO to generate
itric oxide (NO) is a signaling molecule in mammals that is
N
involved in blood pressure control and nerve signal
1
7
transduction. In addition, NO is produced by macrophages as a
typical high-spin {FeNO} adducts with S = 3/2 total spin during
2
6,7
key immune defense agent to kill invading pathogens. For the
turnover. Several conflicting mechanisms have been proposed
latter purpose, inducible NO synthase produces up to micro3-
molar concentrations of NO, which is toxic against microbes.
However, recent research has shown that some pathogens (e.g.,
Helicobacter pylori, Neisseria meningitides, Trichomonas vaginalis,
Salmonella enterica) have evolved defenses against NO toxicity by
expressing flavodiiron nitric oxide reductases (FNORs) that are
for FNORs, starting from either the mono- or dinitrosyl adducts
6,8
of the diferrous form of the active site, but evidence is now
7
building up that the diferrous dinitrosyl, [{FeNO} ] , is in fact
2
the key intermediate for catalysis. Here we present the synthesis
and spectroscopic characterization of the FNOR model complex
[Fe
ture. Excitingly, two-electron reduction of this complex leads to
nearly quantitative formation of N O, making this complex the
(BPMP)(OPr)(NO) ](BPh ) , including its crystal struc-
2
2 4 2
4
,5
able to efficiently remove NO by reducing it to nontoxic N O.
2
This defense mechanism allows these pathogens to proliferate in
the human body, causing harmful infections. FNORs therefore
constitute significant targets for drug development. Despite their
significance for microbial pathogenesis, the mechanism of these
enzymes is not well understood.
2
first functional model for FNORs. Finally, mechanistic
implications for FNORs are discussed.
The diferrous complex [Fe (BPMP)(OPr)](BPh ) (1; see
2
4 2
Scheme 1) was synthesized via previously reported methods with
9
slight modification. Addition of NO gas to an anaerobic solution
FNORs belong to the family of flavodiiron proteins (FDPs),
6
of 1 causes an immediate color change to brown, indicating that
which are usually utilized to protect organisms against O . These
2
[
Fe (BPMP)(OPr)(NO) ](BPh ) (2) has formed. Complex 2
enzymes contain an N-terminal non-heme diiron active site,
which is in close proximity of the C-terminal flavin
mononucleotide (FMN) binding domain of an adjacent subunit.
The non-heme diiron centers of FNORs show His/carboxylate
2 2 4 2
Received: October 3, 2012
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/ja309782m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Figure 2. UV-visible absorbance spectra of 1, 2, and the corresponding
reduction product generated concomitant with N O production.
2
Figure 1. Crystal structure of 2 showing two NO molecules bound at the
diiron core. The two BPh4 counterions have been omitted, along with
−
Recorded at room temperature in CH Cl .
2 2
all H-atoms, for clarity.
exhibits long-term stability in solution under an NO atmosphere;
however, in the absence of an NO atmosphere, 2 slowly loses NO
1
over several days in solution. H NMR and FT-IR spectra
measured in CDCl and CH Cl , respectively, show negligible
3
2
2
decomposition within 1 h in room-temperature solution (see
Supporting Information (SI), Figures S1 and S2). Two
R,X
analogous monoiron complexes, [Fe(BMPA-PhO )(NO)]-
t
(
OTf), with R = Me, X = CH Cl and R = X = Bu (see Scheme 1),
2
were also synthesized (see SI) to compare the reactivity of
analogous mono- and dinuclear nitrosyl complexes (see below).
Structural characterization of 2 reveals a diiron dinitrosyl motif
in which each iron center is coordinated by two pyridine units, a
single NO moiety, and shared phenolate and propionate bridges
to generate a pseudo-octahedral geometry at each iron center
Figure 3. Percent N
CH Cl (blue) and dissolved NO (red) in the presence of cobaltocene.
O formation at various reaction times for 2 in
2
2
2
coupling constant J = −23 cm− has been reported, which
indicates quite strong exchange coupling between the Fe centers.
