Ferric Heme-Nitrosyl Complexes: Kinetically Robust or Unstable Intermediates?

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Ferric Heme-Nitrosyl Complexes: Kinetically Robust or Unstable

Intermediates?

Ashley B. McQuarters,^†Jeﬀ W. Kampf,^†E. Ercan Alp,^‡Michael Hu,^‡Jiyong Zhao,^‡

and Nicolai Lehnert*^,†

^†Department of Chemistry and Department of Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States

^‡Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, United States

S

* Supporting Information

ABSTRACT: We have determined a convenient method for

the bulk synthesis of high-purity ferric heme-nitrosyl

complexes ({FeNO}⁶in the Enemark−Feltham notation);

this method is based on the chemical or electrochemical

oxidation of corresponding {FeNO}⁷precursors. We used this

method to obtain the ﬁve- and six-coordinate complexes

[Fe(TPP)(NO)]⁺(TPP²⁻= tetraphenylporphyrin dianion)

and [Fe(TPP)(NO)(MI)]⁺(MI = 1-methylimidazole) and

demonstrate that these complexes are stable in solution in the

absence of excess NO gas. This is in stark contrast to the

often-cited instability of such {FeNO}⁶model complexes in

the literature, which is likely due to the common presence of halide impurities (although other impurities could certainly also

play a role). This is avoided in our approach for the synthesis of {FeNO}⁶complexes via oxidation of pure {FeNO}⁷precursors.

On the basis of these results, {FeNO}⁶complexes in proteins do not show an increased stability toward NO loss compared to

model complexes. We also prepared the halide-coordinated complexes [Fe(TPP)(NO)(X)] (X = Cl⁻, Br⁻), which correspond to

the elusive, key reactive intermediate in the so-called autoreduction reaction, which is frequently used to prepare {FeNO}⁷

complexes from ferric precursors. All of the complexes were characterized using X-ray crystallography, UV−vis, IR, and nuclear

resonance vibrational spectroscopy (NRVS). On the basis of the vibrational data, further insight into the electronic structure of

these {FeNO}⁶complexes, in particular with respect to the role of the axial ligand trans to NO, is obtained.

Due to the signiﬁcance of ferric heme-nitrosyl complexes in

biology, it is important to understand their basic properties and

reactivity. Synthetic model complexes are a great tool to study

the fundamental properties of these complexes and in

comparison to the proteins, identify the diﬀerent roles that

the protein matrix plays in modulating their geometric and

electronic structures, properties, and reactivities. In the case of

{FeNO}⁶complexes, proteins usually show NO-binding

constants in the 10³−10⁵range,¹²⁻¹⁵whereas corresponding

model complexes are oftentimes only stable in the presence of

excess NO in solution. It is therefore believed that the protein

matrix plays a signiﬁcant role in stabilizing these species. As our

work presented in this paper shows, this prominent conclusion

is in fact incorrect.

INTRODUCTION

■

Nitric oxide (NO) is a toxic gas that has surprisingly important

roles in biological systems. In mammals, NO plays a crucial role

in nerve-signal transduction, vasodilation, and immune

response by white blood cells.¹⁻³NO is produced in vivo by

the nitric oxide synthase (NOS) family of enzymes.^4,5In

mammals, NO produced by endothelial NOS in the

cardiovascular system is sensed by and subsequently activates

soluble guanylate cyclase (sGC), and in this way, it modulates

arterial vasodilation.⁶Interestingly, certain blood-sucking

insects such as Rhodnius proxlixus (known as the “kissing

bug”) and Cimex lectularius (known as the “bed bug”) take

advantage of NO’s vasodilating eﬀect. When these insects bite a

victim, they use small NO-carrier proteins called nitrophorins

(Nps) to inject NO into the bite to increase their blood meal.⁷

Nps use ferric heme-nitrosyl complexes, or {FeNO}⁶in the

Enemark−Feltham notation (where the superscript 6 repre-

sents the number of iron d electrons plus the unpaired

electrons in the π* orbitals of NO),⁸for NO transport. Ferric

heme-nitrosyl complexes are also important intermediates in

dissimilatory denitriﬁcation.⁹⁻¹¹Heme {FeNO}⁶complexes in

proteins are generally six-coordinate (6C) and contain either a

neutral N-donor ligand such as histidine (His) or an anionic S-

donor ligand such as cysteinate (Cys) as a trans ligand to NO.

The generally applied method for the preparation of ferric

heme-NO complexes in the literature is the reaction of ferric

heme precursors with weakly coordinated anions, [Fe(Porph)-

−

(X)] (X = BF₄, ClO₄, etc.; Porph = general porphyrin²⁻

ligand), with excess NO gas as shown in Scheme 1. This results

in the formation of the respective ﬁve-coordinate (5C) ferric

heme-nitrosyl complexes, [Fe(Porph)(NO)]X, and in the

Received: June 14, 2017

A

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a

we demonstrate that addition of halide to solutions of our

{FeNO}⁶complexes indeed leads to fast NO loss.

Scheme 1. Synthesis of Iron NO Complexes

Furthermore, as previously discussed, {FeNO}⁷complexes

are frequently synthesized by reductive nitrosylation (or

commonly referred to as “autoreduction”) of ferric precursors.

In many cases, this reaction is started from a ferric chloride

complex, [Fe(Porph)(Cl)], that is reacted with excess NO gas

in the presence of base (such as methanol). In the ﬁrst step of

the proposed mechanism, the ferric chloride complex binds NO

to form an intermediate, [Fe(Porph)(NO)(Cl)], which then

reacts with the base resulting in a ferrous porphyrin complex

(see Scheme 2). This autoreduction has been studied in detail,

a

MI = 1-methylimidazole. The oval represents the porphyrin ligand.

a

Scheme 2. Proposed Reductive Nitrosylation Mechanism

presence of an N-donor ligand (L), 6C complexes [Fe(Porph)-

(NO)(L)]X are formed. These {FeNO}⁶model systems are

typically handled, stored, and redissolved under NO-saturated

conditions due to their inherent propensity to lose NO from

the iron center.¹⁶⁻²⁰Also, using this method, the formed

iron(III)−NO complexes react with any trace water (or bases

such as methanol) in the presence of excess NO gas to form

corresponding ferrous heme-NO, or {FeNO}⁷, complexes. This

process is called reductive nitrosylation and has been studied in

detail in the literature.²¹⁻²³This frequently results in isolated

mixtures of {FeNO}^6/7complexes. Despite these diﬃculties,

several 5C and 6C ferric heme-nitrosyl complexes with the

OEP²⁻(octaethylporphyrin²⁻) coligand were prepared and

crystallized using this approach and characterized by UV−vis

(in an NO atmosphere) and IR spectroscopy (in the solid

state).^{17,18,24−26}Besides OEP²⁻, another synthetic porphyrin

frequently applied in model complex studies is TPP²⁻

(tetraphenylporphyrin²⁻). In the latter case, however, there

are only four crystal structures of {FeNO}⁶complexes available:

[Fe(TPP)(NO)(H₂O)]⁺,^19,27[Fe(TpivPP)(NO)(NO₂)],²⁵

[Fe(TPP)(NO)(CO₂CF₃)],²⁸and [Fe(TPP)(NO)(ROH)]⁺.²⁰

Since model complex studies performed in our and many other

laboratories are heavily based on TPP²⁻and its deriva-

tives,^16,29−40this represents a surprising knowledge gap in the

literature.

a

Also known as autoreduction.

but there has been limited characterization of the elusive

chloride-bound {FeNO}⁶intermediate.⁴¹To learn more about

this process, we synthesized the complex [Fe(TPP)(NO)(Cl)]

and characterized it using diﬀerent spectroscopic methods. We

found that this complex loses NO from the iron center very

easily and needs to be handled under an NO-saturated

atmosphere at all times. Finally, we also characterized this

species using X-ray crystallography, which represents the ﬁrst

crystal structure of any [Fe(Porph)(NO)(Cl)] complex

reported to this date.

In this work, we ﬁrst prepared the {FeNO}⁶complexes

[Fe(TPP)(NO)]⁺and [Fe(TPP)(NO)(MI)]⁺(MI = 1-

methylimidazole) using the established literature method, and

we fully characterized these compounds using UV−vis, IR,

NMR, and nuclear resonance vibrational spectroscopy (NRVS).

In addition, we obtained the ﬁrst crystal structures for these

complexes. We further devised a convenient route to synthesize

these {FeNO}⁶complexes via chemical oxidation of the

corresponding {FeNO}⁷precursor [Fe(TPP)(NO)], which

can be accomplished by both chemical and electrochemical

means as shown in Scheme 1. Using this method of preparation

results in a surprisingly solution-stable 5C {FeNO}⁶complex

that very slowly loses NO over a long period of time. With this

notable stability, we also show that the oxidation of

[Fe(TPP)(NO)] in the presence of ∼1 equiv MI generates

the 6C {FeNO}⁶complex [Fe(TPP)(NO)(MI)]⁺in pure form.

The {FeNO}⁶complexes generated via chemical oxidation have

identical spectroscopic features to those made via the

traditional method of preparation. The remarkable stability of

the {FeNO}⁶complexes against NO loss observed here diﬀers

from many previous reports in the literature. The accelerated

NO loss in the latter cases is probably due to halide impurities

(although other impurities could certainly also play a role), and

EXPERIMENTAL PROCEDURES

■

All reactions were performed under inert conditions using Schlenk

techniques. The preparation and handling of air-sensitive materials was

carried out under a dinitrogen atmosphere in an MBraun glovebox

equipped with a circulating puriﬁer (O₂, H₂O < 0.1 ppm). Nitric oxide

(Cryogenic Gases Inc., 99.5%) was puriﬁed by passage through an

ascarite II column (NaOH on silica) followed by a cold trap at −80 °C

to remove higher-order nitrogen oxide impurities. ¹⁵N¹⁸O-Nitric oxide

(Sigma-Aldrich) was used without further puriﬁcation. All solvents

(including deuterated solvents) and 1-methylimidazole (MI) were

distilled from CaH₂under dinitrogen and then degassed via ﬁve

freeze−pump−thaw cycles. Tetrabutylammonium hexaﬂuorophos-

phate was recrystallized from ethanol. The puriﬁed solvents were

stored over appropriately sized activated molecular sieves in the

glovebox until used. 1,1′-Diacetylferrocene was purchased from Fisher

Scientiﬁc and used without any further puriﬁcation. [Fe(TPP)(Cl)],⁴²

[Fe(TPP)(X)]⁴³where X = PF₆or SbF₆and [thianthrene][X]⁴⁴

−

where X = BF₄or SbF₆were synthesized as previously reported.

⁵⁷Fe complexes were synthesized in the same way as the natural

abundance isotopes complexes, using ⁵⁷FeCl₂dimethanol salt as the

iron source. All {FeNO}⁶complexes that are prepared from NO gas

were precipitated under an NO atmosphere and then isolated in a

dinitrogen atmosphere (in a glovebox).

Root-Mean-Square Deviation Determination. The root-mean-

square deviation (RMSD) is calculated by the following equation:⁷

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0.1099 and wR2 = 0.2727 [based on I > 2σ(I)], R1 = 0.1244 and wR2

= 0.2791 for all data. Additional details are presented in Tables S2−S6

and are given as Supporting Information.

