Electronic structure and biologically relevant reactivity of low-spin {FeNO}8 porphyrin model complexes: New insight from a bis-picket fence porphyrin

Article abstract of DOI:10.1021/ic400977h

Because of HNO's emerging role as an important effector molecule in biology, there is great current interest in the coordination chemistry of HNO and its deprotonated form, the nitroxyl anion (NO^-), with hemes. Here we report the preparation of four new ferrous heme-nitroxyl model complexes, {FeNO}⁸ in the Enemark-Feltham notation, using three electron-poor porphyrin ligands and the bis-picket fence porphyrin H₂[3,5-Me-BAFP] (3,5-Me-BAFP^2- = 3,5-methyl-bis(aryloxy)-fence porphyrin dianion). Electrochemical reduction of [Fe(3,5-Me-BAFP)(NO)] (1-NO) induces a shift of ν(N-O) from 1684 to 1466 cm^-1, indicative of formation of [Fe(3,5-Me-BAFP)(NO)]^- (1-NO^-), and similar results are obtained with the electron-poor hemes. These results provide the basis to analyze general trends in the properties of ferrous heme-nitroxyl complexes for the first time. In particular, we found a strong correlation between the electronic structures of analogous {FeNO}⁷ and {FeNO}⁸ complexes, which we analyzed using density functional theory (DFT) calculations. To further study their reactivity, we have developed a new method for the preparation of bulk material of pure heme {FeNO}⁸ complexes via corresponding [Fe(porphyrin)]^- species. Reaction of [Fe(To-F ₂PP)(NO)]^- (To-F₂PP^2- = tetra(ortho-difluorophenyl)porphyrin dianion) prepared this way with acetic acid generates the corresponding {FeNO}⁷ complex along with the release of H₂. Importantly, this disproportionation can be suppressed when the bis-picket fence porphyrin complex [Fe(3,5-Me-BAFP)(NO)]^- is used, and excitingly, with this system we were able to generate the first ferrous heme-NHO model complex reported to date. The picket fence of the porphyrin renders this HNO complex very stable, with a half-life of ～5 h at room temperature in solution. Finally, with analogous {FeNO}⁸ and {FeNHO}⁸ complexes in hand, their biologically relevant reactivity toward NO was then explored.

Full text of DOI:10.1021/ic400977h

Article
pubs.acs.org/IC
Electronic Structure and Biologically Relevant Reactivity of Low-Spin
{FeNO}8 Porphyrin Model Complexes: New Insight from a Bis-Picket
Fence Porphyrin
Lauren E. Goodrich,† Saikat Roy,† E. Ercan Alp,‡ Jiyong Zhao,‡ Michael Y. Hu,‡ and Nicolai Lehnert*,†
†Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
‡Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, United States
S
* Supporting Information
ABSTRACT: Because of HNO’s emerging role as an important
effector molecule in biology, there is great current interest in the
coordination chemistry of HNO and its deprotonated form, the
nitroxyl anion (NO−), with hemes. Here we report the
preparation of four new ferrous heme-nitroxyl model complexes,
{FeNO}8 in the Enemark−Feltham notation, using three
electron-poor porphyrin ligands and the bis-picket fence
porphyrin H2[3,5-Me-BAFP] (3,5-Me-BAFP2− = 3,5-methyl-
bis(aryloxy)-fence porphyrin dianion). Electrochemical reduc-
tion of [Fe(3,5-Me-BAFP)(NO)] (1-NO) induces a shift of
ν(N−O) from 1684 to 1466 cm−1, indicative of formation of
[Fe(3,5-Me-BAFP)(NO)]− (1-NO−), and similar results are
obtained with the electron-poor hemes. These results provide
the basis to analyze general trends in the properties of ferrous heme-nitroxyl complexes for the first time. In particular, we found
a strong correlation between the electronic structures of analogous {FeNO}7 and {FeNO}8 complexes, which we analyzed using
density functional theory (DFT) calculations. To further study their reactivity, we have developed a new method for the
preparation of bulk material of pure heme {FeNO}8 complexes via corresponding [Fe(porphyrin)]− species. Reaction of [Fe(To-
F2PP)(NO)]− (To-F2PP2− = tetra(ortho-difluorophenyl)porphyrin dianion) prepared this way with acetic acid generates the
corresponding {FeNO}7 complex along with the release of H2. Importantly, this disproportionation can be suppressed when the
bis-picket fence porphyrin complex [Fe(3,5-Me-BAFP)(NO)]− is used, and excitingly, with this system we were able to generate
the first ferrous heme-NHO model complex reported to date. The picket fence of the porphyrin renders this HNO complex very
stable, with a half-life of ∼5 h at room temperature in solution. Finally, with analogous {FeNO}8 and {FeNHO}8 complexes in
hand, their biologically relevant reactivity toward NO was then explored.
“Intermediate I”, obtained upon reaction of the initial Fe(III)-
NO adduct with NAD(P)H, is characterized by a Soret band
absorption at 444 nm10 and Fe-NO stretching frequency of 543
cm−1.12 Reaction with a second equivalent of NO then
produces N2O and closes the catalytic cycle. Density functional
theory (DFT) and quantum mechanics/molecular mechanics
(QM/MM) calculations13,14 support the idea that the direct
hydride transfer from NAD(P)H to the ferric nitrosyl complex
generates a corresponding Fe(II)-NHO species, which could
react directly with the second NO molecule. Alternatively, the
Fe(II)-NHO complex could undergo protonation to a formally
Fe(IV)-NHOH(-) (or Fe(III)-NHOH(radical)) structure prior
to reaction with the second equivalent of NO. The computa-
tional results in fact predict that the Fe(II)-HNO species is
basic because of the presence of the axial cysteinate ligand and,
thus, will be easily protonated,13,14 but experimental insight into
1. INTRODUCTION
Nitric oxide (NO) is toxic to cells at relatively low (μM)
concentrations,1 and it was therefore surprising when it was
discovered in the 1980s that this diatomic is actually a signaling
molecule in mammals at nM concentrations. In addition,
macrophages produce NO at higher (local) concentration for
the purpose of immune defense.2−5 Because of its toxicity, both
the biosynthesis of this molecule and its degradation are tightly
regulated in mammalian organisms to prevent cellular damage.
In denitrifying fungi that reduce nitrate to nitrous oxide (N2O)
for anaerobic respiration, the toxic metabolite NO is removed
from the cell by the heme enzyme Cytochrome P450 nitric
oxide reductase (P450nor) which catalyzes the reaction:6,7
2 NO + NAD(P)H + H+ → N2O + H2O. In this case, a ferric
nitrosyl intermediate is formed first,8,9 which is then reduced by
NAD(P)H in a two-electron process to form a ferrous nitroxyl
(NO− or HNO in aqueous environments) intermediate,10 an
{FeNO}8 or {FeNHO}8 complex in the Enemark−Feltham
notation.11 From stopped flow measurements on P450nor, this
Received: April 19, 2013
© XXXX American Chemical Society
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this issue is lacking. So despite all of these studies, the key
question remains whether the nitroxyl intermediate of P450nor
(Intermediate I) is singly or doubly protonated and what the
significance of the protonation state is for the reactivity of this
species with NO. Without ferrous heme-nitroxyl model
complexes in hand, this is difficult to ascertain because of our
inability to precisely control protons, and proton dependent
reactions, in protein (aqueous) environments.
Ferrous nitroxyl intermediates have also been proposed in
the catalytic cycle of bacterial nitric oxide reductase (NorBC).
NorBC, like P450nor, reduces two equivalents of NO to N2O,
but instead of a single heme active site as found in P450nor,
NorBC contains a bimetallic heme/non-heme active site.15
While the exact mechanism of NO reduction by NorBC is not
known, one proposal (the “cis-heme b3” mechanism) suggests
that all of the NO chemistry happens exclusively at the heme b3
site.16 The cis-heme b3 mechanism proposes that NO first binds
the heme b3 to form a ferrous heme-nitrosyl. A recent DFT
paper indicates that reduction of this species to generate an
{FeNO}8 complex is crucial for the following reaction with NO
to form the N−N bond and create a hyponitrite-level
intermediate.17 As there is currently no experimental evidence
to support the key N−N bond forming step of this hypothesis,
elucidation of the reaction of free NO with Fe(II)-NO−
complexes is of key importance to further validate the chemical
basis for this mechanism.
In this paper, we report the spectroscopic and electronic-
structural properties of four new {FeNO}8 porphyrin
complexes prepared either through the one-electron reduction
of their {FeNO}7 precursors or through corresponding
[Fe(porphyrin)]− species, which is advantageous for the
preparation of pure bulk material. This new library of
{FeNO}8 model complexes with different porphyrin ligands
allowed us to establish a close correlation of the vibrational
properties of the {FeNO}8 complexes and their {FeNO}7
precursors, demonstrating for the first time that their electronic
structures are closely related. These results are further analyzed
using DFT calculations. The {FeNO}8 complexes were then
investigated for reactivity toward (a) NO gas and (b) weak
acids to generate HNO complexes. Application of the new bis-
picket fence porphyrin 5,10,15,20-tetrakis(2,6-bis(3,5-
dimethylphenoxy)phenyl) porphyrin, H2[3,5-Me-BAFP], al-
lowed for a breakthrough in these reactivity studies, as the
bulky picket fence of this porphyrin efficiently prevented the
previously observed disproportionation of the putative HNO
adduct and intermolecular cross-reactions with NO. In
particular, our results indicate a dramatic stabilization of
heme-bound HNO with the new picket-fence porphyrin
compared to the simple TPP2− and OEP2− ligands reported
previously. Finally, initial results with respect to the biologically
relevant reactivity of {FeN(H)O}8 complexes with NO are
reported that shine some light on proposed mechanisms for
NO reductases.
In summary, mechanistic proposals have been put forth for
both P450nor and NorBC where heme-bound nitroxyl
intermediates of type {FeN(Hn)O}8 (n = 0−2) react with
NO. To systematically evaluate this proposed reactivity as a
function of the protonation state of the {FeNO}8 intermediate,
model complexes are required that allow for a precise control of
the proton (acid) concentration in solution. The most suitable
approach to generate Fe(II)-nitroxyl model complexes is to
start from a stable ferrous heme-nitrosyl adduct {FeNO}7. The
{FeNO}7 precursor can be reduced by one-electron to an
{FeNO}8 species or ferrous nitroxyl complex, which could
subsequently be protonated to generate the {FeNHO}8 species.
Initial studies by Kadish and co-workers demonstrated
reversible one-electron reduction of five-coordinate ferrous
heme-nitrosyls, utilizing TPP2− and OEP2− ligands, to generate
Fe(II)-NO− species as shown by UV−vis spectroelectrochem-
istry.18,19 Ryan and co-workers provided further vibrational
characterization of both [Fe(TPP)(NO)]− and [Fe(OEP)-
(NO)]−.20,21 Finally, using an extremely electron-poor
porphyrin, H2TFPPBr8, Doctorovich and co-workers have
isolated and characterized the corresponding five-coordinate
Fe(II)-NO− complex, obtained by reduction of the Fe(II)-NO
starting material by cobaltocene.22 Unfortunately, no reactivity
of these complexes with NO has been reported, and all
attempts at protonation of the formed Fe(II)-NO− species to
form a Fe(II)-HNO complex have resulted in regeneration of
the corresponding Fe(II)-NO complex, presumably via
disproportionation of an intermediately formed HNO
adduct.20,22 Ryan and co-workers also reported H2 generation
during this process.20 No other details about the reactivity of
these species are known, and because only very few {FeNO}8
porphyrin model complexes have been prepared to date, their
electronic structures are not well understood. Note that the
only well characterized ferrous HNO complexes known to date
were prepared by Farmer and co-workers in the myoglobin
active site, and with human, soy and clam hemoglobin.23,24
2. EXPERIMENTAL SECTION
Synthesis. All reactions (except when noted otherwise) were
performed under an inert gas atmosphere using dried and distilled
solvents. Handling of air-sensitive samples was carried out under an N2
atmosphere in an MBraun glovebox equipped with a circulating
purifier (O2, H2O <0.1 ppm). Nitric oxide gas (Cryogenic Gases Inc.,
99.5%) was passed through ascarite and then through a cold trap at
−80 °C prior to usage to remove higher nitrogen oxide impurities.
Nitric oxide-15N18O (Aldrich, 98% 15N, 95% 18O) was used without
further purification. 1-methylimidazole (MI) was distilled and
degassed prior to use. Phosphazene base, P1-tBu-tris(tetramethylene),
and glacial acetic acid were purchased from Aldrich and freeze−
pump−thawed prior to use. Ammonia analysis was carried out using
the phenolate assay originally developed by Russel.25,26
Tetrakis-5,10,15,20-(per-pentafluorophenyl)porphyrin, H2[Tper-
F5PP], and tetrakis-5,10,15,20-(o-difluorophenyl)porphyrin, H2[To-
F2PP], were synthesized and purified as previously reported.27,28
Tetrakis-5,10,15,20-(2,6-dinitro-4-tert-butylphenyl)porphyrin, H2[To-
(NO2)2-p-tBuPP], was prepared by BF3−OEt catalyzed condensation
of 2,6-dinitro-4-tert-butylbenzaldehyde29 and pyrrole in CH2Cl2 as
reported previously.30 The porphyrin ligand H2[3,5-Me-BAFP] was
prepared according to modified literature procedures as described
below.31,32 Iron(III) chloride porphyrin complexes were prepared
from the porphyrin ligand and excess FeCl2 in refluxing
dimethylformamide (DMF)33 or tetrahydrofuran (THF) (see below;
as needed). Five-coordinate ferrous porphyrin nitrosyls were prepared
by reductive nitrosylation of the corresponding iron(III) chloride
complexes.34 A representative procedure for iron insertion and
reductive nitrosylation is provided below. [57Fe(3,5-Me-BAFP)(NO)]
and [57Fe(3,5-Me-BAFP)(15N18O)] for nuclear resonance vibrational
spectroscopy (NRVS) measurements were prepared in the same
manner as the unlabeled complexes using 57FeCl2 for the initial
metalation.35
2,6-Bis(3′,5′-dimethylphenoxy)benzaldehyde. A 3.4 g portion
of potassium methoxide, 12.5 mL of dry benzene, and 6.1 g of 3,5-
dimethylphenol (50 mmol) were added to a 100 mL Schlenk flask.
The mixture was allowed to stir under Ar(g) for 1 h. After 1 h,
benzene and methanol were removed via a Schlenk line. 12.5 mL of
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dry pyridine was added, and the mixture was brought to a reflux. Then,
3.2 g of 2,6-dibromobenzaldehyde31 and 0.19 g of copper(I) chloride
were added quickly to the mixture. The reaction was kept at reflux
under Ar(g) for 17 h. After 17 h, the mixture was added to 37 mL of
ice water, and conc. hydrochloric acid was added until the solution
became acidic. The reaction mixture was extracted with 20 mL of
CH2Cl2, and the organic layer was washed with H2O, saturated
aqueous NaHCO3, and H2O. The mixture was concentrated to an oil
using a rotary evaporator. The oil was chromatographed twice on silica
with CH2Cl2 as the eluent. The fractions containing the desired
product were rotary evaporated to a light yellow solid. Yield: 2.301 g
UV−vis (THF): 434 (ε = 130800 M−1 cm−1), 550 (6600 M−1 cm−1)
nm.
Generation of 1-NO−. Soret Band Concentration. Under inert
atmosphere, ∼1 mg of [Fe(3,5-Me-BAFP)] was dissolved in 30 mL of
0.1 M TBAP in THF. Using a two compartment bulk electrolysis cell
outfitted with a UV−visible dip (immersion) probe (described below),
the sample was reduced at −1.9 V vs Ag wire to obtain [Fe(3,5-Me-
BAFP)]− (1−). The applied potential was turned off, and 100 μL
NO(g) was added. Formation of 1-NO− was monitored by UV−
visible spectroscopy and, upon completion (∼2 min) the solution was
sparged for 10 min with N2(g) to remove all free NO.
1
Q-Band Concentration. Under inert atmosphere, 12 mg of [Fe(3,5-
Me-BAFP)] was dissolved in 30 mL of 0.1 M TBAP in THF. Using a
two compartment bulk electrolysis cell outfitted with a UV−visible dip
(immersion) probe (described below), the sample was reduced at −1.9
V vs Ag wire to obtain [Fe(3,5-Me-BAFP)]− (1−). The applied
potential was turned off, and 2 mL of NO(g) was added. Formation of
1-NO− was monitored by UV−visible spectroscopy and, upon
completion (∼4 min), the solution was sparged for 10 min with
N2(g) to remove all free NO.
(55%). H NMR (400 MHz, CDCl3): 10.57 (s, 1H); 7.30 (t, 1H);
6.80 (s, 2H); 6.69 (s, 4H); 6.56 (d, 2H); 2.30 (s, 12H); see Supporting
Information, Figure S29.
3,5-Methyl-Bis(Aryloxy)-FencePorphyrin, H2[3,5-Me-
BAFP].32 A 1.135 g portion of 2,6-bis(3,5-dimethylphenoxy)-
benzaldehyde, 325 mL of CH2Cl2, and 2.5 mL of absolute ethanol
were placed in a 500 mL round-bottom flask (RBF) and sparged with
Ar(g). A 0.25 mL portion of pyrrole was then added via syringe, and
the solution was stirred for 5 min. Then, 0.16 mL of boron trifluoride
diethyletherate was added via syringe, and the solution was stirred in
the dark for 1 h at room temperature. After 1 h, 0.56 g of
dichlorodicyano-benzoquinone and 0.16 mL of triethylamine were
added, and the reaction was stirred for an additional hour. The
reaction mixture was then evaporated to dryness, chromatographed on
silica with 100% CH2Cl2, and the obtained solid was recrystallized
Generation of 2-NO−. Under inert atmosphere, ∼1 mg of [Fe(To-
F2PP)] was dissolved in 30 mL of 0.1 M TBAP in THF. Using a two
compartment bulk electrolysis cell outfitted with a UV−visible dip
(immersion) probe (described below), the sample was reduced at −1.6
V vs Ag wire to obtain [Fe(To-F2PP)]− (2−).The applied potential was
turned off, and 100 μL of NO(g) was added. Formation of 2-NO− was
monitored by UV−visible spectroscopy and, upon completion (∼4
min), the solution was sparged for 10 min with N2(g) to remove all
free NO.
Physical Measurements. Infrared spectra were obtained from
KBr disks on a Perkin-Elmer BX spectrometer at room temperature.
Resolution was set to 2 cm−1. Proton magnetic resonance spectra were
recorded on a Varian Inova 400 MHz instrument. Electronic
absorption spectra were measured using an Analytical Jena Specord
600 instrument at room temperature. Electron paramagnetic
resonance spectra were recorded on a Bruker X-band EMX
spectrometer equipped with an Oxford Instruments liquid nitrogen
cryostat. EPR spectra were typically obtained on frozen solutions using
20 mW microwave power and 100kHz field modulation with the
amplitude set to 1 G. Sample concentrations employed were ∼1 mM.
Nuclear resonance vibrational spectroscopy (NRVS) data were
obtained as described previously36 at beamline 3-ID-XOR of the
Advanced Photon Source (APS) at Argonne National Laboratory. This
beamline provides about 2.5 × 109 photons/sec in ∼1 meV bandwidth
(= 8 cm−1) at 14.4125 keV in a 0.5 mm (vertical) × 0.5 mm
(horizontal) spot. Samples were loaded into 4 × 7 × 1 mm copper
NRVS cells. The final spectra represent averages of 6 scans. The
program Phoenix was used to convert the NRVS raw data to the
Vibrational Density of States (VDOS).37,38
Cyclic voltammograms (CVs) were recorded with a CH instru-
ments CHI660C electrochemical workstation using a three compo-
nent system consisting of a platinum or glassy carbon working
electrode, a platinum auxiliary electrode, and an Ag wire pseudorefer-
ence electrode. CVs were measured in 0.1 M tetrabutylammonium
perchlorate (TBAP) solutions in THF or 1,2-dichloroethane.
Potentials are reported vs the measured Fc/Fc+ couple. IR
spectroelectrochemistry (∼4 mM) was performed using a solution
IR cell with CaF2 windows as previously described.21 Electrodes
consist of an 8 × 10 mm Pt mesh (100 mesh, 99.9%, Aldrich) for the
working, 3 × 10 mm Pt mesh for the counter, and Ag wire (0.1 mm
diameter, 99.9%, Aldrich) as a pseudoreference electrode. UV−vis
spectroelectrochemistry (∼0.5 mM) was performed in a OTTLE
cell.39 Electrodes consist of an 8 × 10 mm Pt mesh (100 mesh, 99.9%,
Aldrich) for the working, 3 × 20 mm Pt mesh for the counter, and Ag
wire (1 mm diameter, 99.9%, Aldrich) as a pseudoreference electrode.
Bulk electrolysis (at mM porphyrin concentrations) was performed in
a two compartment setup where the carbon felt working electrode and
Ag wire reference electrode are separated from the carbon felt counter
electrode by a fine frit. The counter electrode compartment contains
solvent and electrolyte. Electrolyte (TBAP of TBAPF6) was 0.1 M. All
1
from CH2Cl2/MeOH. Yield: 349 mg (27%). H NMR (400 MHz,
CDCl3): 8.86 (s, 8H); 7.59 (t, 4H); 7.06 (d, 8H); 6.13 (s, 16H); 6.02
(s, 8H); 1.66 (s, 48H); −2.98 (s, 2H); see Supporting Information,
Figure S30. LCT MS: m/z 1576.1 (M+1). UV−vis (CH2Cl2): 424,
517, 554, 593 nm.
[Fe(3,5-Me-BAFP)(Cl)]. A 270 mg portion of H2[3,5-Me-BAFP] in
60 mL of dry THF was brought to a reflux. Once refluxing, 0.22 g of
FeCl2 was quickly added, and the reaction was allowed to reflux for 3
h. The solution was evaporated to dryness and the crude material
chromatographed on silica with 100% CH2Cl2 (to remove free base
porphyrin) and 98:2 CH2Cl2:MeOH to elute the ferric porphyrin. The
product band was evaporated, the resulting solid was redissolved in
CH2Cl2 and washed with ∼1 M HCl. The organic layer was first dried
with Na2SO4, and the solvent was then removed under reduced
pressure to yield a dark purple powder. Yield: 186 mg (66%). LCT
MS: m/z 1629.8 (M-Cl). UV−vis (CH2Cl2): 374, 424, 510, 584, 667
nm.
[Fe(3,5-Me-BAFP)(NO)] (1-NO). A 325 mg portion of [Fe(3,5-
Me-BAFP)(Cl)] was dissolved in 9 mL of CH2Cl2 and 0.9 mL of
MeOH. The solution was exposed to excess NO(g) and stirred at
room temperature for 30 min. The resulting nitrosyl complex was
precipitated with the addition of 24 mL of MeOH and stored at −30
°C overnight. The resulting solid was filtered under inert atmosphere
and dried for 2 min under reduced pressure. Yield: 253 mg (78%). FT-
IR: v(N−O) 1684 cm−1. [Fe(3,5-Me-BAFP)(15N18O)] was prepared
with 15N18O using the same procedure. FT-IR: v(15N−18O) 1614
cm−1. UV−vis (1,2-DCE): 413, 478, 554 nm. UV−vis (THF): 422 (ε
= 78800 M−1 cm−1), 479 (8480 M−1 cm−1), 555 (5280 M−1 cm−1) nm.
Crystallization of [Fe(3,5-Me-BAFP)(NO)]. In the glovebox, ∼2
mg [Fe(3,5-Me-BAFP)(NO)] was dissolved in 1 mL of THF and
placed in a 7 mm glass tube. 5 mL of MeOH were carefully layered on
the THF solution, and the setup was left under an inert atmosphere.
After 8 days, crystals suitable for X-ray analysis were collected.
[Fe(3,5-Me-BAFP)]. A 600 mg portion of 1-Cl was dissolved in 50
mL of CH2Cl2 and stirred with excess 4 M NaOH (aq.) for 5 h. The
organic layer was washed with water twice, dried with sodium sulfate
and evaporated to dryness to yield [Fe(3,5-Me-BAFP)(OH)]. [Fe(3,5-
Me-BAFP)(OH)] was heated to 70 °C in 40 mL of dry, degassed
toluene with 3 mL of ethanethiol under inert atmosphere. After 4 h,
the reaction was evaporated to dryness via Schlenk line. The resulting
solid was redissolved in a minimum volume of degassed toluene,
layered with hexanes, and placed at −30 °C overnight to precipitate.
The resulting bright purple solid was collected. Yield: 570 mg (97%).
C
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bulk electrolysis experiments were performed at room temperature in
the glovebox (O2 < 0.1 ppm).
X-ray crystallography measurements were performed on a Rigaku
RAXIS SPIDER diffractometer with an imaging area detector using
graphite monochromated Cu−Kα radiation (1.5406 Å). A red prism
crystal of C108H92O9N5Fe × 0.5THF having approximate dimensions
of 0.29 × 0.09 × 0.05 mm was mounted on a MitiGen cryoloop. The
data collection was made at 95 K. The data were processed with
SADABS. An empirical absorption correction was applied to the
structure. The structure was solved and refined with the Bruker
SHELXTL (vs 2008/3) software package. The non-hydrogen atoms
were refined anisotropically, and the hydrogen atoms were refined
using the riding model. See Supporting Information, Table S1 for
crystallographic data and measurement parameters.
DFT Calculations. All geometry optimizations and frequency
calculations were performed with the program package Gaussian 0340
using the BP8641,42 functional and TZVP43,44 basis set. Molecular
orbitals were obtained from B3LYP42,45,46/TZVP single point
calculations on the BP86/TZVP optimized structures using
ORCA.47 In all Gaussian calculations, convergence was reached
when the relative change in the density matrix between subsequent
iterations was less than 1 × 10−8. Molecular orbitals were plotted with
the program orca_plot included in the ORCA package and visualized
using GaussView.
Determination of Binding Constants. To quantify 1-methyl-
imidazole (MI) binding to five-coordinate ferrous heme-nitrosyls, the
binding constant can be calculated for the reaction:
Figure 1. Crystal structure of [Fe(3,5-Me-BAFP)(NO)] (1-NO),
hydrogen atoms are omitted for clarity. Key bond lengths and angles
are provided in Table 1 and Supporting Information, Table S3.
Thermal ellipsoids are shown at 30% probability.
[Fe(TPP*)(NO)] + MI ⇄ [Fe(TPP*)(MI)(NO)]
(1)
where TPP*(2−) is a phenyl-substituted tetraphenylporphyrin deriva-
tive. The titration of MI against the five-coordinate complex 1-NO was
followed by UV−visible spectroscopy, see Supporting Information,
Figure S19, and Keq can then be calculated from the equation
possible positions (four on each side of the porphyrin plane).34
Around 250 K, [Fe(TPP)(NO)] undergoes a phase transition
from tetragonal to triclinic, and NO is now limited to two
unique positions, one on each face of the porphyrin plane.52
Excitingly, the eight phenoxy groups of the porphyrin ligand in
1-NO direct packing of the molecules in the crystal in a way
that further limits the Fe-NO unit to a single orientation (but
note that the presence of a more disordered phase at high
temperature cannot be ruled out). The steric encumbrance of
this bulky porphyrin also appears to direct the position of NO
relative to the Fe−Npyrrole bonds: in the case of 1-NO, the N−
O unit is located directly above one of the Fe−Npyrrole bonds.
This is in contrast to other five-coordinate ferrous heme-
nitrosyls where the O-atom is positioned toward a meso-
carbon; for example, in [Fe(TPP)(NO)] the N−O unit is
rotated 44° from the closest Fe−Npyrrole bond.52 This difference
results in a unique core asymmetry in 1-NO. In this complex,
the Fe−Npyrrole bond which is aligned with the N−O unit
(1.969 Å) is significantly shorter than the remaining three
bonds (1.997, 1.992, and 2.005 Å). Typically, when the N−O
unit points toward a meso-carbon of the porphyrin ligand, two
Fe−Npyrrole bonds are shorter (in the direction of NO) than the
remaining two bonds.53 In the case of [Fe(OEP)(NO)], the
short Fe−Npyrrole bond lengths are 1.989 and 1.993 Å, and the
long bonds are 2.017 and 2.016 Å.54
⎡
⎤
MI
ΔE
−1
[MI] = cTΔε
− Keq
⎢
⎣
⎥
⎦
(2)
which was originally developed by Drago and co-workers.48−50 Here,
cT corresponds to the total concentration of porphyrin complexes, cT =
c(6C) + c(5C), Δε is the difference in extinction coefficients, Δε =
ε(6C) − ε(5C), and 5C and 6C abbreviate the five- and six-coordinate
complexes, respectively. UV−vis absorption measurements are
performed at different concentrations of MI ([MI]) and the change
in absorbance (ΔE) is measured. A plot of [MI] versus [MI]/ΔE then
−1
gives Keq
.
3. RESULTS AND ANALYSIS
3.1. Preparation and Characterization of {FeNO}7
Complexes. [Fe(3,5-Me-BAFP)(NO)] (1-NO), a {FeNO}7
complex in the Enemark−Feltham notation,11 was prepared by
reductive nitrosylation of [Fe(3,5-Me-BAFP)(Cl)]. The
identity of 1-NO has been confirmed by X-ray crystallography
at 95 K. The crystal structure shows two equivalent molecules,
A and B, in the unit cell that differ marginally in their structural
parameters. Figure 1 shows a side view of one of the two
molecules. As listed in Table 1, the Fe−NO and N−O bond
lengths (for molecule A) are 1.71 and 1.15 Å, respectively. The
Fe−N−O angle is 146° and the Fe-atom is displaced from the
heme plane by 0.35 Å toward NO, typical of five-coordinate
ferrous heme-nitrosyls.51 Additionally, the crystal structure
clearly demonstrates that the eight phenoxy-groups of the bis-
picket fence porphyrin do, in fact, create a sterically hindered
binding pocket for axial ligands, NO derivatives in our case.
Interestingly, this is the first crystal structure of a five-
coordinate ferrous heme-nitrosyl with a TPP2− derivative as
coligand that shows a single conformation of the Fe-NO unit. It
has been shown previously by Scheidt and co-workers that at
293 K the NO unit in [Fe(TPP)(NO)] is disordered over eight
The {FeNO}7 complex 1-NO shows an electron para-
magnetic resonance (EPR) spectrum typical of a S = 1/2 five-
coordinate ferrous heme-nitrosyl with g-values of 2.10, 2.06,
and 2.01 in toluene (see Supporting Information, Figure S1). A
well-defined 3-line hyperfine pattern is observed on the smallest
g-value, gz from the 14N nuclear spin (I = 1) of bound NO. In
THF, however, the three-line hyperfine on gz begins to migrate
toward gy, indicating possible binding of THF at 77 K to form a
six-coordinate nitrosyl with bound THF in solution. IR spectra
D
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Table 1. Crystallographic Parameters ([Å] and [deg]) of Selected Five-Coordinate Ferrous Porphyrin Nitrosyls
a
b
complex
T [K]
ΔFe−NO
ΔN−O
∠Fe−N−O
ΔFe−Npyrrole
ΔFe−Npyrrole
ΔFe
ref.
c
[Fe(3,5-Me-BAFP)(NO)] (1-NO), A
95
1.712
1.150
146.27
1.969
1.997
1.992
2.005
1.975
1.989
2.002
2.008
1.991(8)
0.35
t.w.
[Fe(3,5-Me-BAFP)(NO)] (1-NO), B
95
1.714
1.142
146.52
1.993(5)
0.37
t.w.
52
52
54
[Fe(TPP)(NO)]
[Fe(TPP)(NO)]
[Fe(OEP)(NO)], A
33
1.740
1.721
1.722
1.164
1.107
1.167
144.5
149.6
144.4
1.999
2.001
2.004
0.20
0.23
0.29
293
130
1.989
1.993
2.017
2.016
2.000
1.999
2.017
2.023
54
[Fe(OEP)(NO)], B
130
1.731
1.168
142.7
2.010
0.27
a
b
c
Average of all four Fe−Npyrrole bond lengths Iron displacement from the 24 atom mean porphyrin plane This work
in KBr show a clear nitric oxide stretching frequency, v(N−O),
of 1684 cm−1 which shifts to 1614 cm−1 upon 15N18O isotope
labeling as shown in Supporting Information, Figure S2.
Furthermore, utilizing nuclear resonance vibrational spectros-
copy the Fe-NO stretching frequency, ν(Fe-NO), of 1-NO is
found at 518 cm−1 which shifts by 16 cm−1 to lower energy in
1-15N18O (Supporting Information, Figure S3). The Fe−N−O
bending mode is unable to be assigned because of noise in the
1-NO spectrum and overlap with other Fe-centered vibrations
in the 380 cm−1 region.
Table 2. Fe-NO and N−O Stretching Frequencies of
Selected Five- and Six-Coordinate {FeNO}7 and {FeNO}8
Iron Porphyrin Nitrosyls
complex
ν(N−O) ν(Fe−NO)
ref.
{FeNO}7
five-coordinate
[Fe(OEP)(NO)]
1671
1684
1687
1693
1697
1699
1727
522
518
60
[Fe(3,5-Me-BAFP)(NO)] (1-NO)
[Fe(To-F2TPP)(NO)] (2-NO)
[Fe(To-(NO2)2-p-tBuPP)(NO)] (4-NO)
[Fe(TPP)(NO)]
t.w.
t.w.
t.w.
58
Three additional “electron-poor” {FeNO}7 porphyrin
complexes have been synthesized for the purpose of this
study. [Fe(To-F2PP)(NO)] (2-NO), [Fe(Tper-F5PP)(NO)]
(3-NO), and [Fe(To-(NO2)2-p-tBuPP)(NO)] (4-NO) all
show typical N−O stretching frequencies for five-coordinate
ferrous heme-nitrosyls (Table 2; 2-NO and 3-NO were
previously reported, see ref 55). The ν(N−O) for 2-NO, 3-
NO, and 4-NO in a KBr matrix are 1687, 1699, and 1693 cm−1
respectively. EPR spectroscopy indicates that all three
complexes are low-spin Fe(II)-NO species with S = 1/2
ground states. For 3-NO and 4-NO, the EPR spectrum shows
the usual case where a well-defined three-line hyperfine pattern
532
[Fe(Tper-F5TPP)(NO)] (3-NO)
[Fe(TFPPBr8)(NO)]
t.w.
22
six-coordinate
[Fe(3,5-Me-BAFP)(THF)(NO)]
(1THF-NO)
1661
t.w.
[Fe(3,5-Me-BAFP)(MI)(NO)] (1MI-NO)
[Fe(To-F2TPP)(MI)(NO)] (2MI-NO)
1630
1636
1641
t.w.
t.w.
t.w.
[Fe(To-(NO2)2-p-tBuPP)(MI)(NO)]
(4MI-NO)
[Fe(TPP)(MI)(NO)]
1630
1649
437
36,59
[Fe(Tper-F5TPP)(MI)(NO)] (3MI-NO)
{FeNO}8
[Fe(OEP)(NO)]−
t.w.
is observed on the smallest g-value, gz, that stems from the 14
N
1441
1466
1473
1482
21
nuclear spin (I = 1) of bound NO (Supporting Information,
Figure S4; Az = 47 MHz in both spectra). Interestingly, in the
case of 2-NO, this hyperfine interaction is now resolved on all
three g-values. This is a unique case and correspondingly, the
experimental spectrum and simulation generated using the
program Spin Count are provided in Figure 2. The g-values are
2.11, 2.04, and 2.00similar to both 3-NO, 4-NO, and other
five-coordinate ferrous heme systems.16 The hyperfine coupling
constants for Ax, Ay, and Az are 39, 46, and 47 MHz,
respectively.
3.2. Spectroelectrochemical Reduction of Five-Coor-
dinate Ferrous Heme-Nitrosyls. The cyclic voltammogram
of 1-NO shows a quasi-reversible reduction at −1.78 V vs Fc/
Fc+ in THF (Figure 3). This reduction potential is 310 and 190
mV more negative than those for the one-electron reduction of
the previously characterized complexes [Fe(TPP)(NO)] and
[Fe(OEP)(NO)], respectively (Table 3).18,19 To characterize
[Fe(3,5-Me-BAFP)(NO)]− (1-NO−)
[Fe(To-F2PP)(NO)]− (2-NO−)
t.w.
t.w.
t.w.
[Fe(To-(NO2)2-p-tBuPP)(NO)]−
(4-NO−)
[Fe(TPP)(NO)]−
1496
549
20
[Fe(Tper-F5TPP)(NO)]− (3-NO−)
[Fe(TFPPBr8)(NO)]−
∼1500
1550
t.w.
22
this reduction further, infrared spectroelectrochemical measure-
ments were performed in thin layer cells. As shown in Figure 4,
upon one-electron reduction of 1-NO in 1,2-DCE-d4 the ν(N−
O) band at 1684 cm−1 of the {FeNO}7 starting complex
decreases in intensity as a new band at ∼1466 cm−1 appears.
While this N−O stretching vibration of the {FeNO}8 complex
(1-NO−) is partially masked by a porphyrin ligand band,
15N18O labeling shifts this band into an open window of the IR
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Figure 2. EPR spectrum of [Fe(To-F2PP)(NO)] (2-NO). The three-
line hyperfine pattern on all g-values originates from the nuclear spin
of the 14N-atom (I = 1) of NO. The simulated spectrum was generated
using the program SpinCount. Fit parameters are gx = 2.109, gy =
2.0375, gz = 2.003, Ax = 39 MHz, Ay = 46 MHz, Az = 47 MHz, sgx (g-
strain) = 0.0025, sgy = 0.0035, and sgz = 0.002.
Figure 4. Infrared spectra for the spectroelectrochemical reduction of
[Fe(3,5-Me-BAFP)(NO)] (top, 1-NO) and [Fe(3,5-Me-BAFP)-
(15N18O)] (middle, 1-15N18O). Difference spectra are provided in
Supporting Information, Figure S8. The asterisk (*) indicates poor
subtraction of a porphyrin band at 1450 cm−1. The estimated isotope
shift (by DFT) of the N−O stretch in the NO− complex is 61 cm−1,
indicating that the 1450 cm−1 feature in the reduced compound is too
high in energy to be the v(15N−18O) stretch.
nitrosyls typically show significantly lower N−O stretching
frequencies (∼1300 cm−1). This suggests a strongly NO ligand-
centered reduction for the low-spin non-heme NO adducts and
a more metal based reduction for the heme systems. Previous
DFT calculations from our group have shown that for the heme
complexes this corresponds to an electronic structure that is
intermediate between low-spin Fe(II)-NO− ↔ Fe(I)NO·.13
UV−visible spectroelectrochemical measurements in an
OTTLE cell were used to further characterize the one electron
reduced complex 1-NO− as shown in Figure 5. As the potential
is swept reductively from −0.4 V to −1.8 V vs Ag wire at 10
mV/s, there is essentially no change in the Soret band at 413
nm when 1,2-DCE is used as solvent, but dramatic changes are
observed in the Q-band region (see Figure 5, top). The band at
478 nm decreases in intensity while a new band appears at 523
nm upon reduction. The clean isosbestic point at 504 nm is
indicative of a clean conversion from 1-NO to 1-NO− without
further intermediates. The spectral changes observed for the
reduction of 1-NO are in agreement with the reduction of
[Fe(TPP)(NO)] reported previously.20 Importantly, this does
not correspond to a reduction of the porphyrin ligand, as this is
accompanied by a dramatic loss of intensity of the Soret band
not observed here. As an illustration of a porphyrin-centered
reduction the spectroelectrochemical reduction of [Fe(3,5-Me-
BAFP)] in THF is provided in the Supporting Information,
Figure S11. For 1-NO, this porphyrin reduction (correspond-
ing to a two-electron reduction of the {FeNO}7 complex) is not
accessible in 1,2-DCE.
Figure 3. Cyclic voltammograms for [Fe(3,5-Me-BAFP)(NO)] (1-
NO) in THF at various scan rates.
Table 3. Half Wave Potentials (in V vs. Fc/Fc+) for the First
Reduction of Ferrous Porphyrin Nitrosyls
[Fe(P)(NO)]/
complex
[Fe(OEP)(NO)]
solvent
[Fe(P)(NO)]− ref.
CH2Cl2
THF
−1.59
−1.78
−1.18
−1.18
18
[Fe(3,5-Me-BAFP)(NO)] (1-NO)
[Fe(To-F2TPP)(NO)] (2-NO)
t.w.
t.w.
t.w.
1,2-DCE
1,2-DCE
[Fe(To-(NO2)2-p-tBuPP)(NO)]
(4-NO)
[Fe(TPP)(NO)]
CH2Cl2
THF
−1.42
−1.47
−1.13
−0.65
18
18
t.w.
22
[Fe(Tper-F5TPP)(NO)] (3-NO)
1,2-DCE
CH2Cl2
[Fe(TFPPBr8)(NO)]
spectrum at ∼1400 cm−1. Importantly, this reduction is
chemically completely reversible: upon reoxidation, as shown
in Supporting Information, Figures S6 and S7, complex 1-NO
is regenerated. Natural abundance NO and 15N18O difference
spectra for 1-NO and 1-NO− are provided in the Supporting
Information, Figure S8, to further confirm the assignment of
the N−O stretching frequency of 1-NO−.
Upon dissolving 1-NO in THF, the Soret band shifts by 9
nm from 413 to 422 nm, indicating binding of THF in this
system. Solution IR spectra support formation of [Fe(3,5-Me-
BAFP)(NO)(THF)] with a new ν(N−O) band at 1661 cm−1.
Additionally, the EPR spectrum of 1-NO in THF indicates
weak binding of THF as discussed above (Supporting
Information, Figure S1). However, upon one-electron reduc-
tion of this species (Figure 5, bottom), the resulting spectrum
overlays perfectly with the data obtained for the reduction of
As listed in Table 2, the ν(N−O) frequency of 1-NO− is
consistent with previously reported values for five-coordinate
{FeNO}8 porphyrin systems, where ν(N−O) is observed
between 1440−1550 cm−1. In contrast, low-spin non-heme iron
F
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548 nm, increase in absorbance. The reduction is chemically
completely reversible. The formation of 2-NO− is also
completely reversible in IR experiments in 1,2-DCE-d4 as
shown in Figure 6 and Supporting Information, Figures S13−
Figure 6. Infrared spectra for the spectroelectrochemical reduction of
[Fe(To-F2PP)(NO)] (top, 2-NO) and [Fe(To-F2PP)(15N18O)]
(bottom, 2-15N18O). Difference spectra are provided in Figure 8.
S15. Using spectroelectrochemical IR measurements, a new
ν(N−O) band corresponding to 2-NO− is observed at 1471
cm−1, which shifts to 1405 cm−1 upon 15N18O isotope labeling.
Similar experiments were also performed for 3-NO and 4-NO,
and the corresponding ν(N−O) values for these species and
corresponding one-electron reduced complexes are provided in
Table 2. For 3-NO− a ν(N−O) band of ∼1500 cm−1 was
observed (Supporting Information, Figure S16) and for 4-NO−
a N−O stretching frequency of 1482 cm−1 was identified
(Supporting Information, Figure S17). The N−O stretching
frequencies for 1-NO− to 4-NO− are in agreement with
previous literature values as listed Table 2 and further
illustrated in Figure 7. Importantly, the N−O stretching
Figure 5. UV−visible absorption spectra for the spectroelectrochem-
ical reduction of [Fe(3,5-Me-BAFP)(NO)] (1-NO, red to green),
obtained by sweeping from −0.4 V to −1.8 V vs Ag wire at a rate of 10
mV/s in a 0.1 M TBAP solution in dry (top) 1,2-DCE and (bottom)
THF. The reaction is chemically completely reversible upon sweeping
from −1.8 V to −0.4 V vs Ag wire (inset).
five-coordinate 1-NO in 1,2-DCE. This strongly suggests that
the reduction product of six-coordinate [Fe(3,5-Me-BAFP)-
(NO)(THF)] is five-coordinate [Fe(3,5-Me-BAFP)(NO)]−
(1-NO−). As such, this indicates that the thermodynamic σ-
trans effect of NO− is actually stronger than that of NO·, which
is further discussed below.56,57 As in the IR spectroelec-
trochemical measurements, this reduction is chemically fully
reversible as shown in the inset of Figure 5. Finally, all attempts
at chemical reduction (sodium anthracenide, KC8) and
isolation of 1-NO− at room- and low-temperature were
unsuccessful.
Figure 7. Comparison of N−O stretching frequencies in {FeNO}7 and
{FeNO}8 porphyrin complexes.
The spectroelectrochemical reductions of 2-NO, 3-NO, 4-
NO were also performed. The first half-wave reduction
potentials of 2-NO and 4-NO are −1.18 V vs Fc/Fc+, and
E1/2 for the first reduction of 3-NO is slightly more positive at
−1.13 V vs Fc/Fc+ (Table 3). The UV−visible spectral changes
upon reduction of 2-NO are quite similar to those of 1-NO, see
Supporting Information, Figure S12. A decrease is observed in
the band at 472 nm as two bands in the Q-region, at 519 and
frequencies of the {FeNO}8 complexes show a surprisingly strong,
direct correlation with the ν(N−O) frequencies in the
corresponding {FeNO}7 precursors. This indicates strongly
correlated electronic structures in corresponding {FeNO}7
and {FeNO}8 pairs, and this is discussed in detail in Section 3.4.
3.3. The trans Effect of NO− in Low-Spin {FeNO}8
Complexes. Kadish and Ryan studied previously the reduction
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of {FeNO}7 complexes in the presence of pyridine (py) or
other N-donor ligands.19 In particular, it was shown by Choi
and Ryan that the py binding constant to [Fe(TPP)(NO)] of
0.7 M−1 drops to ≤0.03 M−1 in [Fe(TPP)(NO)]−,56 providing
a first indication that the NO− ligand actually has a stronger
trans effect than NO. In our investigations, we used 1-
methylimidazole (MI) as a model for His in proteins. The
corresponding binding constants of MI for our {FeNO}7
complexes, determined as described in the Experimental
Section, are listed in Table 4. These results show that the
confirms these findings: the observed stretching frequency
upon reduction of [Fe(To-F2PP)(MI)(15N18O)] at 1405 cm−1
is exactly identical to 2-15N18O−. The reduction is chemically
completely reversible (see Supporting Information, Figures S21
and S22) and after reoxidation the starting six-coordinate
complexes, 2MI-NO and 2MI-15N18O are regenerated.
Increasing the amount of MI to 170 equiv still shows
formation of N−O stretching frequencies at 1473 and 1405
cm−1 for the natural abundance isotopes and 15N18O
complexes, respectively. Using this, we can estimate the upper
limit of the MI binding constant to 2-NO− to be 0.2 M−1.
Using the Van’t Hoff equation at 298.15 K this corresponds to
an unfavorable Gibbs free energy, ΔG, of +1 kcal/mol for MI
binding as listed in Table 4. This Keq is calculated assuming
10% conversion to 2MI-NO− at 170 equiv of MIwhich is
likely an overestimate. As a result, the actual Keq for MI binding
to 2-NO is, in reality, significantly lower than 0.2 M−1. DFT
geometry optimizations and calculated N−O stretching
frequencies of [Fe(P)(MI)(NO)] and [Fe(P)(MI)(NO)]−
support the strengthened thermodynamic σ-trans effect of
NO− in {FeNO}8 porphyrin complexes compared to NO in the
{FeNO}7 analogues. The BP86/TZVP calculated Fe−NMI
bond length in [Fe(P)(MI(NO)]− is 2.45 Å (see Figure 9),
close to nonbonding compared to 2.18 Å for [Fe(P)(MI)-
(NO)] (see Table 5). Hence, NO− has in fact the strongest
trans effect when compared to NO, CO, O2, and other
diatomics in ferrous heme complexes!
Table 4. Equilibrium Constants, Keq [M−1], and Free
Reaction Energies, ΔG [kcal/mol], for the Reaction of
[Fe(TPP*)(NO)] + MI ⇄ [Fe(TPP*)(MI)(NO)]
complex
[Fe(TPP)(NO)]
Keq
26
ΔG
ref
−1.9
−2.6
−3.9
−4.5
≫ +1
58
[Fe(3,5-Me-BAFP)(NO)] (1-NO)
[Fe(To-(NO2)2-p-tBuPP)(NO)] (4-NO)
[Fe(To-F2PP)(NO)] (2-NO)
76
t.w.
t.w.
58
714
2055
≪ 0.2
[Fe(To-F2PP)(NO)]− (2-NO−)
t.w.
largest Keq for MI binding is found for 2-NO, 2055 M−1,58
which is therefore most favorable to (potentially) observe MI
binding to an {FeNO}8 complex. With the addition of 50 equiv
of MI, the N−O stretching frequency for the six-coordinate
{FeNO}7 complex, 2MI-NO, is observed at 1636 cm−1. This
feature decreases upon one-electron reduction and a new band
at 1473 cm−1 appears, corresponding to the {FeNO}8 complex.
This is the same ν(N−O) as observed for 2-NO−, suggesting a
loss of MI upon formation of the reduced product. As shown in
Figure 8, the spectra obtained by reduction of 2-NO and 2MI-
NO are identical, demonstrating formation of five-coordinate 2-
NO− in both cases. According to BP86/TZVP calculated N−O
stretching frequencies of five-coordinate [Fe(P)(NO)]− and
six-coordinate [Fe(P)(MI)(NO)]−, binding of MI to [Fe(P)-
(NO)]− should shift ν(N−O) to lower energy by at least 15
cm−1 as shown in Table 5. 15N18O isotope labeling further
3.4. Electronic Structure of {FeNO}8 Porphyrin
Complexes in Comparison to the Analogous {FeNO}7
Species. The new spectroscopic data reported in this study for
the complexes 1-NO− to 4-NO− significantly broaden our
knowledge base of corresponding {FeNO}8 heme complexes
(see Table 2) and, for the first time, allow for a detailed
correlation of the electronic structures of analogous {FeNO}7
and {FeNO}8 species. As shown in Figure 7, there is a
surprisingly strong correlation between the N−O stretching
frequencies of analogous {FeNO}7 and {FeNO}8 complexes.
This implies that the nature of the singly occupied molecular
orbital (SOMO) that is occupied with a second electron upon
reduction of the complexes from {FeNO}7 to {FeNO}8 does
not change to a significant degree in this process; that is,
whatever the composition of this MO is in the {FeNO}7
complex is preserved in the {FeNO}8 case. This further implies
that the properties of the {FeNO}8 complexes investigated here
in detail actually provide insight into the nature of the SOMO
in the {FeNO}7 precursors, and in this way, into the electronic
structures of both the {FeNO}7 and {FeNO}8 complexes.
Based on previous work,57 detailed descriptions of the
electronic structures of five- and six-coordinate ferrous heme-
nitrosyls, {FeNO}7, have been obtained. In these complexes,
iron is in the +2 oxidation state and low-spin, leading to a
[t2]6[e]0 electron configuration of the metal. NO is a radical
with one unpaired electron, which causes the resulting Fe(II)-
NO adduct to have a total spin of S = 1/2. Hence, from a
theoretical point of view, the spin-unrestricted scheme has to be
applied to analyze bonding in the {FeNO}7 complexes, which
distinguishes between majority (α) and minority (β) spin MOs.
In the five-coordinate case, strong donation from the singly
occupied π* orbital of NO that is located in the Fe−N−O
plane (α−π*h (h = horizontal) in the spin-unrestricted
Figure 8. NO − 15N18O IR difference spectra for the spectroelec-
trochemical reduction of [Fe(To-F2PP)(NO)] in the absence (A:
{FeNO}7, C: {FeNO}8) and presence (B: {FeNO}7, D: {FeNO}8) of
MI.
2
formalism) into the empty dz orbital of iron is observed,
leading to the formation of a strong Fe-NO σ bond. The
SOMO that results from this interaction is the bonding
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Table 5. BP86/TZVP Calculated Geometric and Vibrational Parameters of Five- and Six-Coordinate {FeNO}7 and {FeNO}8
Heme Complexes (P2− = porphine dianion)
geometric parameters [Å] [deg]
vibrational frequencies [cm−1
]
complex
ΔFe−NNO
ΔN−O
ΔFe−NMI
ΔFe−Nporph
∠Fe−N−O
ν(Fe−NO)
ν(N−O)
five-coordinate
[Fe(P)(NO)]
[Fe(P)(NO)]−
1.704
1.786
1.179
1.206
2.019
2.011
146
125
595
1703
1533
568/(428)
six-coordinate
[Fe(P)(MI)(NO)]
[Fe(P)(MI)(NO)]−
1.734
1.805
1.186
1.210
2.179
2.451
2.021
2.015
140
124
609
1662
1518
543/(434)
responsible for the activation of the NO sensor soluble
guanylate cyclase.62,63
Although basic agreement has been achieved in the literature
on the overall electronic structure description of ferrous heme-
nitrosyls as described above, the specific details are still
controversial. The reason for this is that DFT methods are
generally not very accurate in describing the properties of the
Fe−N−O unit in these complexes.57,62,64 In particular, the spin
density distribution, that is, the distribution of the unpaired
electron of NO over the Fe-NO unit, is strongly affected by the
chosen DFT method,65 as documented nicely by Pierloot and
co-workers.66,67 Whereas gradient-corrected functionals gen-
erally lead to metal-based spin (>60% spin density on iron),
hybrid functionals give a more unified distribution of the spin
density over the whole Fe−N−O unit.57,67 It is therefore most
important to compare calculated properties to experiment to
better assess the quality of the calculated results, in particular
spectroscopic properties are a good way to gauge the quality of
quantum-chemical calculations. In principle, EPR g values and
hyperfine coupling constants (especially those of the
coordinated 14N atom of NO) should be a good experimental
probe for the spin density distribution in ferrous heme-
nitrosyls. In this case, it has been shown that gradient-corrected
functionals59,68,69perform slightly better than hybrid func-
tionals59 for the calculation of g tensors and hyperfine coupling
constants. These types of calculations, however, generally show
quite large deviations from experiment and are also strongly
dependent on the geometry and the applied basis set.70 Thus, it
is difficult to judge the quality of the overall description solely
based on comparisons of EPR parameters.65 On the other hand,
calculated Fe-NO vibrational frequencies and force constants,
which directly reflect the strength of the Fe-NO bond, show
very clear trends when comparing the results from calculations
using gradient-corrected and hybrid functionals. Here, gradient-
corrected functionals tend to overemphasize electron delocal-
ization and, as demonstrated now for many cases,57 lead to an
overestimation of metal−ligand covalencies, and hence, bond
strengths. This is reflected by the calculated Fe-NO stretching
frequencies in five- and six-coordinate ferrous heme-nitrosyls,
predicted at about 600 cm−1, whereas experimentally, these are
observed at 515−530 and ∼440 cm−1, respectively (see Table
2). In contrast, B3LYP predicts the Fe-NO stretch at 540−580
and ∼420 cm−1 for the five- and six-coordinate {FeNO}7
complexes, respectively, which is in much better agreement
with experiment (see also ref 57). The overestimate of the
covalency of the Fe-NO bond with the gradient-corrected
functionals goes along with a significant quenching of the spin
density on the NO ligand.
Figure 9. Model system [Fe(P)(MI)(NO)]−, where P = porphine2−
and MI = 1-methylimidazole, and applied coordinate system. The
structure shown is calculated with BP86/TZVP.
Scheme 1. Molecular Orbitals Proposed to Be Responsible
for the σ-trans Effect of NO in Six-Coordinate Ferrous Heme
Complexes
2
2
combination of α−π*h and α-dz , labeled π*h_dz in Scheme 1,
left. Based on experimentally calibrated DFT (B3LYP)
calculations,58,59 this leads to a complete delocalization of the
unpaired electron of NO, with resulting spin densities of about
50% on Fe and 50% on NO.60 In addition, strong π-
backbonding is observed between the unoccupied π*v orbital
of NO (v = vertical, orthogonal the Fe−N−O plane) and the
dyz orbital of iron (in the applied coordinate system where the z
axis is aligned with the heme-normal, and the Fe−N−O unit is
in the xz plane). For a more detailed analysis see ref 57, 60.
Additional contributions to the backbond are observed between
the unoccupied β−π*h orbital of NO and β-dxz of iron. Upon
coordination of an N-donor ligand (imidazole or His) in trans
position to NO, a distinct weakening of the Fe-NO σ bond is
observed. This induces a distinct drop in the Fe-NO force
constant and corresponding Fe-NO stretching frequency in the
six-coordinate case as observed experimentally.58,61 In addition,
the underlying σ-trans interaction between NO and imidazole
leads to weak binding of imidazole in trans position to NO (Keq
is usually <50 M−1).57 This mechanism, the strong (thermody-
namic) σ-trans effect of NO in low-spin {FeNO}7 complexes, is
Because of this, the spin density distributions calculated with
hybrid functionals (see above) can overall be expected to be
more reliable, and hence, these should be in closer agreement
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Scheme 2. Electronic Structure of Low-Spin {FeNO}7 and
{FeNO}8 Complexes
with the electronic structures of the real complexes. Calculated
Fe-NO binding energies further support these conclusions.
Hybrid functionals are in fact able to predict quite accurate Fe-
NO binding energies when the important van-der-Waals
interactions are included.71 In contrast, gradient-corrected
functionals greatly overestimate NO binding energies,71 in
agreement with the overestimation of the Fe-NO bond
strengths in these cases.
Importantly, the experimental properties of the analogous
{FeNO}8 complexes investigated here provide further key
insight into the properties of the SOMO in {FeNO}7 systems.
Whereas previous DFT results characterize the SOMO of
contributions of this MO are in fact invariant to the one-
electron reduction. This is further illustrated in Scheme 2.
Third, previous work by Ryan and co-workers on [Fe(TPP)-
(NO)] has shown that the Fe-NO stretching frequency
increases in {FeNO}8 compared to the analogous {FeNO}7
complexes (see Table 2).20 This is because the one-electron
reduction leads to an increase in σ bonding by double
occupation of the SOMO (accompanied by a reduction in π
backbonding between β−π*h and β-dxz), causing a noticeable
strengthening of the Fe-NO bond upon reduction. This finding
disagrees with the idea that the SOMO is strongly π
antibonding in nature; in this case, occupation of this MO
should lead to a distinct weakening of the Fe-NO bond, and a
significant drop in the Fe-NO stretching frequency, which is
not observed experimentally. In conclusion, all these findings
provide strong evidence that the SOMO of {FeNO}7
complexes is predominantly σ-bonding in nature, whereas the
admixture of π*h_dxz character is small.
ferrous heme-nitrosyls as the bonding combination of α−π*h
58,72
2
2
and α-dz , π*h_dz , as described above (see Scheme 1, left),
it was recently proposed that for the corresponding six-
coordinate complexes, this orbital should be considered the
antibonding combination between α−π*h and α-dxz, resulting in
a SOMO that is strongly π-antibonding in nature as illustrated
in Scheme 1, right.73 This would de facto eliminate the Fe-NO
σ bond in six-coordinate {FeNO}7 complexes. However, there
are several experimental observations that argue against this
notion.65 First, the strong thermodynamic σ-trans effect of NO
requires the presence of a distinct σ bond; in comparison, the
strongly π backbonding ligand CO does not mediate much of a
trans effect.62,74 Second, the results of this study demonstrate
that adding an electron to the SOMO of six-coordinate
{FeNO}7 complexes leads to a further increase of the trans
effect as discussed above, which is evident from a further,
dramatic decrease of the MI binding constant in the {FeNO}8
case. As inferred from the strong correlation of ν(N−O)
discussed above (see Figure 7), DFT calculations further
confirm that this is not due to a change in the nature of the
SOMO, but simply caused by the addition of one electron to
this MO. As shown in Figure 10 and Table 6, the charge
3.5. Reactivity of {FeNO}8 Complexes. Bulk Electrolysis
in the Presence of Acid. Initial attempts at protonation of
Fe(II)-NO− heme complexes were focused on bulk electrolysis
of the corresponding {FeNO}7 complexes (at mM concen-
trations) in the presence of several equivalents of acetic acid
(see also ref 75). For example, reduction of 2-NO in THF at
room temperature at −0.9 V vs Ag wire resulted in a ferrous
product by UV−visible and EPR spectroscopy. The product,
however, does not show any isotope sensitive IR bands in the
1700−1200 cm−1 region as would be expected for a ferrous
nitrosyl or nitroxyl product complex, and analysis of the
reaction head space did not show the presence of any N2O.
Interestingly, coulometry indicated that the reaction continued
to progress well past one equivalent of electrons. In fact, the
current did not stabilize until ∼5 equiv of electrons were
passed. This implies that the formed Fe(II)-NHO complex is
further reduced under our electrolysis conditions. As proposed
previously for heme systems,76,77 we expect our reaction to
proceed as follows
2
Figure 10. Key π*h_dz /dxz molecular orbitals of (left) [Fe(P)(MI)-
(NO)] and (right) [Fe(P)(MI)(NO)]− which defines the thermody-
namic σ-trans effect in ferrous porphyrin systems. Calculated with
B3LYP/TZVP on BP86/TZVP optimized structures.
Fe(II)−NO(radical) + e− → Fe(II)−NO−
(3)
Fe(II)−NO− + H+ → Fe(II)−NHO
(4)
a
Table 6. Charge Contributions of Key σ Bonding Orbitals
for [Fe(P)(MI)(NO)]0/1−
Fe(II)−NHO + 2e− + 2H+ → Fe(II)−NH2OH
(5)
Fe(II)−NH2OH + 2e− + 2H+ → Fe(II) + NH3 + H2O
Fe
d
NO
s+p
NMI
s+p
complex
orbital
label
(6)
2
[Fe(P)(MI)(NO)]
[Fe(P)(MI)(NO)]−
⟨120⟩
⟨122⟩
π*h_dz /dxz
27
30
58
57
2
1
where the reduction potentials of the intermittently formed
Fe(II)-NHO and Fe(II)-NH2OH complexes observed here are
more positive than that of 2-NO (in agreement with ref 75).
Indeed, ammonia analysis using Russell’s hypochlorite-
phenol25,26 method yielded ∼1 equiv of NH3 in the product
mixture. As such, we propose our product to be a ferrous heme
2
π*h_dz
a
Calculated with B3LTP/TZVP for BP86/TZVP optimized structures
(P2− = porphine dianion).
J
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Figure 11. UV−vis spectra for the one-electron reduction of [Fe(To-F2PP)] (2, blue) to [Fe(To-F2PP)]− (2−, purple), shown at left, and
subsequent reaction with 100 μL of NO(g) (right, green) in THF at room temperature.
complex with bound ammonia or water. Similar reactivity was
observed for the reduction of 1-NO in the presence of acetic
acid. Based on these observations, we speculate that ferrous
hemes could act as catalysts for the electrochemical reduction
of NO to NH3 and water, similar to assimilatory nitrite
reductases.75 However, because of the unfavorable reduction
potential of the first step (eq 3), these catalysts would not be
very energy efficient. Furthermore, this procedure can therefore
not be applied for the preparation of ferrous heme-HNO
complexes.
As protonation of the formed {FeNO}8 complexes in the
presence of an applied potential results in further reduction of
the generated Fe(II)-HNO species, it is essential to separate the
reduction of the {FeNO}7 complex from the protonation of the
Figure 12. UV−visible spectra for the reaction of [Fe(To-F2PP)-
resulting {FeNO}8 species, see eqs 3 and 4 above. To
accomplish this task, bulk electrolysis of 2-NO was performed
in the absence of acid to first convert all of the material into 2-
NO−. This step could then be followed by addition of acid.
However, the reduction of 2-NO is unfortunately unreliable
and often leads to significant decomposition of 2-NO−, so this
process is also not feasible for practical applications.
(NO)]− (2-NO−, green) with 5 equiv of acetic acid in THF at room
temperature. The resulting spectrum (red) is in agreement with
formation of [Fe(To-F2PP)(NO)] (2-NO).
study, but may correspond to a hyponitrite type intermediate.
2-NO− reacts further with free NO in solution to form 2-NO,
as evident from UV−visible spectroscopy. To combat this
reaction, as soon as 2-NO− is generated, the solution is sparged
with inert gas to remove free NO from the reaction vessel. The
further reaction of 2-NO− with NO suggests that 2-NO−
actually reduces free NO, forming 2-NO and NO−, the latter
then decomposes in an unknown fashion. In fact, the reduction
potential of free NO is −0.8 V vs SHE,80 more positive than the
resting potential of 2-NO−. This result indicates that the
reaction of NO with deprotonated Fe(II)-NO− complexes does
not induce N−N bond formation, which therefore likely
requires the presence of protons (see discussion in ref 17).
Therefore, the protonation of 2-NO− was explored next.
Generation of the First Ferrous Heme-HNO Model
Complex. Addition of acetic acid to 2-NO− in THF resulted
in the formation of 2-NO, as shown in Figure 12. EPR
spectroscopy of the reaction mixture shows the characteristic S
= 1/2 signal, indicative of a low-spin ferrous heme-nitrosyl
complex (data not shown). This is similar to the reactivity of
[Fe(TPP)(NO)]− and [Fe(TFPPBr8)(NO)]− with acid where
the corresponding {FeNO}7 complex and 0.5 equiv of H2 are
reported as products20,22
Generation of {FeNO}8 Complexes via Iron(I) Intermedi-
ates. Because of these difficulties, we devised an alternate route
based on the observation that ferrous hemes can be reversibly
reduced by bulk electrolysis (eq 7).78 The generated, formally
iron(I), species (this actually corresponds to a reduction of the
porphyrin ligand as discussed below) could then in principle be
reacted with NO, resulting in the desired {FeNO}8 complex:
[Fe(porphyrin)] + e− → [Fe(porphyrin)]−
[Fe(porphyrin)]− + NO → [Fe(porphyrin)(NO)]−
(7)
(8)
Here, the porphyrin ligand stores the electron necessary for
reduction of the Fe-NO unit. This approach has been applied
to [Fe(To-F2PP)] (2) and the resulting in situ UV−visible
spectra are provided in Figure 11. Upon one-electron reduction
in THF, the sharp Soret band at 422 nm decreases in intensity
while new broad features at 364 and 386 nm appear (Figure 11,
left). This drastic decrease in intensity is characteristic of a loss
in porphyrin conjugation indicating that the product complex is
formally a Fe(II)-porphyrin•− (porphyrin radical) species.79
Upon addition of 100 μL of NO gas to 2−, see Figure 11
(right), the desired 2-NO− is generated. Interestingly, if only 10
μL of NO gas are added, a unique species with a Soret band at
416 nm is observed (Supporting Information, Figure S24). The
nature of this species (likely a ferrous complex) requires further
2[Fe(porphyrin)(NHO)]
→ 2[Fe(porphyrin)(NO)] + H2
(9)
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Figure 13. UV−vis spectra for the one-electron reduction of [Fe(3,5-Me-BAFP)] (0.2 mM 1, blue) to [Fe(3,5-Me-BAFP)]− (1−, red), shown at left,
and subsequent reaction with 100 μL of NO (g) in THF at room temperature resulting in formation of [Fe(3,5-Me-BAFP)(NO)]− (1-NO−, right,
green). Inset: same reactions at ∼0.02 mM heme concentrations.
This result is perhaps not surprising as To-F2PP2−, similar to
TPP2− and TFPPBr82−, lacks the steric protection needed to
prevent this disproportionation of the Fe-NHO unit. In this
sense, the reaction of the bis-picket fence porphyrin complex
[Fe(3,5-Me-BAFP)(NO)]− (1-NO−) with acid is of extreme
interest.
undergoes decomposition to 2-NO. To further confirm the
identity of the heme HNO complex, 2 equiv of phosphazene
base (P1-tBu-tris(tetramethylene)) was reacted with the
presumable formed 1-NHO. Upon addition of base, see Figure
15, UV−visible spectroscopy shows essentially complete
To this end, bulk electrolysis of [Fe(3,5-Me-BAFP)] (1) in
THF was performed at room temperature. The in situ UV−
visible spectra for the one-electron reduction to 1− are reported
in Figure 13 (left). EPR spectroscopy (Supporting Information,
Figure S25) shows a sharp isotropic spectrum with a g-value of
1.99 for 1− indicating porphyrin ring reduction to an S = 1/2
complex. Addition of 100 μL of NO to 1− at room temperature
results in formation of 1-NO− with a Soret band at 415 nm
(consistent with spectroelectrochemical measurements), as
shown in Figure 13 (right). Subsequent reaction of 1-NO−
with ∼1.4 equiv of acetic acid indicates formation of a new
complex, [Fe(3,5-Me-BAFP)(NHO)] (1-NHO), with the
Soret band at 426 nm and the Q-band observed at 545 nm,
see Figure 14. Excitingly, this species does not correspond to 1-
NO, which instead shows a Soret band of 422 nm in THF. In
addition, gas IR analysis of the reaction headspace after addition
of acid indicates no formation of N2O (Supporting
Information, Figure S26), which proves that the complex
does not simply lose HNO after protonation. This is in contrast
to the protonation of 2-NO−, discussed above, which instantly
Figure 15. UV−visible spectrum for the reaction of 0.2 mM [Fe(3,5-
Me-BAFP)(NO)]− (1-NO−, red) with 1.4 equiv of acetic acid (blue)
to generate 1-NHO followed by deprotonation of 1-NHO with 2
equiv of phosphazene base, P1-tBu-tris(tetramethylene) (BTPP, black)
to regenerate 1-NO−.
reformation of 1-NO−. Further addition of acetic acid then
results in ∼quantitative reformation of 1-NHO. Finally, the
UV−vis spectral properties of 1-NHO are similar to Farmer’s
Mb(II)-NHO complex.23 Both compounds show typical
ferrous porphyrin absorption spectra with Soret band positions
at 423 (Mb-NHO complex) and 426 nm (1-NHO), further
supporting the idea that 1-NHO in fact contains a bound HNO
ligand.