Although 2 shows two slightly nonequivalent Fe−N−O units,
this complex is characterized by only one prominent N−O
1
(
see Figure 1). The two FeNO units within each molecule are
geometrically distinct, with Fe−N(O) distances of 1.774 and
1
Fe2, respectively. This is due to a nonsymmetric coordination of
.796 Å and Fe−N−O angles of 155.5° and 144.7° for Fe1 and
−
1
−1
stretching feature at 1760 cm , which shifts to 1686 cm with
the BPMP ligand, where one NO moiety is trans to a tertiary
15
18
N O. Nuclear resonance vibrational spectroscopy measure-
amine (shorter Fe −NO bond) while the other is trans to the N-
1
5
7
atom of a pyridine moiety (longer Fe −NO bond). Fe−O
ments on [ Fe
2 2 4 2
(BPMP)(OPr)(NO) ](BPh ) display one
2
−1
distances to the bridging phenolate moiety are 2.02 and 2.06 Å,
with the longer Fe−O distance correlating with the more linear
FeNO unit. The Fe−O bonds to the propionate moiety are also
nonequivalent, at 1.997 and 2.067 Å, where the longer distance
again corresponds to the more linear FeNO unit. The only other
structurally characterized non-heme diiron dinitrosyl complex is
isotope-sensitive feature at 487 cm which is assigned to the
Fe−NO stretch. These N−O and Fe−NO frequencies agree
well with previously published results for S = 3/2 high-spin
57
7
12
mononuclear non-heme {FeNO} complexes. Importantly, the
observation of single N−O and Fe−NO stretching vibrations for
7
2 indicates that the two {FeNO} units in this complex are
[
Fe (Et-HPTB)(O CPh)(NO) ](BF ) (3), reported by Lip-
electronically not very strongly coupled.
Importantly, complex 2 does not mediate NO reduction and
2
2
2
4 2
10
pard and co-workers. In comparison to 2, 3 displays equivalent
Fe−N(O) bonds at 1.750 Å and significantly less bent Fe−N−O
units with an average angle of 167°.
The UV-visible absorption spectrum of 2 shown in Figure 2
displays a broad charge-transfer transition at 410 nm, in
accordance with typical absorption features associated with
N O formation in solution, in agreement with previous
2
observations for 3 and the dinitrosyl adducts of methane
monooxygenase and ribonucleotide reductase, which are also not
8b
proficient in NO reduction. Excitingly, however, two-electron
reduction of 2 by either chemical or electrochemical means
7
8b
7
non-heme {FeNO} complexes. Both {FeNO} units (each S =
/2) in 2 are antiferromagnetically coupled, which is typically
causes rapid production of N O, as observed by IR spectroscopic
2
3
analysis of the reaction headspace (see Figure S9). Integration of
7
observed for [{FeNO} ] dimers in both enzymes and the above-
the N O stretching feature against a calibration curve generated
2
2
11
mentioned model complex 3. Correspondingly, EPR spectros-
copy at 4 K in frozen CH Cl solution reveals that 2 is EPR-silent.
Given the relatively small Fe−Fe distance of ∼3.5 Å and the
presence of bridging ligands, the observed magnetic (electron spin)
coupling between the iron centers is not surprising. For 3, a
from known N O gas standards reveals nearly quantitative N O
2
2
formation after only 1 min in the presence of cobaltocene as
reductant (see Figure 3). These results are obtained for a variety
2
2
of chemical reductants including KC , decamethyl chromocene,
8
and cobaltocene. By monitoring the corresponding metallocene
B
dx.doi.org/10.1021/ja309782m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
oxidation, UV-visible absorption measurements show the initial
reduction of 2 to be rapid, occurring within mixing time (∼20 s;
see Figure S16). Hence, the reduction of 2 is not rate limiting for
1
N O generation. As mentioned above, H NMR and solution IR
2
measurements show 2 to be stable with respect to NO loss over a
matter of hours in solution (see SI). The rapid reactivity upon the
addition of reductant thus confirms that 2 is the active species
that mediates N O generation. Reaction of NO gas with
2
cobaltocene under similar conditions (as a control) leads to
much slower N O formation, as shown in Figure 3, and a
2
maximum N O yield of only ∼40% under our experimental
2
conditions. This further indicates that the reduction of NO to
N O is in fact mediated by complex 2.
2
As a further control, the complex [Fe(BMPA-PhO)(NO)]-
(OTf) was synthesized as a monomeric analogue to 2 (see
Scheme 1, R = Me, X = CH Cl, and SI). This complex displays a
2
Figure 4. Sweep-rate-dependent cyclic voltammetry showing the
−1
broad ν(N−O) band at 1782 cm (Figure S7). Importantly, this
+
irreversible reduction of 2 in CH Cl vs Fc/Fc . Data taken at 50 and
2
2
monomeric analogue of 2 does not generate N O upon reduction
2
100 mV/s show two consecutive scans, leading to the generation of the
with a slight excess of cobaltocene (reaction conditions are
analogous to the reduction of 2). However, we found this
complex difficult to obtain in pure form, and in order to ensure
new signal at ∼ −0.56 V.