1

N

RMSD =

(dist)²

∑

[Fe(TPP)(NO)]BF₄. A brown block-like crystal of [Fe(TPP)(NO)]-

BF₄of dimensions 0.22 × 0.12 × 0.12 mm was mounted on the

diﬀractometer. A total of 2028 images were collected with an

oscillation width of 1.0° in ω. The exposure times were 5 s for the low-

angle images and 30 s for high-angle images. Rigaku d*trek images

were exported to CrysAlisPro for processing and corrected for

absorption.^48,50The integration of the data yielded a total of 64 490

reﬂections to a maximum 2θ value of 139.14° of which 15 452 were

independent and 14 777 were greater than 2σ(I). The ﬁnal cell

constants (Table S7) were based on the xyz centroids of 30 783

reﬂections above 10σ(I). Analysis of the data showed negligible decay

during data collection. The structure was solved and reﬁned with the

Bruker SHELXTL (version 2014/6) software package,⁴⁹using the

where N corresponds to the number of atoms that constitute the mean

heme plane and dist is the distance (in Å) of a speciﬁc atom to the

mean heme plane. The RMSD can be calculated from the 25-atom

core displacement or the 4-atom meso carbon displacement.

Physical Measurements. Infrared spectra were obtained from

KBr disks on PerkinElmer BX and GX spectrometers at room

temperature. Measurements in solution were performed in a cell

equipped with two CaF₂windows on the same instruments. Electronic

absorption spectra were measured using an Analytical Jena Specord

S600 instrument at room temperature. In situ UV−vis measurements

were taken with a Hellma quartz immersion probe with a 10 mm path

length. Electron paramagnetic resonance spectra were recorded on a

Bruker X-band EMX spectrometer equipped with Oxford Instruments

liquid nitrogen and helium cryostats. EPR spectra were typically

obtained on frozen solutions using 20 mW microwave power and 100

kHz ﬁeld modulation with the amplitude set to 1 G. Sample

concentrations were ∼1−3 mM. Proton and ﬂuorine NMR spectra

were recorded on a Varian MR 400 MHz instrument or a Varian

NMRS 500 MHz spectrometer at room temperature. Cyclic

voltammograms were obtained using a CH instruments CHI600E

electrochemical workstation with a three-component electrochemical

cell consisting of a glassy carbon working electrode, platinum counter

electrode, and silver-wire pseudoreference electrode. All potentials

were corrected to Fc/Fc⁺. UV−vis and IR spectroelectrochemical

(SEC) measurements were performed using custom-built thin-layer

electrochemical cells as previously described.³⁰All electrochemical and

spectroelectrochemical measurements were carried out in the presence

of 0.1−0.3 M tetrabutylammonium hexaﬂuorophosphate. Nuclear

resonance vibrational spectroscopy (NRVS) was carried out as

previously described⁴⁵at beamline 3-ID-XOR at the Advanced Photon

Source (APS) at Argonne National Laboratory. This beamline

provides about 2.5 × 10⁹photons/sec in ∼1 meV bandwidth (8

cm⁻¹) at 14.4125 keV in a 0.5 (vertical) × 0.5 mm (horizontal) spot.

Samples were loaded into 4 × 7 × 1 mm copper cells. The ﬁnal spectra

represent averages of four scans. The program Phoenix was used to

convert the NRVS raw data to the vibrational density of states

(VDOS).^46,47All of the {⁵⁷FeNO}⁶/{⁵⁷Fe¹⁵N¹⁸O}⁶complexes were

pure as determined by IR spectroscopy. The resonance Raman (rR)

measurements were performed using the 413.13 nm excitation line

from a Kr⁺ion laser (Spectra Physics Beam Lok 2060-RS). Raman

spectra were recorded at 77 K using an Acton two-stage TriVista 555

monochromator connected to a liquid-nitrogen-cooled CCD camera

(Princeton Instruments Spec-10:400B/LN). The total exposure time

of the samples to the laser radiation was 3 min using 1−2

accumulations, and typical laser powers were in the 20−30 mW range.

Crystal Structure Determination. All crystals were measured on

a Rigaku AFC10K Saturn 944+ CCD-based X-ray diﬀractometer

equipped with a low-temperature device and a Micromax-007HF Cu-

target microfocus rotating anode (λ = 1.54187 Å) operated at 1.2 kW

power (40 kV, 30 mA). The X-ray intensities were measured at 85 K

with the detector placed at a distance of 42.00 mm from the crystals.

[Fe(TPP)(NO)(MI)]PO₂F₂. A black needle of [Fe(TPP)(NO)(MI)]-

PO₂F₂of dimensions 0.22 × 0.05 × 0.02 mm was mounted on the

diﬀractometer. A total of 4053 images were collected with an

oscillation width of 1.0° in ω. The exposure time was 5 s for the low

angle images, 30 s for high angle. The integration of the data yielded a

total of 71 715 reﬂections to a maximum 2θ value of 136.44° of which

9081 were independent, and 6991 were greater than 2σ(I). The ﬁnal

cell constants (Table S1) were based on the xyz centroids of 18 806

reﬂections above 10σ(I). Analysis of the data showed negligible decay

during data collection. The data were processed with CrystalClear 2.0

and corrected for absorption.⁴⁸The structure was solved and reﬁned

with the Bruker SHELXTL (version 2008/4) software package,⁴⁹

using the space group P1 with Z = 2 for the formulae FeC₄₈H₃₄N₇O,

−

space group P1 with Z = 4 for the formulae FeC₄₄H₂₈N₅O, BF₄, and

2.25(CH₂Cl₂). All non-hydrogen atoms were reﬁned anisotropically

with the hydrogen atoms placed in idealized positions. Full matrix

least-squares reﬁnement based on F²converged at R1 = 0.0714 and

wR2 = 0.1969 [based on I > 2σ(I)], R1 = 0.0732 and wR2 = 0.1995 for

all data. Additional details are presented in Table S8−S12 and are

given as Supporting Information.

[Fe(TPP)(NO)(Cl)]. A brown prism of [Fe(TPP)(NO)(Cl)] of

dimensions 0.16 × 0.10 × 0.10 mm was mounted on the

diﬀractometer. A total of 2028 images were collected with an

oscillation width of 1.0° in ω. The exposure times were 1 s for the low-

angle images and 4 s for the high-angle images. Rigaku d*trek images

were exported to CrysAlisPro for processing and corrected for

absorption.^48,50The integration of the data yielded a total of 13 642

reﬂections to a maximum 2θ value of 137.91° of which 870 were

independent and 867 were greater than 2σ(I). The ﬁnal cell constants

(Table S13) were based on the xyz centroids of 6524 reﬂections above

10σ(I). Analysis of the data showed negligible decay during data

collection. The structure was solved and reﬁned with the Bruker

SHELXTL (version 2014/6) software package,⁴⁹using the space

group I4/m with Z = 2 for the formula FeC₄₄H₂₈N₅OCl. All non-

hydrogen atoms were reﬁned anisotropically with the hydrogen atoms

placed in idealized positions. The chloro and nitrosyl ligands are

disordered 50/50% on both faces of the porphyrin. Full matrix least-

squares reﬁnement based on F²converged at R1 = 0.0492 and wR2 =

0.1506 [based on I > 2σ(I)] and R1 = 0.0492 and wR2 = 0.1506 for all

data. Additional details are presented in Tables S14−S19 and are given

as Supporting Information.

Synthesis of [Fe(η⁵-C₅H₄COMe)₂][BF₄]. In the glovebox, 115 mg

(0.426 mmol) of 1,1′-diacetylferrocene and 129 mg (0.426 mmol) of

[thianthrene][BF₄] were dissolved in 7 mL of dichloromethane, which

caused a light-blue solid to precipitate from the reaction mixture. The

reaction was allowed to stir for ∼30 min and, at this point, was vacuum

ﬁltered through a frit to give a teal blue powder. This powder was

washed with dichloromethane and hexanes and then stored in the

glovebox freezer. Yield: 93 mg (0.261 mmol, 61%). UV−vis (CH₂Cl₂):

655 nm. Anal. Calcd for C₁₄H₁₄BF₄FeO₂: C, 47.11; H, 3.95; N, 0.00.

Found: C, 46.69; H, 3.58; N, 0.00. No peaks from the oxidant are

observed in the ¹H NMR spectrum. ¹⁹F{¹H} NMR (CD₂Cl₂, 377

MHz): −168.341 (s). IR (KBr): ν(CO) = 1693 cm⁻¹, ν(BF₄) = 1054

cm⁻¹.

Synthesis of [Fe(η⁵-C₅H₄COMe)₂][SbF₆]. In the glovebox, 72 mg

(0.267 mmol) of 1,1′-diacetylferrocene and 118.6 mg (0.262 mmol) of

[thianthrene][SbF₆] were dissolved in 5 mL of dichloromethane,

which caused a light-blue solid to precipitate from the reaction

mixture. The reaction was allowed to stir for ∼30 min and, at this

point, was vacuum ﬁltered through a frit to give a teal blue powder.

This powder was washed with dichloromethane and hexanes and then

stored in the glovebox freezer. Yield: 59 mg (0.117 mmol, 44%). UV−

vis (CH₂Cl₂): 655 nm. Anal. Calcd for C₁₄H₁₄F₆FeO₂Sb: C, 33.24; H,

2.79; N, 0.00. Found: C, 33.28; H, 2.71; N, 0.00. No peaks from the

oxidant are observed in the H NMR spectrum. ¹⁹F{¹H} NMR (471

−

1

PO₂F₂, CH₂Cl₂, and H₂O. All non-hydrogen atoms were reﬁned

anisotropically with the hydrogen atoms placed in idealized positions.

MHz): −123.96 (m). IR (KBr): ν(CO) = 1701 cm⁻¹, ν(SbF⁶) = 659

Full matrix least-squares reﬁnement based on F²converged at R1 =

cm⁻¹

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Synthesis of [Fe(TPP)(NO)]. A 219 mg (0.242 mmol) portion of

[Fe(TPP)(SbF₆)] was dissolved in 8 mL of dichloromethane and 1

mL of methanol. Then, the solution was exposed to NO gas, which

caused the solution to turn bright red in color. The solution was

brought carefully into the glovebox and allowed to stir overnight. The

following day, 30 mL of methanol was added to the solution, which

was then stored at −33 °C in the glovebox freezer. After 2 days, the

reaction mixture was vacuum ﬁltered in the glovebox through a frit,

and the resulting dark-purple powder was washed with hexanes. Yield:

145 mg (0.208 mmol, 86%). UV−vis (CH₂Cl₂): 404, 476, 538, 612

color from brown-red to bright red. EPR and solution IR data were

recorded on the same sample to ensure full conversion and complete

nitrosylation of the iron complex. No NO gas was added to the gas

head space in these experiments. UV−vis (CH₂Cl₂): 370, 411, 550 nm.

¹H NMR (CD₂Cl₂, 400 MHz): δ = 7.25 (s); 7.46 (s); 7.72 (s); 7.88

(s). ¹⁹F{¹H} NMR (471 MHz): −125.83 (br, m). IR (CH₂Cl₂):

ν(NO) = 1850 cm⁻¹.