The formed 1-NHO complex is EPR silent and decays at a
slow rate to 1-NO as evidenced by EPR, see Supporting
Information, Figure S27, and UV−visible spectroscopy. Spin
quantification of the EPR spectra (Supporting Information,
Figure S28) indicate that after 2 h (0.2 mM, room temperature)
27% of the formed 1-NHO is decomposed to 1-NO and after
20 h the decomposition is essentially complete (90% of 1-NO
are detected). This is consistent with UV−visible spectroscopy.
Thus, for the first time, we are able to delay the
disproportionation of bound HNO through the use of a bis-
picket fence porphyrin to generate the first stable ferrous heme-
Figure 14. UV−visible spectra for the reaction of [Fe(3,5-Me-
BAFP)(NO)]− (0.2 mM 1-NO−, green) with 5 equiv of acetic acid in
THF at room temperature. The resulting spectrum is shown in purple.
Inset: same reaction at ∼0.02 mM heme concentration.
L
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HNO model complex reported to this date. With this complex
in hand, detailed reactivity studies on a heme-bound HNO
complex can be performed in the future. Here, we explored
whether a ferrous heme-HNO complex is reactive toward NO,
as proposed recently in a DFT study on the mechanism of
P450nor.14 Surprisingly, addition of low equivalents of NO to
1-NHO at room temperature resulted in immediate formation
of 1-NO as evident from UV−vis spectroscopy, where HNO is
presumably displaced by the stronger ligand NO. No N−N
bond formation results from the reaction of 1-HNO with NO,
indicative that ferrous heme-HNO complexes are not
competent intermediates in NO reductases. To further solidify
this conclusion, analogous investigations on six-coordinate
HNO complexes with an axial MI ligand are necessary, which
are currently in progress. The biological implications of these
results are discussed in the next section.
perform reactivity studies, a preparation of bulk material of the
reduced complexes is necessary, which has been generally
found difficult. We were able to achieve this via a completely
new synthetic approach that starts from the four-coordinate
ferrous precursor, [Fe(porphyrin)], which can be bulk-reduced
by one electron to yield the stable (under rigorous exclusion of
O2), formally iron(I) complex [Fe(porphyrin)]−. Importantly,
this species can then be reacted with NO to generate bulk
material of pure {FeNO}8 complexes as shown in this work.
The reactivity of the resulting nitroxyl complexes was then
tested. First, the complex [Fe(To-F2PP)(NO)]− reacts with
acetic acid to generate the corresponding {FeNO}7 complex
and 0.5 equiv of H2, as previously reported for similar species,
because of disproportionation of an intermittently formed
HNO complex. However, the corresponding sterically hindered
3,5-Me-BAFP2− complex shows unique reactivity, effectively
blocking the disproportionation of bound HNO and generating
the first ferrous heme-HNO model complex, [Fe(3,5-Me-BAFP)-
(NHO)]. This HNO complex is stable in solution at room
temperature for several hours and can be further deprotonated
with phosphazene base to regenerate the corresponding
[Fe(3,5-Me-BAFP)(NO)]− species. Hence, the bis-picket
fence porphyrin provides a unique stabilization for the heme-
bound HNO that has not been observed with any other
porphyrin.
4. CONCLUSIONS
In this work, the new bis-picket fence porphyrin ferrous-nitrosyl
complex [Fe(3,5-Me-BAFP)(NO)] has been prepared and
crystallographically characterized, showing a unique conforma-
tion of the Fe-NO unit. This complex, along with three ferrous
heme-nitrosyls with electron-poor porphyrins, is then used to
generate a series of four new ferrous heme-nitroxyl, {FeNO}8,
complexes. The N−O stretching frequency of the resulting,
reduced complex [Fe(3,5-Me-BAFP)(NO)]− is observed at
1466 cm−1. With the electron-poor hemes, the nitroxyl
complexes show higher values for ν(N−O) in the 1470−
1500 cm−1 range. With these new nitroxyl complexes in hand,
the number of well-characterized heme {FeNO}8 complexes is
now increased to seven, which, for the first time, allows for the
systematic analysis of trends in the properties of these species
(see Table 2). Importantly, a very strong correlation of ν(N−
O) between the {FeNO}7 precursors and the resulting
{FeNO}8 species is observed, which indicates that the nature
of the SOMO that becomes double occupied when the
{FeNO}7 complex is reduced (see Scheme 2) does not change
in the {FeNO}8 species. In other words, whatever the nature of
this MO is in the {FeNO}7 complex is preserved in the
{FeNO}8 species, and this result is further supported by DFT
calculations. We further investigated whether a six-coordinate
{FeNO}8 complex can be generated using the six-coordinate
precursor [Fe(To-F2PP)(MI)(NO)], which has a very high
affinity for MI.58 However, upon one-electron reduction, this
complex forms the five-coordinate species [Fe(To-F2PP)-
(NO)]−. We estimate the binding constant, Keq, for MI binding
to [Fe(To-F2PP)(NO)]− to be ≪0.2 M−1, at least 4 orders of
magnitude smaller than that of MI binding to [Fe(To-
F2PP)(NO)], in good agreement with previous results for py
binding to [Fe(TPP)(NO)]−.56 These results demonstrate that
NO− has the strongest trans effect when compared to NO, CO,
and O2 in ferrous heme systems. The increase of the trans effect
upon one-electron reduction of the {FeNO}7 complex, in
combination with the strong correlation of the electronic
structures of analogous {FeNO}7 and {FeNO}8 complexes
mentioned above, provides further direct experimental proof
that the SOMO in ferrous heme-nitrosyls is in fact Fe-NO σ-
bonding in nature. This finding, although quite established in
the literature, has recently been challenged.73
Finally, the reactivity of {FeN(H)O}8 complexes toward NO
was tested. First, reaction of [Fe(To-F2PP)(NO)]− with NO
results in reduction of free NO to NO− and oxidation of the
ferrous nitroxyl complex to [Fe(To-F2PP)(NO)], and a similar
process is seen for [Fe(3,5-Me-BAFP)(NO)]−. Second,
reaction of [Fe(3,5-Me-BAFP)(NHO)] with NO leads to
displacement of the HNO ligand by NO, generating [Fe(3,5-
Me-BAFP)(NO)], but no direct N−N bond formation is
observed, which would generate a ferric heme-hyponitrite
complex. These results have important biological implications.
The lack of reactivity of the heme-HNO complex with NO with
respect to N−N bond formation indicates that a ferrous heme-
HNO complex is likely not catalytically competent for N−N
bond formation and N2O generation in Cyt. P450nor. This
provides strong evidence, as proposed originally,13,14 that a
second protonation of the HNO complex is required to achieve
hyponitrite formation. With respect to NorBC, recent
computational studies have indicated that {FeNO}8 and in
particular, {FeNHO}8 species should react with NO to induce
N−N bond formation and hyponitrite generation,17 but
experimentally, we do not observe this reaction for either one
of these complexes, casting doubt on the mechanistic proposal
of a cis-heme b3 mechanism81,82 for this enzyme.
Future studies will focus on the characterization and isolation
of [Fe(3,5-Me-BAFP)(NHO)] (1-NHO) and the generation of
a corresponding six-coordinate complex with bound MI. The
latter species can then be used to explore whether the presence
of the axial ligand could influence the reactivity of the HNO
complex with NO. Please note in this regard that the isolation
of nitroxyl complexes after bulk electrolysis is difficult because
of the large amounts of electrolyte present in solution, which
also interferes with all attempts to crystallize 1-NHO. New
procedures are currently being developed to achieve this
without decomposition of the complex. This work is in
progress.
The studies into the spectroscopic properties and electronic
structures of ferrous heme-nitroxyl complexes mentioned above
are all based on spectroelectrochemical measurements.
However, to prepare a ferrous heme-HNO complex and to
M
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(20) Choi, I. K.; Liu, Y.; Feng, D. W.; Paeng, K. J.; Ryan, M. D. Inorg.
ASSOCIATED CONTENT
■
S
* Supporting Information
Further spectroscopic characterization (EPR, IR) of 1-NO−4-
NO, spectroelectrochemical data for the reduction of 1, 1-NO−
4-NO and 2MI-NO and the corresponding 15N18O-labeled
complexes, UV−vis titration of 1-NO and 4-NO with MI,
spectroscopic data for the generation and slow decomposition
of 1-NHO, H NMR data for the synthesis of H2[(3,5-Me-
BAFP)], and the complete ref 40. This material is available free
of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: lehnertn@umich.edu.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported by the National Science Foundation
(CHE-0846235 to N.L.). We acknowledge Dr. Jeff Kampf
(University of Michigan) for his X-ray crystallographic analysis
of [Fe(3,5-Me-BAFP)(NO)] (1-NO), and funding from NSF
Grant CHE-0840456 for X-ray instrumentation. We particularly
thank Dr. Michael D. Ryan (Marquette University) for helpful
discussions on spectroelectrochemical techniques, and Ms.
Ashley McQuarters for determining the extinction coefficients
of [Fe(3,5-Me-BAFP)(NO)] and [Fe(3,5-Me-BAFP)].
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