of the reduction product by atmospheric O is slow, indicating a
2
7
that monomeric {FeNO} complexes are not capable of efficient
coordinatively saturated structure. However, under weakly acidic
conditions, which would be expected to disrupt an oxo bridge via
protonation, air oxidation is quite rapid (see Figure S15). X-ray
crystallography shows that the reaction product after acetic acid
treatment corresponds to the diferrous complex with two acetate
bridges, [Fe (BPMP)(Ac) ](BPh ) , which is isostructural to the
N O generation, we further synthesized the complex [Fe-
2
t
t
(
BMPA- Bu PhO)(NO)](OTf) (see Scheme 1, R = X = Bu). IR
2
and, in particular, EPR data (see Figures S19−S21) confirm that
7
this complex is a pure, monomeric {FeNO} species. Treatment
of this complex with reductant does not lead to N O generation
2
2
2
4 2
9
c
after 15 min of reaction time, whereas N O generation is
2
analogous bis-propionate complex reported before.
complete and quantitative with 2 after 1 min (see Figure 3). This
further demonstrates that the diiron structure of 2 is essential for
Electrochemical potentials have been previously reported for
Fe (BPMP)(OPr) ](BPh ) , which is the bis-propionate-
[
2
2
4 2
9
a
efficient N O formation. In agreement with this, no N O
2
2
bridged analogue of 1. Cyclic voltammetry measurements on
in CH Cl under a N atmosphere show two similar features
7
generation upon reduction of mononuclear {FeNO} species has
been reported in the literature.
1
2
2
2
II II
II III
II III
III III
associated with the Fe Fe /Fe Fe and Fe Fe /Fe Fe
couples at 486 and 1198 mV vs Fc/Fc , respectively (see Figure
+
Interestingly, N O production via reduction of 2 is not a
2
proton-dependent process and occurs in the absence of any
S17). The large splitting between the two redox waves is
indicative of a strong electronic interaction (coupling) between
the iron centers. Curiously, however, the nitrosyl adduct 2+
displays a single, irreversible reduction wave at −1.1 V vs Fc/Fc
(∼ −0.5 V vs SHE). Upon reduction, a weak new feature appears
at −0.56 V, which corresponds to the reaction product after
external acid source. This finding is quite surprising, as the
−
+
formation of water, i.e., 2NO + 2e + 2H → N O + H O, could
2
2
be envisioned as a thermodynamic driving force. As such,
extreme care was taken to synthesize compound 2 in the strict
absence of H O and using minimal alcohol throughout the entire
2
metalation, nitrosylation, and reduction process (see SI). All
denitrosylation of 2, consistent with N O formation upon
2
solvents were distilled from CaH , and all solid reagents were
electrochemical reduction of 2 (Figure 4). Here, the scan-rate-
dependent return of the −1.1 V reduction peak is due to diffusion
of 2 to the electrode surface. Importantly, upon bulk two-electron
reduction of 2 at −1050 mV (passing exactly 2 equivalents of
2
vacuum-dried prior to use. Protic solvents are also not seen in the
crystal lattice by XRD. Additionally, reduction of 2 in the
presence of 4 equiv of acetic acid does not cause a noticeable
increase in the reaction rate. This indicates that a sufficiently large
charge), N O is detected in the gas headspace in high yield,
2
thermodynamic driving force is available from N O generation
further demonstrating that 2 is able to mediate NO reduction
chemistry.
These results have direct mechanistic implications for FNORs
and support the so-called “super-reduced” mechanism of NO
reduction by these enzymes. This points to a specific role for
the flavin cofactor, located in direct proximity to the diiron active
site, for the catalytic mechanism of FNORs. We propose that
direct two-electron reduction of the [{FeNO} ] intermediate of
FNORs by the flavin cofactor is the central step of the catalytic
mechanism that facilitates N O formation as shown in Scheme 2.