Addition of MI. Under an inert atmosphere, 6.6 mg of

[Fe(TPP)(NO)] (0.0094 mmol) was dissolved with ∼0.75 μL of

MI in 3 mL of dichloromethane. The solution was added to 5.7 mg of

solid [Fe(η⁵-C₅H₄COMe)₂][SbF₆] (0.0113 mmol) and agitated until

all of the solid had dissolved, forming [Fe(TPP)(NO)(MI)]SbF₆. The

solution changed color from brown-red to bright red. EPR and

solution IR data were recorded on the same sample to ensure full

conversion and complete nitrosylation of the iron complex. No NO

gas was added to the gas head space in these experiments. Note:

Samples of [Fe(TPP)(NO)(MI)]⁺generally contain a small amount

of [Fe(TPP)(NO)]SbF₆, which is evident from a small solution-IR

1

nm. H NMR (CD₂Cl₂, 400 MHz): δ = ∼ 6.0(br, β pyrrole H); 7.46

(s, para-H); 7.80 (br, ortho-H); 8.26 (s, meta-H). Peaks were assigned

based on a previous literature report.³¹Anal. Calcd for C₄₄H₂₈FeN₅O

with 1 molecule of methanol: C, 73.98; H, 4.41; N, 9.59. Found: C,

73.99; H, 4.17; N, 9.78. IR (KBr): ν(NO) = 1696 cm⁻¹.

Synthesis of [Fe(TPP)(NO)]SbF₆. A 65 mg (0.0720 mmol) portion

of [Fe(TPP)(SbF₆)] was dissolved in 4 mL of dichloromethane. Then,

the solution was exposed to NO gas, which caused the solution to turn

bright red in color. The solution was brought carefully into the

glovebox and was layered with 20 mL of hexanes and stored at −33 °C

in the glovebox freezer overnight. The next day, a purple solid was

ﬁltered oﬀ in the glovebox through a frit. The complex was stored in

the glovebox freezer. Yield: 59 mg (0.0631 mmol, 88%). UV−vis

1

band at 1850 cm⁻¹. UV−vis (CH₂Cl₂): 430, 544, 580 nm. H NMR

(CD₂Cl₂, 400 MHz): δ = 2.06 (s); 3.90 (s); 4.68 (s); 7.26 (s); 7.49

(s); 7.83 (s); 8.14 (s); 9.16 (s). IR (CH₂Cl₂): ν(NO) = 1920 cm⁻¹.

Crystallization of [Fe(TPP)(NO)(MI)]PO₂F₂. [Fe(TPP)(NO)(MI)]-

PF₆(10 mg) (see discussion below regarding the counterion) was

dissolved in 0.2 mL of NO-saturated dichloromethane and placed in a

5 mm diameter glass tube and sealed with a septa. The solution was

then carefully layered with 2 mL of NO-saturated hexanes and placed

in a −33 °C glovebox freezer. After 3 days, black needles suitable for

X-ray analysis were collected.

1

(CH₂Cl₂): 370, 411, 550 nm. H NMR (CD₂Cl₂, 500 MHz): δ =

7.70−7.876 (m). ¹⁹F{¹H} NMR (471 MHz): −126.99 (br, m). IR

(KBr): ν(NO) = 1850 cm⁻¹and ν(SbF₆) = 656 cm⁻¹.

Synthesis of [Fe(TPP)(NO)(MI)]SbF₆. A 63 mg (0.0697 mmol)

portion of [Fe(TPP)(SbF₆)] with 5.6 μL of MI (0.0662 mmol) added

was dissolved in 3 mL of dichloromethane. Then, the solution was

exposed to NO gas, which caused the solution to turn bright red in

color. The solution was brought carefully into the glovebox and was

layered with 16 mL of hexanes and stored at −33 °C in the glovebox

freezer overnight. The next day, a purple solid was ﬁltered oﬀ in the

glovebox through a frit. The complex was stored in the glovebox

freezer. Yield: 45 mg (0.0442 mmol, 64%). UV−vis (CH₂Cl₂): 430,

544, 580 nm. IR (KBr): ν(NO) = 1918 cm⁻¹and ν(SbF₆) = 658 cm⁻¹.

The corresponding PF₆⁻complex (used for crystallization below) was

prepared in a similar manner.

It should be noted that in the crystal structure of this complex, the

−

counterion is not the PF₆anion and was determined to be PO₂F₂₋

through a series of ³¹P- and ¹⁹F-NMR experiments. The PO₂F₂

counterion is formed during the methathesis reaction of the ferric

−

chloride complex, [Fe(TPP)(Cl)], with AgPF₆to form the ferric PF₆

complex and AgCl. Interestingly, the PF₆⁻anion further undergoes an

iron-catalyzed hydrolysis (in the presence of trace amounts of water),

−

resulting in a mixture of PF₆and PO₂F₂counterions. This reaction

has been noted in previous reports.^51,52Initially, we did not observe

−

the PO₂F₂counterion in the bulk material of [Fe(TPP)(PF₆)]

because it is bound to the iron center, and the paramagnetism of the

iron center masks the signals in the NMR spectra. To counteract this

problem, excess (>2 equiv) MI was added to the solution of the

Synthesis of [Fe(TPP)(NO)(Cl)]. A 104 mg (0.148 mmol) portion of

[Fe(TPP)(Cl)] was dissolved in 11 mL of dichloromethane. Then, the

solution was exposed to NO gas, which caused the solution to turn

brown-red in color. The solution was brought carefully into the

glovebox and was layered with 30 mL of hexanes and stored at −33 °C

in the glovebox freezer overnight. The next day, the resulting solid was

ﬁltered oﬀ in the glovebox through a frit. The complex was stored in

the glovebox freezer. Yield: 40 mg (0.0545 mmol, 37%). UV−vis

(CH₂Cl₂): 430, 544, 582 nm. IR (KBr): ν(NO) = 1880 cm⁻¹.

Synthesis of [Fe(TPP)(NO)(X)] using [TBA][X] (X = Cl⁻, Br⁻, I⁻).

[Fe(TPP)(SbF₆)] (61.3 mg, 0.068 mmol) was dissolved in 4 mL of

dichloromethane and then exposed to NO gas, causing the solution to

turn bright red in color. Then, the solution was carefully brought into

the glovebox, and 22 mg of tetrabutylammonium bromide (0.0682

mmol), dissolved in 0.3 mL of dichloromethane, was injected into the

solution, which was then allowed to stir for ∼30 min. Over time, the

solution turned more brown-red in color, and at that point, the

solution was layered with 15 mL of hexanes and left in the glovebox

freezer. Several hours later, the solution was vacuum ﬁltered to give a

purple solid coated in a white patina (the white patina is

[TBA][SbF₆]). IR (KBr): ν(NO) = 1870 cm⁻¹. The procedure with

tetrabutylammonium chloride (ν(NO) = 1880 cm⁻¹) and tetrabuty-

lammonium iodide was carried out in a similar manner. However, with

[TBA][I], the ferric NO complex is reduced by one electron, forming

[Fe(TPP)(NO)] (ν(NO) = 1696 cm⁻¹), I₂, and [TBA][SbF₆].

Typically, ∼1.0−1.3 equiv of the halide salt were used to do these

experiments.

−

iron(III)−PF₆complex to remove the PO₂F₂ligand from the iron

−

center (via formation of [Fe(TPP)(MI)₂]⁺and free PO₂F₂and

−

PF₆), which allowed us to observe the signals from both the PF₆⁻and

−

PO₂F₂counterions in the ¹⁹F- and ³¹P NMR spectra (see Figures

S4 −S5).

Crystallization of [Fe(TPP)(NO)(Cl)]. [Fe(TPP)(Cl)] (55.6 mg) was

dissolved in 6 mL of dichloromethane. Then, the solution was exposed

to NO gas and brought carefully into the glovebox. Next, ∼0.2 mL of

the solution was transferred to a 5 mm diameter glass tube and layered

with 2 mL of dimethoxyethane (DME) and placed in a −33 °C

glovebox freezer. After 4 days, brown prisms suitable for X-ray analysis

were collected.

Crystallization of [Fe(TPP)(NO)]BF₄. A portion of [Fe(TPP)(NO)]

(15.5 mg, 22.2 mmol) was dissolved in 3.5 mL of dichloromethane. To

this solution, 9.9 mg of solid [DAcFc][BF₄] (27.7 mmol) was added,

and the solution was agitated until the oxidant was completely

dissolved. In a 5 mm diameter glass tube, 0.2 mL of the oxidized

solution was carefully layered with 2 mL of hexanes and placed in a

−33 °C glovebox freezer. After 3 days, brown block-like crystals

suitable for X-ray analysis were collected. Excess NO gas was not

required.

RESULTS AND ANALYSIS

■

Preparation of {FeNO}⁶Complexes using NO Gas.

Using standard literature procedures,^16,17we reacted ferric

porphyrins with the TPP²⁻coligand, for example [Fe(TPP)-

(SbF₆)], with excess NO gas to form the corresponding ﬁve-

coordinate (5C) {FeNO}⁶complex, [Fe(TPP)(NO)]SbF₆. In

Chemical Oxidation of [Fe(TPP)(NO)]. Under an inert atmosphere,

6.6 mg of [Fe(TPP)(NO)] (0.0094 mmol) was dissolved in 3 mL of

dichloromethane. The solution was added to 5.7 mg of solid [Fe(η⁵-

C₅H₄COMe)₂][SbF₆] (0.0113 mmol) and agitated until all of the solid

had dissolved, forming [Fe(TPP)(NO)]SbF₆. The solution changed

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Figure 1. Left: Comparison of the UV−vis spectra of the precursor, [Fe(TPP)(SbF₆)] (black), the {FeNO}⁷complex [Fe(TPP)(NO)] (blue), and

the {FeNO}⁶complex [Fe(TPP)(NO)]SbF₆(purple, redissolved under non-NO-saturated conditions), isolated from the reaction of the iron(III)-

SbF₆complex with excess NO gas in dichloromethane. All spectra recorded at room temperature. Right: Overlay of the IR spectra of the precursor,

[Fe(TPP)(SbF₆)] (black), [Fe(TPP)(NO)] (blue), and [Fe(TPP)(NO)]SbF₆(purple) measured in KBr pellets.

the solid state, this compound has an N−O stretching

frequency of 1850 cm⁻¹as shown in Figure 1 (right), which

is consistent with the analogous OEP²⁻complex (N−O

stretching frequency is 1862 cm⁻¹).¹⁹The dissolution of the

solid {FeNO}⁶complex in dichloromethane (Figure 1, left)

results in a UV−vis spectrum with a split Soret band at 370 and

oxidation of this complex is chemically reversible. Additionally,

IR and UV−vis SpectroElectroChemical (SEC) studies of the

5C {FeNO}⁷complex showed clean formation of the oxidized

product, for example [Fe(TPP)(NO)]⁺, at least on the CV time

scale. In the presence of ∼1 equiv of N-donor ligands (such as

pyridine), [Fe(TPP)(NO)(Py)]⁺and analogous complexes

were formed. However, attempts to obtain {FeNO}⁶heme

complexes on the preparative scale by bulk electrolysis of the

{FeNO}⁷precursors resulted in direct decomposition, generat-

ing a ferric porphyrin complex with a weakly coordinating

anion (determined by UV−vis spectroscopy).⁵³In fact, there is

only one report in the literature (from 1995) that describes the

successful generation of {FeNO}⁶complexes via oxidation of

{FeNO}⁷precursors.⁵⁴Since these pioneering studies, there

have been no other attempts to synthesize {FeNO}⁶complexes

via one-electron oxidation of an {FeNO}⁷precursor. With this

in mind, we investigated the one-electron oxidation of the

{FeNO}⁷complex [Fe(TPP)(NO)], which itself was synthe-

sized by reductive nitrosylation of [Fe(TPP)(SbF₆)] in a

solution of methanol/dichloromethane that was exposed to

excess NO gas. This reaction generates [Fe(TPP)(NO)] in

pure form as determined by IR and ¹H NMR spectroscopy (see

Figure S2). Next, we measured the CV of the {FeNO}⁷

complex as shown in Figure 2. The CV shows the

{FeNO}^7/8reduction at an E_1/2value of −1.37 V vs Fc/Fc⁺

in CH₂Cl₂, which is chemically reversible and consistent with

the literature value of −1.42 V (vs Fc/Fc⁺in CH₂Cl₂).³⁰The

one-electron oxidation of the {FeNO}⁷complex is observed at

an E_1/2value of +0.302 V vs Fc/Fc⁺in dichloromethane

(+0.932 V vs NHE). The {FeNO}^7/6oxidation is also

chemically reversible, and our results are consistent with the

literature (E_1/2= +0.356 V vs Fc/Fc⁺in CH₂Cl₂).⁵³In order to

determine whether oxidation indeed produces the {FeNO}⁶

complex, [Fe(TPP)(NO)]⁺, we then carried out the corre-

sponding UV−vis and IR SEC studies on the {FeNO}⁷

complex. We can reversibly form the 5C {FeNO}⁶complex

by SEC, and the spectroscopic data are consistent with the

literature reports (see Figure S16 for UV−vis SEC and Figure

S17 for IR SEC) as well as the material that we obtained by

1

411 nm and a Q-band at 550 nm. The H NMR spectrum of

the redissolved solid contains a multiplet of peaks from 7.70−

7.88 ppm as shown in Figure S6, which is indicative of a

diamagnetic iron heme complex, demonstrating that the

complex is pure (NO loss results in a paramagnetic species).

However, monitoring the solution over time by in situ UV−vis

and solution IR experiments shows that the complex is not

stable and shows signs of decomposition into a ferrous heme-

nitrosyl complex after 2 h (Figure S24). The Fe−NO unit is

reduced by a single electron, and the source of the electron is

likely an impurity or potentially the solvent. Next, we

synthesized the six-coordinate (6C) ferric heme-nitrosyl

−

complex [Fe(TPP)(NO)(MI)]X (X = PF₆, SbF₆) and

characterized this complex by UV−vis and IR spectroscopy.

The UV−vis spectrum was obtained by reacting the ferric

precursor complex with excess NO gas in the presence of ∼1

equiv of MI, resulting in features at 430, 544, and 580 nm (see

Figure S7). The UV−vis spectrum of this {FeNO}⁶complex is

generally similar to that of [Fe(TpivPP)(NO)(NO₂)],

previously reported by Scheidt and co-workers.²⁵The IR of

our 6C complex in the solid state shows a very intense N−O

stretching frequency at 1918 cm⁻¹and no feature at 1676 cm⁻¹,

which would indicate the presence of an {FeNO}⁷impurity

(see Figure S8). The dissolution of this {FeNO}⁶complex in

dichloromethane (see Figure S7), monitored by UV−vis

spectroscopy, shows decomposition of the complex into a

ferric bis-imidazole complex, [Fe(TPP)(MI)₂]SbF₆, and the

ferric complex [Fe(TPP)(SbF₆)] via NO loss.

Preparation of {FeNO}⁶Complexes via {FeNO}⁷

Oxidation. To overcome the problems with autoreduction

and purity, we investigated an alternative route to synthesize

ferric NO complexes that was inspired by previous studies by

Kadish and co-workers.⁵³In their work, cylic voltammetry

(CV) experiments on iron−NO complexes, such as [Fe(TPP)-

(NO)], were performed, and it was found that the one-electron

−

reaction of [Fe(TPP)(X)] (X = PF₆, SbF₆) with NO gas (see

above and Table 1). In particular, the IR SEC data show the

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investigated whether the bulk generation of these complexes is

possible via one-electron chemical oxidation of the correspond-

ing {FeNO}⁷precursor. The UV−vis spectra in Figure 3 show

the titration of [Fe(TPP)(NO)] (in CH₂Cl₂solution) with the

chemical oxidant 1,1′-diacetylferrocenium hexaﬂuoroantimo-

nate (E_1/2= +0.49 V vs Fc/Fc⁺in CH₂Cl₂),⁵⁵abbreviated as

[DAcFc][SbF₆] (dissolved in dimethoxyethane (DME)), at

room temperature. The reaction is complete with ∼1 equiv of

the oxidant, and the UV−vis data of the product exhibit

features at 372, 404, and 550 nm that are identical to those of

the UV−vis SEC-generated species (see overlay in Figure 4).

The addition of excess ferrocene to the {FeNO}⁶solution

reduces the complex back to the starting ferrous NO complex,

as shown in the UV−vis spectra in Figure 4. The 5C {FeNO}⁶

complex can also be generated at higher concentrations (2−6

mM range) for characterization by solution IR spectroscopy.

For these experiments, the {FeNO}⁷complex, [Fe(TPP)-

(NO)], was dissolved in dichloromethane, and the resulting

solution was then added to a slight excess of the solid oxidant,

[DAcFc][SbF₆], causing the solution to change color from

orange-brown to bright red. The {FeNO}⁷precursor exhibits

an intense N−O stretching vibration in dichloromethane at

1676 cm⁻¹, which shifts to 1850 cm⁻¹(Δ = 174 cm⁻¹) upon

oxidation as shown in Figure 5. Intense C−O stretching bands

from the [DAcFc]^0/+oxidant are also observed in the IR

spectrum at 1672 and 1702 cm⁻¹, respectively. Finally, we

followed the conversion of the {FeNO}⁷precursor to the

{FeNO}⁶species by EPR spectroscopy. EPR spectra at 4 K

show that upon addition of the oxidant to the [Fe(TPP)(NO)]

solution, an EPR-silent species is generated with a very minor

high-spin ferric impurity that shows g values of ∼5.7 (<5% from

spin integration against [Fe(TPP)(Cl)]).

Figure 2. CV of ∼6.6 mM [Fe(TPP)(NO)] in CH₂Cl₂at room

temperature. The working electrode was a glassy carbon electrode, and

the counter electrode was a platinum electrode with an Ag wire

pseudoreference. Tetrabutylammonium hexaﬂuorophosphate was used

as the electrolyte (∼0.1 M).

N−O stretching vibration at 1676 cm⁻¹in the starting

{FeNO}⁷complex (in CH₂Cl₂), which decreases in intensity

upon oxidation as a new band at 1850 cm⁻¹appears.

Additionally, the UV−vis and IR SEC oxidations were

completed in the presence of ∼1 equiv of MI (as a model

for His in proteins). Whereas ferrous heme-NO complexes

have a strong σ trans eﬀect, leading to low binding constants of

N-donor ligands to 5C ferrous heme-nitrosyls,³¹the corre-

sponding ferric complexes do not show this eﬀect.¹⁴Hence, in

the SEC experiments, oxidation of the 5C {FeNO}⁷complex

generates the corresponding 6C complex [Fe(TPP)(NO)-

(MI)]⁺in the presence of MI (UV−vis and IR SEC data for

these transformations are shown in Figures S19,S20). In the IR

SEC experiments, the N−O stretching vibration of [Fe(TPP)-

(NO)] at 1676 cm⁻¹disappears upon oxidation as new bands

at 1920 and 1850 cm⁻¹appear (see Figure S20). The band at

1850 cm⁻¹corresponds to a small amount of the 5C {FeNO}⁶

complex, [Fe(TPP)(NO)]⁺.

We further reacted the 5C {FeNO}⁶complex with ∼1 equiv

of MI to form the 6C complex [Fe(TPP)(NO)(MI)]⁺. The

UV−vis spectrum of [Fe(TPP(NO)(MI)]⁺, shown in Figure

S27, has features at 430, 544, and 580 nm, which matches the

spectra of the complex made from iron(III)−X with excess NO

gas and 1 equiv of MI, and also the UV−vis SEC-generated data

(see Figure S28). On the basis of the UV−vis data shown in

Figure S27, the [Fe(TPP)(NO)(MI)]⁺complex can also be

formed by addition of ∼1 equiv of MI to the {FeNO}⁷solution,

followed by the addition of [DAcFc][SbF₆] dissolved in DME.

Figure 6 shows a comparison of the UV−vis spectra of all

Bulk Oxidation: A Better Way To Make Pure {FeNO}⁶

Complexes. With spectroscopic handles on 5C and 6C

{FeNO}⁶complexes with the TPP²⁻coligand established, we

a

Table 1. Comparison of Geometric Parameters for Selected {FeNO}⁶Complexes

b

c

complex

Fe−N_p

Fe−N(NO)

Fe−L

N−O

<Fe−N−O

ref

[Fe(TPP)(NO)]BF₄

1.986/1.990

1.994

1.640(3)/1.665(3)

1.644(3)

-

1.153(4)/1.124(4)

1.112(4)

1.148(5)

1.150(2)

0.925(6)

1.135(2)

1.141(3)

1.136(4)

1.209(8)

1.144(3)

1.151(8)

1.187(9)

178.3(3)/177.4(3)

176.9(3)

176.3 (4)

174.4(3)

177.1(7)

177.28(17)

176.9(3)

177.6(3)

180

t.w.

19

[Fe(OEP)(NO)]ClO₄

-

[Fe(TPP)(NO)(MI)]PO₂F₂

[Fe(TPP)(NO)(H₂O)]ClO₄

[Fe(TPP)(NO)(i-C₅H₁₁OH)]ClO₄

[Fe(OEP)(NO)(MI)]ClO₄

[Fe(OEP)(NO)(Pz)]ClO₄

[Fe(OEP)(NO)(Iz)]ClO₄

[Fe(TPP)(NO)(Cl)]

2.001

1.6275(3)

1.652(5)

1.973(3)

2.001(5)

2.063(3)

1.9889(16)

1.988(2)

2.010(3)

2.099(4)

1.998(2)

1.899(6)

2.356(3)

t.w.

19

1.999

2.013

1.776(5)

20

2.003

1.6465(17)

1.627(2)

17

2.004

17

1.996

1.632(3)

17

2.011

1.668(9)

t.w.

25

d

[Fe(TpivPP)(NO)(NO₂)]

1.996

1.671(2)

169.3(2)

175.8(6)

159.6(8)

[Fe(TPP)(NO)(CO₂CF₃)]

2.011

1.618(8)

28

e

[Fe(OEP)(NO)(SR-H₂)]

2.010

1.671(9)

68

a

Values for the bond distances given in Å and for the Fe−N−O angles given in degrees. The numbers given in parentheses are estimated standard

b

deviations. A complete table of bond lengths and angles for crystal structures obtained in this work is given in the Supporting Information. Average

c

d

e

value. All values are given in degrees. TpivPP = meso-tetra(α,α,α,α,o-pivalamidophenyl)porphyrin²⁻. SR-H₂= S-2,6-(CF₃CONH)₂C₆H₃.

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Figure 3. Left: UV−vis spectra following the titration of ∼10 μM [Fe(TPP)(NO)] (black) with the chemical oxidant [DAcFc][SbF₆] (dissolved in

dimethoxyethane) in dichloromethane, forming the ferric NO complex [Fe(TPP)(NO)]⁺(purple). Right: Changes of the absorption at 550 nm

upon addition of the oxidant, showing that the reaction is complete after the addition of one equiv of oxidant.

Figure 4. Left: UV−vis SEC used for the formation of [Fe(TPP)(NO)]⁺(black), compared to [Fe(TPP)(NO)]⁺(purple) generated by chemical

oxidation, both recorded in dichloromethane. These data show that identical species are formed by both methods. Right: UV−vis spectra of

[Fe(TPP)(NO)] before (black) and after addition of the chemical oxidant [DAcFc][SbF₆] (dissolved in dimethoxyethane) in dichloromethane,

leading to the formation of the ferric NO complex, [Fe(TPP)(NO)]⁺(purple). Subsequent reaction of the oxidized species with ferrocene reforms

the {FeNO}⁷precursor (blue).

relevant complexes. For the preparation of 2−6 mM solutions,

the {FeNO}⁷complex, [Fe(TPP)(NO)], was dissolved in

dichloromethane with ∼1 equiv of MI, and the resulting

solution was then added to a slight excess of the solid oxidant,

[DAcFc][SbF₆], causing the solution to change color from

orange-brown to deep red with a pink hue. The {FeNO}⁷

precursor exhibits an intense N−O stretching band in

dichloromethane at 1676 cm⁻¹, which shifts to 1920 cm⁻¹(Δ

= 244 cm⁻¹) upon oxidation in the presence of MI as shown in

Figure S29. The small band at 1850 cm⁻¹can be attributed to

the [Fe(TPP)(NO)]⁺complex. As previously mentioned,

intense C−O stretching bands from the [DAcFc]^0/+oxidant

are also observed in the IR spectrum at 1672 and 1702 cm⁻¹,

respectively. Lastly, the conversion to the {FeNO}⁶complex

was quantiﬁed by EPR spectroscopy. The oxidation product is

EPR silent with only a small (<5%) high-spin ferric heme

impurity (integrated against [Fe(TPP)(Cl)]).

chemical oxidation of iron(II)−NO precursors at room

temperature and characterized all products using diﬀerent

spectroscopic methods.

Stability of the {FeNO}⁶Complex. As mentioned above,

the generally used literature method to synthesize both 5C and

6C ferric NO model complexes frequently results in inherently

unstable species in solution that have a high propensity to lose

NO over time (in the absence of excess NO gas). This is

surprising, considering that analogous 6C ferric heme-NO

complexes in protein active sites (where the proximal ligand is a

histidine or cysteinate) are quite stable under NO-limited

conditions.¹²The instability of the {FeNO}⁶heme model

complexes makes studies of their fundamental properties and

reactivity diﬃcult in solution, since such studies require the

presence of excess NO gas, which by itself is very reactive and

can interfere with the desired reactivity studies. In contrast to

these previous observations, we noticed that the {FeNO}⁶

complexes, when generated via oxidation of the corresponding

{FeNO}⁷precursors, are surprisingly stable. To investigate this

In summary, we have established a convenient method to

make preparative amounts of ferric heme-nitrosyl complexes via

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Figure 5. Left: Solution IR spectra of 1.8 mM [Fe(TPP)(NO)] (black) in CH₂Cl₂and of the reaction product (purple) after the addition of ∼1.30

equiv of [DAcFc][SbF₆] added to the solution. The N−O stretch at 1676 cm⁻¹shifts to 1850 cm⁻¹upon oxidation. The bands at 1702 and 1672

cm⁻¹are the C−O stretching frequencies (ester groups) of the respective DAcFc^0/+reagents. Right: EPR spectra of the same solutions used for the

solution IR experiments (on the left) at 4 K. The black spectrum represents the {FeNO}⁷complex with a broad, isotropic S = 1/2 signal at g = 2.0,

and the purple spectrum represents the solution of the corresponding oxidized species, [Fe(TPP)(NO)]⁺, which is EPR silent and contains a minor

g = 6 signal that spin integrates to <5% of a ferric impurity. The sample was spin integrated against [Fe(TPP)(Cl)] using SpinCount.

cm⁻¹can be attributed to excess oxidant that decomposes over

time. Another possibility is that some amount of {FeNO}⁷

complex is formed over time. This complex shows the N−O

stretch at 1676 cm⁻¹, which could contribute to the intensity

increase of the 1672 cm⁻¹feature. In order to test this further,

EPR spectroscopy was used (Figure S23). In order to ensure

consistency, we used the same solutions for solution IR and

EPR spectroscopy (Figures S21,S23). At cryogenic temper-

atures of ∼4 K, the {FeNO}⁷precursor has a broad S = 1/2

signal with a g value centered at 2.0 (note: when the

temperature is raised to 77 K, the hyperﬁne couplings from

the ¹⁴N atom of NO appear; see Figure S18), and the addition

of a slight excess of [DAcFc][SbF₆] results in a completely

EPR-silent spectrum as shown in Figure S23. Over time (t > 15

h), a small signal around g = ∼ 6 appears in the EPR spectrum,

which is indicative of a hs ferric heme complex resulting from

NO loss. Spin integration of this signal against [Fe(TPP)(Cl)]

shows that it corresponds to <5% ferric complex. No formation

of the {FeNO}⁷complex is observed. In conclusion, the 5C

{FeNO}⁶complex is stable over extended periods of time in

solution as shown by UV−vis, solution IR, and EPR

spectroscopy. Analysis of the UV−vis data in Figure S33

estimates the k_offrate of NO for [Fe(TPP)(NO)]⁺to be ∼4.7

× 10⁻⁵s⁻¹. For the six-coordinate adduct, [Fe(TPP)(NO)-

(MI)]⁺, we monitored the stability of the complex by solution

IR experiments as shown in Figure S34. Over ∼6 h, a decrease

in the intensity of NO band of the complex at 1920 cm⁻¹is

observed, and an increase in the band at 1850 cm⁻¹from the

ﬁve-coordinate complex, [Fe(TPP)(NO)]⁺, is noted. The

imidazole complex, [Fe(TPP)(MI)]⁺, has an extremely high

binding aﬃnity for another MI ligand, resulting in the bis-

imdazole complex, [Fe(TPP)(MI)₂]⁺.⁵⁶This corresponds to

the reaction sequence in eqs 1,2. Analysis of the UV−vis data

after the addition of MI to a solution of [Fe(TPP)(NO)]⁺

results in a signiﬁcantly larger k_offof ∼1.9 × 10⁻⁴s⁻¹(Figure

S35) than that of [Fe(TPP)(NO)]⁺(Figure S33). The

observed rate constant for [Fe(TPP)(NO)(MI)]⁺compares

favorably with ferric heme NO complexes in protein environ-

ments, which have k_offrate constants that range from ∼10¹−

10⁻²s⁻¹(this includes Mb/Hb, NPs, HRP, NOS, and Cyt.

Figure 6. Comparison of the UV−vis spectra of the ﬁve-coordinate

{FeNO}⁶complex [Fe(TPP)(NO)]⁺(green), the six-coordinate

complex [Fe(TPP)(NO)(MI)]⁺(purple), and the [Fe(TPP)(SbF₆)]

precursor (black) in dichloromethane. All spectra recorded at room

temperature.

further, we took aliquots from the oxidation-reaction solutions

and monitored the changes in the IR spectra over a ∼24 h time

period at room temperature. After a few hours, the N−O

stretching band at 1850 cm⁻¹of the 5C complex completely

overlaid with the initial IR spectrum, indicating minimal NO

loss (see Figure S21). After >15 h, the 1850 cm⁻¹band starts to

slowly decrease in intensity; however, this change is very small,

indicating that the {FeNO}⁶complex is quite stable in solution

in the absence of excess NO. At the same time, the intensity of

the C−O stretching band of reduced DAcFc at 1672 cm⁻¹

increases notably. Since excess [DAcFc][SbF₆] oxidant is

present in solution, we tested whether [DAcFc][SbF₆] is stable

in DME (the oxidant is not soluble enough in dichloromethane

for solution IR measurements). The IR spectra in Figure S22

show that the oxidant [DAcFc][SbF₆] decomposes within 2 h

to form DAcFc and other decomposition products. Therefore,

we believe that the increasing intensity of the band at 1672

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Figure 7. Top: Crystal structures of the two diﬀerent 5C {FeNO}⁶complexes in the unit cell of [Fe(TPP)(NO)]BF₄. Bottom: Crystal structure of

[Fe(TPP)(NO)(MI)]PO₂F₂. In all structures, the hydrogen atoms, solvent molecules, and counterions are omitted for clarity. Thermal ellipsoids are

shown at 40% probability.

P450s; Mb denotes myoglobin, Hb denotes hemoglobin, HRP

denotes horseradish peroxidase, and NPs denotes nitro-

phorins).¹²In contrast, previous work with other model

complexes shows much faster k_offrates of ∼10¹−10³s⁻¹(see,

for example, ref 57).

0.10 Å.⁷For example, the completely planar complex

[Fe(TMP)(MI)₂]ClO₄(TMP²⁻= tetramesitylporphyrin) has

a 25-atom core displacement of 0.02 Å,⁵⁸which is 15 times

smaller than that of [Fe(TPP)(NO)]BF₄. The RMSD for the 4-

atom meso carbon displacement of [Fe(TPP)(NO)]BF₄is

0.077 Å. In addition, we crystallized the analogous {FeNO}⁶

complex with MI bound to the iron center. The bulk material,

[Fe(TPP)(NO)(MI)]PF₆, was prepared by reaction of the

ferric precursor with excess NO gas in dichloromethane. The

dissolution of [Fe(TPP)(NO)(MI)]PF₆in dichloromethane,

layered with hexanes under an NO atmosphere, at −33 °C,

results in needle-like crystals. The crystal structure of this

complex is shown in Figure 7. It should be noted that the

k₁

[Fe(TPP)(NO)(MI)]⁺→ [Fe(TPP)(MI)]⁺+ NO

(1)

[Fe(TPP)(MI)]⁺+ [Fe(TPP)(NO)(MI)]⁺

k₂

→ [Fe(TPP)(MI)₂]⁺+ [Fe(TPP)(NO)]⁺

(2)

Crystallographic Studies. With the long-term stability of

[Fe(TPP)(NO)]⁺in solution established, we prepared the 5C

{FeNO}⁶complex as described above from the chemical

oxidant, [DAcFc][BF₄] and then layered the solution with

hexanes and placed it in the −33 °C freezer to obtain X-ray

quality crystals (in the absence of excess NO gas). The crystal

structure of [Fe(TPP)(NO)]BF₄is shown in Figure 7. In the

unit cell, there are two unique {FeNO}⁶complexes with slightly

diﬀerent geometric parameters (see Table 1). The crystal

structure of the complex exhibits a linear Fe−N−O unit (Fe−

N−O angle: 177.4(3)/178.3(3)^o) and Fe−NO bond lengths of

1.640/1.665 Å. These bond lengths are in agreement with the

crystal structure of [Fe(OEP)(NO)]ClO₄, which exhibits an

Fe−NO bond length of 1.644 Å.¹⁹Interestingly, the porphyrin

is completely planar in the crystal structure of the 5C {FeNO}⁶

complex with the OEP²⁻coligand, but the heme plane is ruﬄed

in our crystal structure of [Fe(TPP)(NO)]BF₄. A ruﬄing

distortion is characterized by the rotation of trans pyrrole rings

in the opposite direction around the Fe−N_pyrrolebonds of the

porphyrin coligand. This distortion is commonly observed in

5C ferric porphyrins because the iron sits above the heme

plane. This out-of-plane distortion is measured by the RMSD of

the porphyrin atoms from the heme plane. The 25-atom core

displacement of [Fe(TPP)(NO)]BF₄is 0.31 Å, whereas planar

hemes are deﬁned by a 25-atom core displacement of less than

−

counterion in this structure is PO₂F₂as further discussed in

the Experimental Procedures. This is the ﬁrst crystal structure

of a 6C ferric heme-nitrosyl complex with an N-donor ligand

using TPP²⁻as the porphyrin. In the crystal structure of

[Fe(TPP)(NO)(MI)]PO₂F₂, the porphyrin coligand is com-

pletely planar, unlike the ruﬄed conformation of the heme in

the 5C complex [Fe(TPP)(NO)]BF₄. The complex exhibits a

linear Fe−N−O unit (Fe−N−O angle: 176.3(4)^o), and the

Fe−NO bond length is 1.628 Å, which is similar to other 6C

ferric heme-nitrosyl complexes with neutral N-donor ligands

that exhibit Fe−NO bond lengths ranging from 1.627−1.647 Å

(Table 1).¹⁷It should be noted that this {FeNO}⁶complex has

a Fe−N_MIbond length of 1.973 Å, which is slightly shorter than

those in other neutral N-donor coordinated {FeNO}⁶

complexes with the OEP²⁻coligand, which show Fe−N bond

lengths ranging from 1.996−2.003 Å as shown in Table 1.

Finally, the average Fe−N_pyrrolebond distance is 2.001 Å, similar

to other 6C ferric heme-nitrosyl complexes.¹⁷

Role of Halides for Stability. With a stable 5C {FeNO}⁶

complex in hand, we further investigated the stability of this

complex in the presence of halides. First, we generated the

{FeNO}⁶complex via chemical oxidation of the {FeNO}⁷

precursor with [DAFc][SbF₆], and then reacted it with ∼1

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Figure 8. Left: UV−vis spectra of the ferric precursor, [Fe(TPP)(Cl)] (black) and of the corresponding reaction product after bubbling the solution

with NO(g) to form [Fe(TPP)(NO)(Cl)] (red), in CH₂Cl₂. Right: The reaction product after ﬂushing the solution with argon gas (green), which

results in reformation of the starting material [Fe(TPP)(Cl)] (black).

equiv of tetrabutylammonium chloride (TBACl) and moni-

tored the reaction using in situ UV−vis spectroscopy. Addition

of [TBA][Cl] to the {FeNO}⁶solution results in immediate

formation of the ferric chloride complex [Fe(TPP)(Cl)] and

NO release as shown in Figure S9. The same result is also

obtained when [Fe(TPP)(NO)]⁺is reacted with [TBA][Br]

forming a ferric bromide complex (Figure S9). This is

consistent with previous work by Wayland and co-workers

and shows that the {FeNO}⁶complex, [Fe(TPP)(NO)(Cl)], is

not stable under non-NO-saturated conditions. In this work, it

was observed that SEC-generated [Fe(Porph)(NO)(X)]

complexes (where X = Cl⁻, Br⁻, and I⁻) were unstable and

decomposed into the corresponding [Fe(Porph)(X)] com-

plexes.⁵²However, at low temperature (−70 °C), the addition

of [TBA][Br] to the {FeNO}⁶solution results in the formation

of a new species with a Soret band at 435 nm and the main Q-

band at 550 nm (see Figure S11, top). This species has features

that are consistent with a six-coordinate {FeNO}⁶complex.

When the solution is warmed to room temperature, this species

rapidly decomposes into [Fe(TPP)(Br)] within 5 min as

shown in Figure S11 (bottom).

co-workers carried out the IR SEC oxidation of [Fe(TPP)-

(NO)] in the presence of [TBA][Cl], [TBA][Br], and

[TBA][I], resulting in the potential formation of the

corresponding {FeNO}⁶complexes [Fe(TPP)(NO)(X)]

where X = Cl⁻, Br⁻, and I⁻. The N−O stretching frequencies

of these complexes were reported to occur at 1886, 1883, and

1879 cm⁻¹, respectively.⁵³We generated [Fe(TPP)(NO)(Cl)]

by reacting the ferric chloride complex with excess NO gas.

Under an NO atmosphere, hexanes were added to precipitate

the target complex. Characterization of the isolated complex in

the solid state by IR spectroscopy shows an N−O stretching

frequency of 1880 cm⁻¹(Figure S12, left). This ferric chloride

NO complex shows minimal NO loss over a 3 month time

period in the solid state when stored in the glovebox freezer at

−33 °C (Figure S12, right). The dissolution of the NO

complex results in immediate NO loss from the iron center and

reformation of the ferric chloride precursor. Due to the facile

NO loss from the iron center, the ferric chloride complex,

[Fe(TPP)(Cl)], was reacted with excess NO gas in dichloro-

methane directly in a Schlenk cuvette in order to obtain the

UV−vis spectrum of [Fe(TPP)(NO)(Cl)]. The resulting

spectrum shows features at 430, 544, and 582 nm (Figure 8)

that are similar to the spectral features observed for the 6C

{FeNO}⁶complex [Fe(TPP)(NO)(MI)]⁺(see Figure 6).

Upon ﬂushing the cuvette with argon gas, the ferric chloride

precursor is reformed, indicating again that NO binding is

reversible (Figure 8, right). In addition, under an NO-rich

environment, we were able to grow crystals of [Fe(TPP)-

(NO)(Cl)] and solve the crystal structure of this complex,

which is displayed in Figure 9. It should be noted that the

crystal structure is disordered with respect to the NO and Cl⁻

ligands, which makes the heme plane appear atypically planar.

The crystal structure of this complex exhibits a linear Fe−N−O

unit (Fe−N−O angle: 180°), an Fe−NO bond length of

1.6685 Å, and an Fe−Cl distance of 2.013 Å (the Fe−Cl bond

length in [Fe(TPP)(Cl)] is 2.192 Å).⁶⁰

Quest for the Elusive Complex [Fe(TPP)(Cl)(NO)]The

Critical Intermediate in Reductive Nitrosylation. Ferrous

heme-nitrosyl complexes, {FeNO}⁷, are typically synthesized by

reductive nitrosylation as mentioned in the Introduction. In

reductive nitrosylation, a ferric precursor complex, [Fe(Porph)-

(X)] (where Porph²⁻= a porphyrin²⁻coligand; X⁻

=

−

monoanionic ligand: halide, PF₆, SbF₆, ClO₄, etc.), is

typically reacted with excess NO gas in the presence of a base

(such as methanol).⁵⁹In the ﬁrst step of the proposed

mechanism, a ferric chloride complex, for example, binds NO to

the iron center to form a 6C ferric heme-nitrosyl complex,

[Fe(Porph)(NO)(Cl)], which then reacts with the base to

form HCl, RNO₂, and a ferrous porphyrin complex (see

Scheme 1). The ferrous heme then binds another equivalent of

NO to generate the respective {FeNO}⁷complex.¹²The key

intermediate for this process, [Fe(Porph)(NO)(Cl)], has so far

only been poorly characterized in the literature. In a previous

report from over 30 years ago, Wayland and co-workers

describe the reaction of [Fe(TPP)(Cl)] with excess NO gas to

form [Fe(TPP)(NO)(Cl)], monitored by in situ UV−vis

spectroscopy. The isolated reaction product was characterized

in the solid state by IR spectroscopy.⁴¹Additionally, Kadish and

Finally, we reacted the 5C {FeNO}⁶complex with other

halides to form the corresponding halide-bound complexes,

[Fe(TPP)(NO)(X)], X = Br⁻, I⁻under an NO-saturated

atmosphere. The reaction of [Fe(TPP)(NO)]⁺with [TBA]-

[Br] results in the corresponding bromide-coordinated NO

complex, [Fe(TPP)(NO)(Br)], and [TBA][SbF₆]. The N−O

stretching frequency of [Fe(TPP)(NO)(Br)] is 1870 cm⁻¹as

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Figure 9. Crystal structure of [Fe(TPP)(NO)(Cl)]. The hydrogen

atoms are omitted for clarity, and thermal ellipsoids are shown at 40%

probability.

shown in Figure S11. Interestingly, in our hands, the reaction of

[Fe(TPP)(NO)]⁺with [TBA][I] results in reduction of the

Fe−N−O unit to form the ferrous-heme nitrosyl complex,

[Fe(TPP)(NO)] (N−O stretching frequency = 1696 cm⁻¹), I₂,

and [TBA][SbF₆] (see Figure S14). Furthermore, when a

solution of [Fe(TPP)(NO)]⁺in dichloromethane in the

absence of NO gas is reacted with [TBA][I], there is immediate

formation of the [Fe(TPP)(NO)] complex as shown in Figure

S15, indicating that iodide reduces the {FeNO}⁶complex in an

outer sphere process (since no NO loss is observed).

Nuclear Resonance Vibrational Spectroscopy (NRVS).

To gain further insight into the electronic properties of our new

{FeNO}⁶complexes, we measured their Fe−NO stretching

frequencies applying NRVS. In correlation to the N−O

stretching frequencies, this provides key insight into changes

in Fe−NO bonding along a series of complexes as we and

others have previously shown.^{16,31,61−64}In linear {FeNO}⁶

complexes, the Fe−N−O unit gives rise to one Fe−NO stretch

and two degenerate Fe−N−O linear bends, which, in the case

of corresponding six-coordinate complexes, are very similar in

energy.^16,24This complicates spectral assignments.

Figure 10. Top: NRVS-derived vibrational density of states (VDOS)

for [⁵⁷Fe(TPP)(NO)(Cl)] (black) and the ¹⁵N¹⁸O-labeled complex

(green). Middle: NRVS VDOS for [⁵⁷Fe(TPP)(NO)(MI)]SbF₆

(black) and the ¹⁵N¹⁸O-labeled complex (purple). Bottom: NRVS

VDOS for [⁵⁷Fe(TPP)(NO)]SbF₆(black) and the ¹⁵N¹⁸O-labeled

complex (blue).

dichloromethane in the presence of ∼1 equiv of MI and was

exposed to NO gas. The NO-saturated solution was then

transferred to a quartz EPR tube. In the rRaman spectrum

shown in Figure S36, the oxidation-state marker band (ν₄) is

observed at 1370 cm⁻¹, and the spin-state marker band (ν₂) is

found at 1569 cm⁻¹, both consistent with other ferric heme-NO

complexes.^65,66Interestingly, the ν₄and ν₂bands are identical

to the {FeNO}⁷complex [Fe(TPP)(NO)] (ν₄= 1370, ν₂=

1568 cm⁻¹), indicating that the heme is in the low-spin ferrous

state.³¹In the low-energy region of the spectrum, the natural

abundance isotopes (n.a.i.) complex exhibits a broad band at

∼598 cm⁻¹(see Figure S37, top) that downshifts to 586 cm⁻¹

(Δ = 12 cm⁻¹) upon isotope labeling with ¹⁵N¹⁸O gas (Figure

S37, bottom). In contrast to our complex, six-coordinate ferric

heme-NO adducts in proteins (such as myoglobin) show

resolved Fe−NO stretching and bending modes (measured via

rRaman; see Table 2).⁶³

The NRVS data of the 5C {FeNO}⁶complex, [⁵⁷Fe(TPP)-

(NO)]SbF₆, show the Fe−N−O bends at 393 cm⁻¹, which shift

to 386 cm⁻¹upon ¹⁵N¹⁸O labeling (Δ = 7 cm⁻¹). This ﬁnding

is fully consistent with the results for [Fe(OEP)(NO)]ClO₄,

where the linear Fe−N−O bends are found at 402 cm⁻¹. At

higher energy, there are two peaks observed at 559 and 585

cm⁻¹that are isotope sensitive. As shown in Figure 10, the

spectrum of the ¹⁵N¹⁸O complex exhibits a signiﬁcant decrease

in the intensity of the peak at 585 cm⁻¹, while at the same time,

the peak at 559 cm⁻¹increases in intensity. This implies that

the Fe−NO stretch is located at 585 cm⁻¹and coupled with a

porphyrin-based vibration around 559 cm⁻¹. This is consistent

with work by Scheidt and co-workers on [Fe(OEP)(NO)]ClO₄

where the Fe−NO stretch is observed at 595 cm⁻¹. This would

also explain the very large (apparent) isotope shift (Δ = 26

cm⁻¹) observed for this mode.

We also obtained NRVS data for the 6C complex,

[⁵⁷Fe(TPP)(NO)(MI)]SbF₆, which show one isotope-sensitive

feature at 590 cm⁻¹that downshifts to 573 cm⁻¹upon ¹⁵N¹⁸O

labeling (Δ = 17 cm⁻¹). This complex exhibits a much sharper

Fe−NO band in the NRVS spectrum than that of [Fe(TPP)-

(NO)(MI)]BF₄, as reported by us previously,¹⁶but again, the

Fe−NO stretch and the Fe−N−O bends are not resolved (see

ref 24). In the 6C case, we measured the resonance Raman

(rR) spectrum of the [Fe(TPP)(NO)(MI)]SbF₆complex. For

this purpose, the precursor [Fe(TPP)(SbF₆)] was dissolved in

Finally, in the [⁵⁷Fe(TPP)(NO)(Cl)] complex, the IR

spectrum of the n.a.i. species show two isotope-sensitive

features, the main band at 1880 cm⁻¹and a minor feature at

1829 cm⁻¹(see Figure S40). Isotope labeling with ¹⁵N¹⁸O

results in a downshift of the N−O stretching frequency to 1802

cm⁻¹(Δ = 78 cm⁻¹), whereas the 1829 cm⁻¹feature actually

shifts to higher energy at 1844 cm⁻¹(Δ = 15 cm⁻¹). We believe

this is due to Fermi resonance, since the 1829/1844 cm⁻¹band

is lower in energy than the N−O stretch in the n.a.i. complex,

but higher in energy than the N−O stretch in the ¹⁵N¹⁸O-

labeled complex. The NRVS spectrum contains one isotope-

sensitive band at 563 cm⁻¹, which shifts to 556 cm⁻¹upon

¹⁵N¹⁸O labeling (Δ = 7 cm⁻¹). There is another band at 539

cm⁻¹in the n.a.i. complex that signiﬁcantly increases in

intensity in the ¹⁵N¹⁸O-labeled complex, indicating that the

Fe−NO stretch is coupled to another vibration that is close in

energy. We believe that this feature at 539 cm⁻¹is a Fe-

porphyrin-related vibration that steals some intensity from the

Fe−NO stretch. In summary, we can resolve the Fe−NO

stretch from the linear Fe−N−O bends in the 5C complex,

[Fe(TPP)(NO)]SbF₆; however, both 6C {FeNO}⁶complexes

only show one combined band for these features.

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complexes, immediate denitrosylation of the complexes is

observed. With respect to R. proxlixus nitrophorins, this

indicates that a special stabilization of the {FeNO}⁶complexes

by the protein matrix may not be required as previously

proposed,⁷in particular since the saliva of R. proxlixus contains

excess NO. Once the nitrophorins are injected into the victim’s

blood, the conformational changes of the protein (triggered by

the change in pH) and the presence of histamine that can bind

in the active site might actually be required to induce eﬃcient

NO loss.

Table 2. Comparison of Vibrational Frequencies for Selected

{FeNO}⁶Complexes

v(N−O)

ν/δ(Fe−NO)

complex

cm⁻¹

ref

[Fe(TPP)(NO)]SbF₆

1850

1862

1920

1896

1862

1935

δ: 393, ν: 585

t.w.

19

[Fe(OEP)(NO)]ClO₄

δ: 402, ν: 595

[Fe(TPP)(NO)(MI)]SbF₆

[Fe(TPP)(NO)(MI)]BF₄

[Fe(TPP)(NO)(H₂O)]ClO₄

589

t.w.

16

δ: 586, ν: 578

-

19

[Fe(TPP)(NO)(i-C₅H₁₁OH)]

ClO₄

20

We further characterized our series of {FeNO}⁶complexes

via UV−vis, IR, NMR, and NRVS. The {FeNO}⁶complexes

obtained in this way have identical spectroscopic features to

those prepared via the traditional method shown in Scheme 1.

The complexes are also more stable, which is likely due to less

impurities in the precursor complex, especially halides. We

avoid halide impurities by synthesizing a pure form of the

{FeNO}⁷complex via a reductive nitrosylation reaction from

an iron(III)−X precursor (where X⁻is a weakly coordinating

anion). This approach allows us to handle {FeNO}⁶complexes

in the absence of any excess NO gas, enabling reactivity studies

with reagents that are sensitive to NO gas (for example thiols).

Next, we determined that [Fe(TPP)(NO)(Cl)] can be made

and isolated as a solid material. This complex represents the

proposed key intermediate in the reductive nitrosylation of

[Fe(Porph)(Cl)] and related ferric precursors (see Scheme 2),

which is the most widespread method to prepare ferrous heme-

nitrosyl complexes. Yet, not much is known about the

properties of these types of complexes. [Fe(TPP)(NO)(Cl)]

reversibly binds NO, but quickly loses NO upon dissolution of

the isolated material or removal of excess NO gas from the

reaction vessel. We can also generate the halide-bound

{FeNO}⁶complexes by addition of [TBA][X] (where X =

Cl⁻or Br⁻) to a solution of [Fe(TPP)(NO)]⁺under an NO

atmosphere. However, the reaction of [TBA][I] with [Fe-

(TPP)(NO)]⁺results in one-electron reduction of the Fe−NO

unit to generate the ferrous heme-nitrosyl complex, [Fe(TPP)-

(NO)]. This ﬁnding is not in agreement with a previous

literature report that presents the IR SEC oxidation of

[Fe(TPP)(NO)] in the presence of [TBA][I], which was

thought to result in the generation of [Fe(TPP)(NO)(I)] with

an N−O stretching frequency of 1879 cm⁻¹.⁵³To further

elucidate this issue, we can examine the reduction potentials of

the reported species in CH₃CN: for redox couples Cl⁻/Cl₂,

Br⁻/Br₂, and I⁻/I₂, redox potentials of +0.18, +0.07, and −0.14

V (vs Fc/Fc⁺), respectively,⁵⁵have been determined. Given the

E_1/2value of +0.302 V (vs Fc/Fc⁺) for the {FeNO}⁶complex

(in dichloromethane), the oxidation potential of I⁻to I₂is

roughly ∼440 mV more negative than the [Fe(TPP)(NO)]/

[Fe(TPP)(NO)]⁺couple, which means the complex will easily

oxidize I⁻to I₂. This is also consistent with the fact that the

ferric complex FeI₃does not exist (due to conversion to FeI₂+

I₂). We speculate that the putative complex “[Fe(TPP)(NO)-

(I)]” reported previously is likely the nitro−nitrosyl complex

[Fe(TPP)(NO)(NO₂)], which readily forms in the presence of

O₂, is quite stable, and shows a similar N−O stretch of about

1875−1880 cm⁻¹.¹⁶

[Fe(OEP)(NO)(MI)]ClO₄

[Fe(OEP)(NO)(2-MI)]ClO₄

1921

N/A

-

17

δ: 574, 580 ν:

600

[Fe(OEP)(NO)(Pz)]ClO₄

[Fe(OEP)(NO)(Iz)]ClO₄

Mb(III)−NO

Hb(III)−NO

rNP1(III)−NO

1909

1914

1927

1925

1917

1880

1870

1874

-

17

-

δ: 572, ν: 595

65, 74, 75

65, 76, 77

66, 78

t.w.

594

δ: 578, ν: 591

[Fe(TPP)(NO)(Cl)]

563

[Fe(TPP)(NO)(Br)]

-

t.w.

[Fe(TpivPP)(NO)(NO₂)]

ClO₄

25

[Fe(TPP)(NO)(CO₂CF₃)]

1907

1850

1828

1837

1851

1806

1818

-

28

67, 68

12

a

[Fe(OEP)(NO)(SR-H₂)]

549

510

515

530

528

522

524

b

[Fe(SPorph)(NO)]

c

[Fe(SPoprh-HB)(NO)]

12

P450nor(III)−NO

P450cam(III)−NO

P450cam(III)−NO + camphor

71

70, 71

71, 72

P450cam(III)−NO +

norcamphor

P450cam + adamantanone

1818

1868

520

538

71, 72

71, 73

d

CPO(III)−NO

a

b

SR−H₂= S-2,6-(CF₃CONH)₂C₆H₃; SPorph = meso-a,a,a,a-[o-

[[(acetylthio)methyl]phenoxy]acetamido]phenyl]tris(o-

c

pivalamidophenyl)porphyrin^2‑SPorph-HB = SPorph with proposed

d

hydrogen bonding; CPO denotes chloroperoxidase.

DISCUSSION

■

In this Article, we describe a convenient route to synthesizing

ferric heme-nitrosyl complexes (without anionic axial ligands)

in bulk by chemical and electrochemical oxidation of {FeNO}⁷

precursors in the absence of NO gas, and demonstrate this

approach for complexes with the TPP²⁻ligand. When

generated in this way, the 5C {FeNO}⁶complex is solution

stable and slowly loses NO over time. The k_offrate is ∼4.7 ×

10⁻⁵s⁻¹, which means that the half-life is ∼4 h (see Figure

S33). The addition of ∼1 equiv of MI (1-methylimidazole) to

the solution results in the formation of the 6C complex

[Fe(TPP)(NO)(MI)]⁺, which is also solution stable, but shows

a signiﬁcantly enhanced rate for NO loss (k_off= ∼ 1..9 × 10⁻⁴

s⁻¹with a half-life of ∼1 h; see Figure S35). These rate

constants for NO loss compare favorably with the values

derived for proteins, which fall in the range of 10¹−10⁻²s⁻¹, but

are much smaller than typical values reported for model

complexes, 10¹−10³s⁻¹.¹²This implies that it is not necessarily

the protein matrix that is responsible for the enhanced stability

of {FeNO}⁶complexes in proteins but on the contrary, that

there could also be factors that reduce the stability of these

complexes in model systems. Our results show that one such

source of instability are halide impurities: upon addition of ∼1

equiv of a chloride or bromide source to our {FeNO}⁶

We further obtained the crystal structures for our new series

of {FeNO}⁶complexes. The 5C complex, [Fe(TPP)(NO)]BF₄,

has a linear Fe−N−O unit (Fe−N−O angle: 177.4(3)/

178.3(4)^o) and a similar Fe−NO bond length of 1.640/1.665

Å compared to that of the [Fe(OEP)(NO)]ClO₄complex (see

Table 1). The only notable diﬀerence between these complexes

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is that our 5C {FeNO}⁶complex has a slightly lower N−O