2
without the need for protonation of the remaining oxo unit to
form H O. In the absence of a proton source, NO reduction
2
mediated by the diiron complex 2 might therefore proceed via
II
−
6,8b
formation of a bridging μ-oxo unit, i.e., 2Fe −NO + 2e → N O
2
II
II
+
Fe −O−Fe . The product is believed to take the form of a
ferrous oxo-bridged cluster. While rare, similar complexes have
been reported in the literature. The yellow-brownish color of
the reaction product, along with its absorption spectrum that
lacks the characteristic broad absorption band of the air-oxidized
13
7
2
2
(
mixed-valent) form of 1 around 650 nm, supports this
In this respect, the observed weak electronic interaction between
the two {FeNO} units in 2 is of key significance for this
7
conclusion (see Figures 2 and S14). IR spectroscopy further
shows that the reaction product lacks the NO ligand (see Figure
S13), and H NMR spectra taken at room temperature show
chemical shifts similar to those observed for 1, indicating that the
reaction product also contains magnetically weakly coupled high-
spin ferrous centers (see Figure S12). In contrast to 1, oxidation
mechanism, as this avoids a large shift of the second reduction to
very negative potentials, which would make it inaccessible to the
flavin reductant. Hence, both reductions to generate the central
1
7
2−
[{FeNO} ]2 intermediate (cf. Scheme 2) can be performed at
similar, accessible potentials. Therefore, we propose that this step
C
dx.doi.org/10.1021/ja309782m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Scheme 2. Proposed Mechanism of NO Reduction by FNOR
AUTHOR INFORMATION
Corresponding Author
lehnertn@umich.edu
■
Author Contributions
†
S.Z. and T.C.B. contributed equally to this work.
Notes
The authors declare no competing financial interest.
Scheme 3. Binding Mode of NO to Non-heme Diiron Centers
and Its Effect on Reactivity
ACKNOWLEDGMENTS
■
This work was supported by the NSF (CHE 0846235). ALS
acknowledges support from an NSF Graduate Research
Fellowship (DGE-0718128). We acknowledge Dr. Jeff Kampf
(
University of Michigan) for X-ray crystallographic analysis of 2.
REFERENCES
■
(
1) (a) Lehnert, N.; Berto, T. C.; Galinato, M. G. I.; Goodrich, L. E. In
The Handbook of Porphyrin Science; Kadish, K. M., Smith, K. M., Guilard,
R., Eds.; World Scientific: Singapore, 2011; Vol. 14, p 1. (b) Nitric
Oxide: Biology and Pathobiology; Ignarro, L., Ed.; Academic Press: San
Diego, 2000. (c) Wink, D. A.; Mitchell, J. B. Free Radical Biol. Med. 1998,
2
(
5, 434.
2) Stuehr, D. J.; Gross, S. S.; Sakuma, I.; Levi, R.; Nathan, C. F. J. Exp.
corresponds to a fast two-electron reduction on the basis of our
findings for 2 (see Scheme 2).
Med. 1989, 169, 1011.
(3) (a) Halpenny, G. M.; Heilmann, B.; Mascharak, P. K. Chem.
Biodiversity 2012, 9, 1829. (b) Burney, S.; Tamir, S.; Gal, A.;
Tannenbaum, S. R. Nitric Oxide 1997, 1, 130. (c) Long, R.; Light, B.;
Talbot, J. A. Antimicrob. Agents Chemother. 1999, 43, 403. (d) Ouellet,
H.; Lang, J.; Couture, M.; Ortiz de Montellano, P. R. Biochemistry 2009,
In preliminary studies, we have also investigated the binding of
NO to 1 by stepwise addition of NO, monitoring the reaction by
EPR and IR spectroscopies. These data do not show preferential
formation of a mononitrosyl complex but straightforward
formation of the dinitrosyl 2. This is in contrast to the
preferential formation of a mononitrosyl adduct for deflavinated
Thermogata maritima FDP. In this case, the mononitrosyl adduct
is stabilized by a weak electrostatic interaction with the second
4
(
8, 863.
4) (a) Baek, S.-H.; Rajashekara, G.; Splitter, G. A.; Shapleigh, J. P. J.
Bacteriol. 2004, 186, 6025. (b) Missall, T. A.; Lodge, J. K.; McEwen, J. E.
Eucaryot. Cell 2004, 3, 835. (c) Sarti, P.; Fiori, P. L.; Forte, E.; Rappelli,
P.; Teixeira, M.; Mastronicola, D.; Sanciu, G.; Giuffre, A.; Brunori, M.
́
Cell. Mol. Life Sci. 2004, 61, 618. (d) Stevanin, T. M.; Moir, J. W. B.;
Read, R. C. Infect. Immun. 2005, 73, 3322.
8
b,14
iron center, as shown in Scheme 3, top.