stretching frequency of 1850 cm⁻¹in comparison to the OEP²⁻

analogue at 1862 cm⁻¹(see Table 2).¹⁹The Fe−NO stretching

and bending vibrations are observed at 585 and 393 cm⁻¹,

which are slightly lower in energy than those of the

[Fe(OEP)(NO)]ClO₄complex at 595 and 402 cm⁻¹,

respectively (see Table 2).¹⁹This indicates that the Fe(TPP)

fragment is less electron-rich compared to Fe(OEP), leading to

a weaker Fe−NO π backbond in the former, and correspond-

ingly, a lower Fe−NO stretching frequency. Ferric heme-

nitrosyl complexes with axial neutral N-donors (ie. imidazole,

pyridine, and pyrazole) also have linear Fe−N−O units and

N−O and Fe−NO stretching frequencies in the 1890−1935

cm⁻¹and 580−600 cm⁻¹range.¹¹Our 6C complex [Fe(TPP)-

(NO)(MI)]⁺exhibits a linear Fe−N−O moiety (Fe−N−O

angle: 176.3(4)^o; see Table 1) with N−O and Fe−NO

stretching-frequency values of 1920 and ∼589 cm⁻¹(see

Table 2). Interestingly, ferric heme-nitrosyl species with

histidine ligation, such as myoglobin/hemoglobin and nitro-

phorins, have N−O (1917−1927 cm⁻¹) and Fe−NO stretching

frequencies (591−595 cm⁻¹) similar to those of our 6C N-

donor {FeNO}⁶complex (see Table 2). We have previously

Figure 11. Experimental vibrational stretching frequency correlation of

the Fe−NO versus the N−O stretching frequency based on this work

and published data. 5C {FeNO}⁶complexes [Fe(TPP)(NO)]BF₄and

[Fe(OEP)(NO)]ClO₄are depicted in blue. 6C {FeNO}⁶complexes

with an axial thiolate ligand trans to NO are shown in purple: Cyt.

P450cam^70,71(+camphor, norcamphor, and adamantanone),^71,72Cyt.

P450nor,⁷¹CPO,^71,73and [Fe(OEP)(NO)(SR-H₂)]ClO₄.^67,68[Fe-

(TPP)(NO)(Cl)] is shown in black and 6C {FeNO}⁶complexes with

an axial histidine (Mb,^65,74,75Hb,^65,76,77and rNP^66,78) or MI ligand

trans to NO are shown in red. MI denotes 1-methylimidazole, SR-H₂

denotes the S-2,6-(CF₃CONH)₂C₆H₃ligand, CPO denotes chlor-

operoxidase, Mb denotes myoglobin, Hb denotes hemoglobin, and

rNP denotes nitrophorins from Rhodnius prolixus.

investigated the complex [⁵⁷Fe(TPP)(NO)(MI)]BF₄with BF₄

−

as the counterion (compared to SbF₆⁻used here for NRVS). In

this case, the NRVS data exhibit a broad, ¹⁵N¹⁸O-sensitive

feature at 578/586 cm⁻¹.¹⁶In conjunction with normal

coordinate analysis (NCA) and density functional theory

(DFT) calculations, we assigned the Fe−NO stretch to the

578 cm⁻¹band and the Fe−N−O linear bends to the feature at

586 cm⁻¹. Further work by Scheidt and co-workers utilized

oriented single-crystal NRVS to provide deﬁnitive assignments

of the stretching and bending modes of the Fe−N−O moiety in

the ⁵⁷Fe-labeled complexes [Fe(OEP)(NO)]ClO₄and [Fe-

(OEP)(MO)(2-MI)]ClO₄(2-MI = 2-methylimidazole).²⁴

These data show the Fe−NO stretch at higher energy than

the Fe−N−O linear bends. This implies that for [Fe(TPP)-

(NO)(MI)]⁺, the Fe−NO stretch should likely be identiﬁed

with the mode at 586 cm⁻¹, reversing our previous assignment.

This is supported by the resonance Raman data, which show

the Fe−NO stretch at 598 cm⁻¹with natural abundance Fe in

solution.

π-donors with one dominant lone pair, whereas chloride is the

only isotropic π-donor with two equivalent Cl(p) π-donor

orbitals. Previously, we have shown that thiolates cause a

bending of the Fe−N−O unit by inducing a σ−trans eﬀect on

the bound NO via the population of an Fe−N−O σ*

antibonding orbital.⁶⁹Our results for [Fe(TPP)(NO)(Cl)]

show that this bending is also related to anisotropic π-donation,

which is a fact that has previously been overlooked. Therefore,

the Cl⁻ligand is a moderate σ- and isotropic π-donor that

causes a simultaneous weakening of the Fe−NO and N−O

bonds, as demonstrated by vibrational spectroscopy, but the

isotropic π-donating ability of Cl⁻results in the linearity of the

Fe−N−O unit unlike other anisotropic π-donor ligands. This

emphasizes the importance of (vibrational) spectroscopy over

structural data for the characterization of metal−ligand bond

strength. More work is required to fully understand the

electronic reasons for Fe−N−O bending in thiolate-coordi-

nated ferric heme-NO complexes and the signiﬁcance of this

eﬀect for NO activation in Cyt. P450s (especially Cyt. P450

NO reductase).¹¹

Interestingly, for six-coordinate {FeNO}⁶complexes with

anionic axial ligands, a correlation of the ligand donor strength

and the Fe−N−O bond angle has been derived.⁶⁷For example,

with a weak donor like triﬂuoroacetate, [Fe(TPP)(NO)-

(CO₂CF₃)] exhibits a slightly bent Fe−N−O unit of 176°.²⁸

Upon increasing the donor strength, the Fe−N−O unit bends

in [Fe(TpivPP)(NO)(NO₂)] with an Fe−N−O bond angle of

169°.²⁵Furthermore, in the presence of an S-donor, [Fe-

(OEP)(NO)(SR-H₂)] (where SR-H₂

= S-2,6-

(CF₃CONH)₂C₆H₃), the Fe−N−O moiety is bent even

more at 160°.⁶⁸By comparison, our chloride-bound {FeNO}⁶

complex, [Fe(TPP)(NO)(Cl)], has a linear Fe−N−O unit

(180°), which would make Cl⁻a very weak donor by this

classiﬁcation (similar to the acetate-ligated complex). However,

the axial-ligand donor strength is also correlated with N−O and

Fe−NO stretching frequencies as depicted in Figure 11.

Interestingly, [Fe(TPP)(NO)(Cl)] falls in the region between

the neutral N-donor- and thiolate-ligated complexes on the

graph. This would indicate that the Cl⁻ligand is a moderate

donor but does not explain the fact that the Fe−N−O unit is

completely linear. The most notable diﬀerence between the

anionic ligands mentioned above is that they are all anisotropic

In summary, our approach to synthesize {FeNO}⁶complexes

in the absence of excess NO gas makes future reactivity studies

feasible with reagents that would typically react with free NO

gas in solution. We spectroscopically and structurally

characterized the 5C complex, [Fe(TPP)(NO)]⁺and the 6C

complexes [Fe(TPP)(NO)(MI)]⁺and [Fe(TPP)(NO)(Cl)]

with axial imidazole and halide coordination. The latter

complex is a key intermediate in reductive nitrosylation that

has never been properly characterized. Finally, we determined

the k_offrates for NO loss from the diﬀerent complexes prepared

here, which emphasizes the ability of halides to denitrosylate

{FeNO}⁶complexes.

M

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Inorg. Chem. XXXX, XXX, XXX−XXX

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Article

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ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.inorg-

chem.7b01493.

■

S

The SI includes UV−vis, NMR, NRVS, rRaman, EPR,

and FT-IR spectra (PDF)

Accession Codes

CCDC 1556645−1556647 contain the supplementary crystal-

lographic data for this paper. These data can be obtained free of

charge via www.ccdc.cam.ac.uk/data_request/cif, or by email-

ing data_request@ccdc.cam.ac.uk, or by contacting The

Cambridge Crystallographic Data Centre, 12 Union Road,

Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

Nather, C.; Sulok, C. D.; Lehnert, N. Electronic Structure of Six-

̈

Coordinate Iron(III)-Porphyrin NO Adducts: the Elusive Iron(III)-

NO(radical) State and Its Influence on the Properties of these

Complexes. J. Am. Chem. Soc. 2008, 130, 15288−15303.

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and Properties of Six-Coordinate [Fe(OEP)(L)(NO)]⁺Derivatives:

Elusive Nitrosyl Ferric Porphyrins. J. Am. Chem. Soc. 1999, 121, 5210−

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characterization of nitrosyl complexes of ferric porphyrinates.

Molecular structure of aquonitrosyl(meso-tetraphenylporphinato)iron-

(III) perchlorate and nitrosyl(octaethylporphinato)iron(III) perchlo-

rate. J. Am. Chem. Soc. 1984, 106, 3191−3198.

AUTHOR INFORMATION

Corresponding Author

*E-mail: lehnertn@umich.edu

■

ORCID

Nicolai Lehnert: 0000-0002-5221-5498

Notes

The authors declare no competing ﬁnancial interest.

(20) Yi, G.-B.; Khan, M. A.; Richter-Addo, G. B. Activation of

Thionitrites and Isoamyl Nitrite by Group 8 Metalloporphyrins and

the Subsequent Generation of Nitrosyl Thiolates and Alkoxides of

Ruthenium and Osmium Porphyrins. Inorg. Chem. 1997, 36, 3876−

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Ferrihemoproteins in Buffer Solutions. J. Am. Chem. Soc. 1996, 118,

5702−5707.

ACKNOWLEDGMENTS

■

This work was supported by a grant from the National Science

Foundation (CHE-1464696 to N.L.). A.B.M. acknowledges a

Rackham Merit Fellowship and the Wayne & Carol Pletcher

Graduate Research Fellowship (University of Michigan). This

research used resources of the Advanced Photon Source, a U.S.

Department of Energy (DOE) Oﬃce of Science User Facility

operated for the DOE Oﬃce of Science by Argonne National

Laboratory under Contract No. DE-AC02-06CH11357.

(23) Roncaroli, F.; Videla, M.; Slep, L. D.; Olabe, J. A. New features

in the redox coordination chemistry of metal nitrosyls {M−NO⁺; M−

NO; M−NO⁻(HNO)}. Coord. Chem. Rev. 2007, 251, 1903−1930.

(24) Li, J.; Peng, Q.; Oliver, A. G.; Alp, E. E.; Hu, M. Y.; Zhao, J.;

Sage, J. T.; Scheidt, W. R. Comprehensive Fe−Ligand Vibration

Identification in {FeNO}⁶Hemes. J. Am. Chem. Soc. 2014, 136,

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Characterization, and Structural Studies of Several (Nitro)(nitrosyl)-

iron(III) Porphyrinates: [Fe(Porph)(NO₂)(NO)]. Inorg. Chem. 1999,

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DOI: 10.1021/acs.inorgchem.7b01493

Inorg. Chem. XXXX, XXX, XXX−XXX

Article Doi

Ferric Heme-Nitrosyl Complexes: Kinetically Robust or Unstable Intermediates?

DOI: 10.1021/acs.inorgchem.7b01493

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Article abstract of DOI:10.1021/acs.inorgchem.7b01493

Full text of DOI:10.1021/acs.inorgchem.7b01493

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