Curiously, a similar
binding mode is observed for the mononitrosyl adduct of the O
carrier hemerythrin. Consistent with this, the T. maritima FDP
2
14
(5) Gardner, A. M.; Helmick, R. A.; Gardner, P. R. J. Biol. Chem. 2002,
2
77, 8172.
has been shown to be an O -reducing enzyme, as the NO
2
7
(6) Kurtz, D. M., Jr. J. Chem. Soc., Dalton Trans. 2007, 4115.
reduction rate of this protein is low. Based on these
(7) (a) Silaghi-Dumitrescu, R.; Kurtz, D. M., Jr.; Ljungdahl, L. G.;
observations, we propose that the discriminating factor of
whether an FDP functions as an efficient NO reductase or not
lies in this difference in binding mode (see Scheme 3): whereas
Lanzilotta, W. N. Biochemistry 2005, 44, 6492. (b) Hayashi, T.; Caranto,
J. D.; Wampler, D. A.; Kurtz, D. M., Jr.; Moenne-Loccoz, P. Biochemistry
2
010, 49, 7040.
the O -reducing enzymes stabilize the mononitrosyl adduct and,
2
(8) (a) Blomberg, L. M.; Blomberg, M. R. A.; Siegbahn, P. E. M. J. Biol.
Inorg. Chem. 2007, 12, 79. (b) Berto, T. C.; Speelman, A. L.; Zheng, S.;
Lehnert, N. Coord. Chem. Rev. 2013, 257, 244.
(9) (a) Borovik, A. S.; Papaefthymiou, V.; Taylor, L. F.; Anderson, O.
P.; Que, L., Jr. J. Am. Chem. Soc. 1989, 111, 6183. (b) Suzuki, M.;
Kanatomi, H.; Murase, I. Chem. Lett. Chem. Soc. Jpn. 1981, 1745.
in this way, prevent formation of the dinitrosyl, the NO-reducing
enzymes do not show the semibridging binding mode of NO and,
in this way, promote fast formation of the catalytically relevant
dinitrosyl species, as shown in Scheme 3, bottom. In this respect,
the ability of the O reductases to bind diatomics in a bridging
2
(
c) Borovik, A. S.; Hendrich, M. P.; Holman, T. R.; Munck, E.;
Papaefthymiou, V.; Que, L., Jr. J. Am. Chem. Soc. 1990, 112, 6031.
10) Feig, A. L.; Bautista, M. T.; Lippard, S. J. Inorg. Chem. 1996, 35,
892.
11) (a) Coufal, D. E.; Tavares, P.; Pereira, A. S.; Hyunh, B. H.;
fashion can be envisioned to be advantageous for O activation
2
and reduction.
(
In conclusion, the diiron dinitrosyl adduct 2 has been prepared
and spectroscopically characterized in this work. Reduction of 2
6
(
results in the liberation of N O in nearly quantitative yield, which
2
Lippard, S. J. Biochemistry 1999, 38, 4504. (b) Haskin, C. J.; Ravi, N.;
Lynch, J. B.; Munch, E.; Que, L., Jr. Biochemistry 1995, 34, 11090. (c) Lu,
directly supports the so-called “super-reduced” mechanism of
NO reduction by FNOR enzymes. Future work will focus on
testing the ability of 2 to mediate reduction of NO catalytically in
S.; Libby, E.; Saleh, L.; Xing, G.; Bollinger, J. M., Jr.; Moen
J. Biol. Inorg. Chem. 2004, 9, 818.
12) Berto, T. C.; Hoffman, M. B.; Murata, Y.; Landenberger, K. B.;
Alp, E. E.; Zhao, J.; Lehnert, N. J. Am. Chem. Soc. 2011, 133, 16714.
13) Baran, P.; Boca, R.; Chakraborty, I.; Giapintzakis, J.; Herchel, R.;
̈
ne-Loccoz, P.
15
(
an aqueous environment.
(
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures; UV-visible, IR, NMR, and NRVS spectra
■
Huang, Q.; McGrady, J. E.; Raptis, R. G.; Sanakis, Y.; Simopoulos, A.
Inorg. Chem. 2008, 47, 645.
*
S
(14) Hayashi, T.; Caranto, J. D.; Hirotoshi, M.; Kurtz, D. M., Jr.;
and CVs of 1 and 2; N O headspace analysis; UV-visible, IR, and
2
Moenne-Loccoz, P. J. Am. Chem. Soc. 2012, 134, 6878.
(15) This requires careful optimization of the experimental conditions
NMR spectra of the reaction product after N O formation; and
2
tables of distances and angles and CIF file for 2. This material is
to avoid direct reduction of NO gas by the applied reductant.
available free of charge via the Internet at http://pubs.acs.org.
D
dx.doi.org/10.1021/ja309782m | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX