Synthetic Iron Porphyrins for Probing the Differences in the Electronic Structures of Heme a3, Heme d, and Heme d1

Article abstract of DOI:10.1021/acs.inorgchem.8b02063

A variety of heme derivatives are pervasive in nature, having different architectures that are complementary to their function. Herein, we report the synthesis of a series of iron porphyrinoids, which bear electron-withdrawing groups and/or are saturated at the β-pyrrolic position, mimicking the structural variation of naturally occurring hemes. The effects of the aforementioned factors were systematically studied using a combination of electrochemistry, spectroscopy, and theoretical calculations with the carbon monoxide (CO) and nitric oxide (NO) adducts of these iron porphyinoids. The reduction potentials of iron porphyrinoids vary over several hundreds of millivolts, and the X-O (X = C, N) vibrations of the adducts vary over 10-15 cm^-1. Density functional theory calculations indicate that the presence of electron-withdrawing groups and saturation of the pyrrole ring lowers the π?-acceptor orbital energies of the macrocycle, which, in turn, attenuates the bonding of iron to CO and NO. A hypothesis has been presented as to why cytochrome c containing nitrite reductases and cytochrome cd₁ containing nitrite reductases follow different mechanistic pathways of nitrite reduction. This study also helps to rationalize the choice of heme a₃ and not the most abundant heme b cofactor in cytochrome c oxidase.

Full text of DOI:10.1021/acs.inorgchem.8b02063

Article
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
pubs.acs.org/IC
Synthetic Iron Porphyrins for Probing the Differences in the
Electronic Structures of Heme a , Heme d, and Heme d1
3
Sk Amanullah, Paramita Saha, Rajat Saha, and Abhishek Dey*
Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Kolkata 700032, India
*
S Supporting Information
ABSTRACT: A variety of heme derivatives are pervasive in
nature, having different architectures that are complementary
to their function. Herein, we report the synthesis of a series of
iron porphyrinoids, which bear electron-withdrawing groups
and/or are saturated at the β-pyrrolic position, mimicking the
structural variation of naturally occurring hemes. The effects of
the aforementioned factors were systematically studied using a
combination of electrochemistry, spectroscopy, and theoretical
calculations with the carbon monoxide (CO) and nitric oxide
(
NO) adducts of these iron porphyinoids. The reduction
potentials of iron porphyrinoids vary over several hundreds of
millivolts, and the X−O (X = C, N) vibrations of the adducts
−
1
vary over 10−15 cm . Density functional theory calculations
indicate that the presence of electron-withdrawing groups and saturation of the pyrrole ring lowers the π*-acceptor orbital
energies of the macrocycle, which, in turn, attenuates the bonding of iron to CO and NO. A hypothesis has been presented as to
why cytochrome c containing nitrite reductases and cytochrome cd containing nitrite reductases follow different mechanistic
1
pathways of nitrite reduction. This study also helps to rationalize the choice of heme a and not the most abundant heme b
3
cofactor in cytochrome c oxidase.
INTRODUCTION
to substrate oxidation using high-valent (oxoferryl heme)
■
13
intermediates. Sirohemes (iron-bound isobacteriochlorin)
Tetrapyrrole macrocycles are ubiquitous in nature and are
known to play a central role in most living organisms. Their
importance stems from the unique attributes of tetrapyrroles
for diverse bioenergetic processes by the versatility, concision,
and molecular logic of the tetrapyrrole biosynthetic pathway.
1
4,15
16,17
1
catalyze the reduction of nitrite
and sulfite;
cyto-
Heme d
iron-bound chlorin) is present in some catalases and
1
4,18,19
chrome cd is also involved in nitrite reduction.
1
(
1
9−21
2
peroxidases.
In prokaryotes (mainly in pathogenic
bacteria), cytochrome bd have distinct physiological roles as
All naturally occurring corrinoid, porphyrin, and (bacterio)-
chlorin macrocycles, collectively known as the pigments of life,
investigated so far are biosynthetically derived from
22
terminal oxidases. This clearly demonstrates the structure-
dependent reactivity relationship of porphyrin and porphyr-
ynoid macrocycles that is elegantly present in nature and is yet
to be understood. For example, multi-c-heme-containing nitrite
reductases (CcNiR) are known to be involved in dissimilatory
nitrite reduction to ammonium ion and siroheme-containing
nitrite reductases (CSNiR) are also known to be involved in
assimilatory nitrite reduction to ammonium ion, while heme c
and d containing nitrite reductases (Cd NiR) are involved in
3
uroporphyrinogen III. In higher plants, tetrapyrrole synthesis
occurs in plastids, where it is initiated by reduction of the
glutamyl moiety of glutamyl-tRNA to glutamate-1-semi-
aldehyde (Scheme 1).
glutamate semialdehyde generates 5-aminolevulinic acid
ALA), which is then transformed into uroporphyrinogen III,
4
,5
Intermolecular transamination of
(
1
1
by the catalytic activity of ALA dehydratase, hydroxymethylbi-
dissimilatory nitrite reduction to nitric oxide (Figure
lane synthase, and uroporphyrinogen III synthase (Scheme
14,19
5
−8
1B,C).
In cytochrome c oxidase (CcO) active site, a
1
).
Uroporphyrinogen III represents the first branch point
different kind of heme, heme a (which contains electron-
in the pathway because methylation of this intermediate leads
to the formation of siroheme, vitamin B , and cofactor F430,
3
withdrawing formyl and hydroxyfarnesyl groups), is present in
12
the O binding site (Figure 1A). Recently, the Lu group has
whereas decarboxylation leads toward heme and chlorophyll
2
3
demonstrated how the reduction potential of heme in a
functional model of CcO in myoglobin can be tuned up to
synthesis.
Although these porphyrinoids (porphyrins and porphyrin-
like macrocycles) have similar biosynthetic pathways, they play
distinctly different roles. Hemes (iron-bound protoporphyrin
IX) are commonly involved in a variety of biological
transformations using molecular O , ranging from O transport
23
∼
215 mV. The reason(s) for the different reactivities of these
1
2
Received: July 21, 2018
2
2
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.8b02063
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
3
Scheme 1. Schematic Diagram of the Biosynthetic Pathways of Naturally Occurring Porphyrinoids
9
Figure 1. Crystal structure of the enzyme active sites. (A) CcO, which contains heme a in the oxygen binding site (PDB 1OCR). (B) Nitrite-
3
10
bound pentaheme cytochrome c nitrite reductase, which contains heme c in the nitrite binding site (PDB 2E80). (C) Cytochrome cd nitrite
reductase, which contains heme d in the nitrite binding site (PDB 1NIR). The structures have been redrawn using Chimera 1.2rc software.
1
11
1
2
9
Figure 2. Representative molecules having saturated pyrrole centers: (A) Fe(OEC)Cl; (B) Fe(OEiBC)Cl. (C) Heme d mimic reported by Fujii
et al. (D) Cobalt chlorin reported by Nocera et al. (E) Cobalt chlorins reported by Fukuzumi et al.
1
37
35
38
enzymes is (are) still obscured. In the 1980s, a few groups had
iron octaethylchlorin (FeOEC; Figure 2A) and iron
attempted to understand the effect of saturating the pyrrole
rings by synthetic modeling and computational studies.
Holm and co-workers had synthesized reduced porphyrins like
octaethylisobactriochlorin (FeOEiBC; Figure 2B) by hydro-
24−27
25,28−30
genation of their unsaturated analogues.
They were
successful in attaching an isobacteriochlorin analogue to an
B
DOI: 10.1021/acs.inorgchem.8b02063
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Inorganic Chemistry
Article
iron−sulfur cluster, resulting in a synthetic mimic of the
sodium metal in the presence of benzophenone, until the color of the
solution turned intense blue. MeOH was first dried as toluene using
sodium after that it was distilled from magnesium cake.
3
1
Siroheme sulfite reductase active site. However, these
complexes are very unstable and are readily oxidized back to
iron octaethylporphyrin (FeOEP) and did not even sustain
Instrumentation. All electrochemical experiments were per-
formed using a CH Instruments (model CHI710D electrochemical
analyzer). Biopotentiostat reference electrodes were purchased from
CH Instruments. The absorption spectra were measured in the
Shimadzu spectrograph (UV-2100). The aerobic and anaerobic
cuvettes were purchased from Starna Scientific. The Fourier transform
infrared (FTIR) data were measured on a Shimadzu FTIR 8400S
instrument. The KBr windows for IR spectroscopy were purchased
from Sigma-Aldrich. The anaerobic setup for IR spectroscopy was
purchased from PerkinElmer. All of the NMR spectra were recorded
on a Bruker DPX-300, a Bruker DPX-400, or a DPX-500 spectrometer
at room temperature. The mass spectrometry (MS) spectra are
recorded by a QTOF Micro YA263 instrument. The EPR spectra
were recorded on a X-band JEOL (JES FA200) instrument. Single-
crystal X-ray diffraction (SCXRD) data were collected at 120 K using
radiation on a SMART APEX diffractometer equipped with a CCD
area detector with a graphite monochromator and Mo Kα (λ = 0.7107
Å) radiation. The structures were solved by the Patterson method,
followed by successive Fourier and difference Fourier synthesis. Full-
1
29
routine H NMR experiments. Albeit the seminal work by
Holm and co-workers, iron chlorins have not been investigated
in detail and, unfortunately, most of the effort in this area is
limited to a few groups, and further development in
understanding the role of the structural variations in
porphyrinoids awaits. Their abundance in the active sites of
metalloenzymes involved in the sulfur and nitrogen cycle and
some peroxidases compelled further research in trying to
understand their electronic structure and reactivity. Wei and
Ryan have reported nitric oxide (NO) complexes of FeOEP,
where pyrrole rings are oxidized to pyrrolidone and the NO
−
1
32
stretches of the complexes were lowered by 6−8 cm .
33,34
Recently, Fukuzumi and co-workers
(Figure 2E) and
Nocera and co-workers (Figure 2D) have used cobalt and
nickel chlorin for water splitting, renewing interest in these
35
arcane macrocycles and their potential reactivities. The Bro
group reported porphyrins with one saturation at the meso
̈
ring
2
matrix least-squares refinements were performed on F using
36
SHELXL-97 with anisotropic displacement parameters for all non-
hydrogen atoms. The hydrogen atoms were refined isotropically, and
their locations were determined from a difference Fourier map. All
position, forming isoporphyrin. Fujii et al. reported a model
for heme d in cytochrome cd nitrite reductase by varying keto
1
1
37
groups attached with the porphyrin.
39
40
calculations were carried out using SHELXL 97, SH43ELXS 97,
In this Article, a convenient method has been designed to
make stable iron chlorin analogues using a 1,3-cycloaddition
reaction to form C−C bonds akin to the role of methyl
transferases in biosynthetic pathways to form saturated
41
42
PLATON 99, ORTEP-32, and WinGX system v1.64.
Synthesis. The porphyrins diester porphyrin (DEsP)
tetraphenylporphyrin (TPP) were prepared following reported
procedures. The porphyrins were saturated using a 1,3-cycloaddition
reaction to get the chlorins, i.e., diester chlorin (DEsC) and
tetraphenylchlorin (TPC), respectively.
44,45
and
46
3
porphyrins. Both β-H’s of one of the four pyrroles are
replaced by ester groups for two reasons. First, this allows
regioselective 1,3-cycloaddition reaction (due to the attach-
ment of electron-withdrawing ester groups) with an
azomethine ylide, and, second, it gives a good opportunity to
probe the electronic structure of an iron porphyrin by
introducing two electron-withdrawing groups. This can be
FeDEsP. To a solution of DEsP (50 mg, 0.082 mmol) in 20 mL of
dry degassed THF was added 2,4,6-collidine (21 μL, 0.164 mmol),
and the solution was stirred for 10 min in a glovebox. FeBr (70 mg,
2
0
.328 mmol) was added and the solution stirred overnight. The
reaction mixture was worked up with dilute HCl, followed by the
addition of DCM. The organic layer was washed with a brine solution
and collected. It was dried over anhydrous Na SO and purified by
directly correlated with heme a , which has two electron-
3
2
4
withdrawing groups at the two β positions of the heme in the
active site of CcO to reduce oxygen to water (Figure 1A). The
column chromatography with silica gel (60−120 mesh size) using a
1% MeOH/DCM mixture as the eluent. Yield: 90 ± 2% (45 ± 5 mg).
1
pyrrole with ester groups is saturated to model heme d , which
λ
max (DCM, nm): 412, 575, 612. H NMR (CDCl
3
): δ 82.53,
1
1
1
8
0.51, 76.34 (paramagnetic shift for β-pyrrolic H’s and meso H’s of
Fe -HS complex). ESI-MS (positive-ion mode, MeOH): m/z 659.9
45%; [M − H] ). EPR (1 mM sample in dry THF, 4 K): an axial
signal with g values of 5.88 and 1.97 (Figure S29). Anal. Calcd for
is saturated and contains electron-withdrawing groups (Figure
III
1
1
1
C). The effect of these peripheral modifications on the
+
(
electronic structures of iron porphyrinoids is probed by
employing their NO and carbon monoxide (CO) adducts.
C H FeN O Cl: C, 65.58; H, 4.06; N, 8.05. Found: C, 64.93; H,
3
8
28
4
4
4
.44; N, 8.02 (FeDEsPCl, 0.5H O).
ZnDEsP. To a solution of DEsP (50 mg, 0.082 mmol) in 20 mL of
2
EXPERIMENTAL DETAILS
■
Materials. All reagents used are of the highest grade commercially
available. Iodine, trifluoroacetic acid, 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone, ethanol, aqueous ammonia solution, ceric ammonium
nitrate (CAN), and sarcosine were purchased from Spectrochem Ltd.
Diethyl ether, tetrahydrofuran (THF), acetonitrile (ACN), dichloro-
methane (DCM), and toluene were purchased from Rankem Ltd.
dry THF was added Zn(OAc) ·2H O (60 mg, 0.328 mmol), and and
2 2
the solution was stirred for 3 h at room temperature. The reaction
mixture was worked up with water, followed by the addition of DCM.
The organic layer was washed with a brine solution and collected. It
was dried over anhydrous Na SO and purified by column
2 4
chromatography with silica gel (60−120 mesh size) using a 1%
MeOH/DCM mixture as the eluent. Yield: 90 ± 5% (50 ± 5 mg).
The complex was crystallized by layering hexane over a solution in
Paraformaldehyde, anhydrous ferrous bromide (FeBr ), 2,4,6-
collidine, tetrabutylammonium perchlorate (TBAP), potassium
hexafluorophosphate (KPF ), and all buffers were purchased from
Sigma-Aldrich Chemical Company. Methanol (MeOH) was pur-
chased from Finar Limited as a solution for Leishman Stain. Sodium
sulfate (Na SO ) and zinc acetate [Zn(OAc) ·2H O] were purchased
from Merck and used without any further purification. Unless
otherwise mentioned, all reactions were performed at room
temperature. Column chromatography was performed with silica gel
2
DCM. The crystal data are given below.
6
1
λ
max (DCM, nm): 422, 517, 551, 595. H NMR (CDCl
): δ 1.00 (t,
3
6H), 3.83 (q, 4H), 7.80 (m, 6H), 8.27 (d, 4H), 8.96 (s, 2H), 9.03 (d,
1
3
2H), 9.27 (d, 2H), 9.69 (s, 2H). C NMR (CDCl3, 100 MHz): δ
13.94, 61.41, 105.93, 120.72, 126.82, 127.69, 131.58, 132.63, 132.90,
133.73, 134.80, 142.29, 142.90, 150.05, 151.76, 152.24, 165.58. ESI-
2
4
2
2
+
MS (positive-ion mode, ACN): m/z 668.5 (40%; [M] ), 690.5
+
(mesh size: 60−100, 100−200, and 230−400), basic alumina, and
(100%; [M + Na] ).
neutral alumina. NMR and electron paramagnetic resonance (EPR)
tubes were purchased from Wilmad-LabGlass. THF was dried using
potassium metal in the presence of benzophenone, until the color of
the solution turned intense bluish green. Toluene was dried using
DEsC. Paraformaldehyde (0.1 g, 3.3 mmol) and N-methylglycine
(0.11g, 1.32 mmol) were taken in a 25 mL two-neck round-bottomed
flask equipped with a condenser attached with a Schlenk line. A total
of 5 mL of dry THF was added and refluxed for 20 min until an off-
C
DOI: 10.1021/acs.inorgchem.8b02063
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Inorganic Chemistry
Article
Scheme 2. Synthetic Strategy of the Porphyrins and Chlorins
+
white suspension appeared because of the in situ generation of imine.
A solution of DEsP (0.2 g, 0.33 mmol) in 10 mL of dry THF was
added to the previous reaction mixture and refluxed for 24 h with
constant monitoring by thin-layer chromatography (TLC). After a
period of 5 h, the same amounts of paraformaldehyde and sarcosine
were added until ∼30% conversion to the desired chlorin had taken
place. THF was removed under reduced pressure. Chlorin was
separated by column chromatography with silica gel (60−120 mesh
size). First, DEsP was eluted with 90% DCM/hexane and chlorin with
(50%; [M] ). EPR (1 mM sample in dry THF, 4 K): a rhombic signal
with g values 6.25, 5.87, 5.53, and 1.97 (Figure S29).
ZnDEsC. To a solution of DEsC (50 mg, 0.075 mmol) in THF was
added Zn(OAc) ·2H O (82.1 mg, 0.37 mmol), and the solution was
stirred for 5 h at room temperature. THF was removed under reduced
pressure and purified by column chromatography with silica gel (60−
120 mesh size) using a 4% MeOH/DCM mixture as the eluent. Yield:
2 2
95 ± 2% (45 ± 2 mg).
1
λ
max (DCM, nm): 410, 512, 561, 579, 604. H NMR (CDCl
): δ
3
2
% MeOH/DCM as a greenish-purple oily solid, which was further
1.13 (t, 6H), 4.25 (q, 4H), 2.00 (s, 4H), 2.35 (s, 3H), 7.68 (m, 6H),
8.04 (m, 4H), 8.30 (s, 2H), 8.57 (d, 2H), 8.69 (d, 2H), 8.78 (s, 2H).
purified by crashing out with pentane from a solution in MeOH.
Yield: 40 ± 2% (87 ± 3 mg).
1
3
C NMR (CDCl , 100 MHz): δ 14.1, 14.4, 20.8, 30.7, 42.0, 54.8,
3
−1
λmax (DCM, nm): 418, 517, 550, 591, 658. FTIR (ν, cm ):
61.54, 63.0, 71.1, 72.8, 97.1, 125.3, 127.5, 128.4, 129.6, 133.6, 134.8,
144.5, 147.6, 148.8, 154.7, 159.2, 173.1. ESI-MS (positive-ion mode,
1
1
2
4
2
9
1
704.96, 1683.74 (CO Et group). H NMR (CDCl ): δ −2.22 (s,
2
3
+
H), 1.12 (t, 6H), 2.28 (s, 3H), 3.81 (d, 2H), 4.13 (d, 2H), 4.24 (q,
H), 7.72 (s, 6H), 8.13 (m, 4H), 8.51 (s, 2H), 8.79 (d, 2H), 8.90 (d,
H), 9.17 (s, 2H). C NMR (CDCl 100 MHz): δ 14.1, 61.7, 69.5,
ACN): m/z 725.9 (15%; [M] ).
TPC. Paraformaldehyde (0.1 g, 3.3 mmol) and N-methylglycine
(0.11g, 1.32 mmol) were taken in a round-bottomed flask equipped
with a Dean−Stark apparatus, attached with the Schlenk line. A total
of 60 mL of dry toluene was added and refluxed for 20 min until an
off-white suspension appeared, indicating in situ generation of the
Schiff base. Solid TPP (0.2 g, 0.33 mmol) was added to the previous
reaction mixture and refluxed for 72 h with constant monitoring by
TLC. After a period of 5 h, the same amounts of paraformaldehyde
and sarcosine were added until ∼50% conversion to the desired
chlorin had taken place. Toluene was removed in vacuo and chlorin
separated by column chromatography with silica gel (100−200 mesh
size). First. the unreacted TPP was eluted with DCM and TPC with
some other impurities with 1% MeOH/DCM as a greenish-purple
oily residue. It was then dissolved in THF, and 50 mg of Zn(OAc)2·
1
3
3
,
6.5, 114.2, 119.3, 124.1, 124.2, 124.6, 126.8, 128.7, 132.5, 134.1,
35.7, 139.3, 140.5, 142.2, 147.2, 152.8, 162.1, 172.0. ESI-MS
+
(positive-ion mode, ACN): m/z 664.1 (100%; [M + H] ).
FeDEsC. To a solution of DEsC (50 mg, 0.075 mmol) in 15 mL of
dry degassed THF was added 2,4,6-collidine (20 μL, 0.15 mmol) in a
Schlenk flask equipped with a stopper, and the solution was stirred for
1
flask was brought outside of the glovebox and refluxed for 3 h on the
Schlenk line. After cooling to room temperature, THF was evaporated
in a rotary evaporator and worked up with DCM. The organic layer
was washed with a brine solution and collected. It was dried over
0 min in a glovebox. FeBr (64 mg, 0.3 mmol) was added, and the
2
anhydrous Na SO4 and purified by column chromatography with
2
silica gel (60−120 mesh size) using 4% MeOH/DCM mixture as the
2H O was added and stirred overnight. THF was removed in vacuo
2
eluent. Yield: 87 ± 2% (42 ± 2 mg).
and ZnTPC separated by column chromatography with basic alumina.
ZnTPC was eluted with 2% MeOH/DCM as a greenish-purple solid.
Acidic workup with dilute HCl removed zinc from the cavity. The
1
λmax (DCM, nm): 406, 548, 656. H NMR (CDCl ): δ 83.93,
3
1
1
8
0.78, 68.09 (paramagnetic shift for β-pyrrolic H’s and meso H’s of
Fe -HS complex). ESI-MS (positive-ion mode, ACN): m/z 717.3
III
organic layer was dried over activated MgSO , filtered, and evaporated
4
D
DOI: 10.1021/acs.inorgchem.8b02063
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Inorganic Chemistry
Article
to obtain a greenish-purple solid, which was immediately stored in a
FTIR Data Collection. The samples are drop-casted onto the KBr
window inside the glovebox and fitted in the anaerobic setup. The cell
is brought out of the box and data collected.
glovebox. Yield: 4−5% (10 ± 1 mg).
1
λ
(DCM, nm): 419, 518, 545, 594, 647. H NMR (CDCl ): δ
max
3
−
1.74 (br s, 2H), 1.44 (s, 3H), 2.00 (s, 4H), 7.67−7.70 (m, 12H),
UV−Vis Absorption Data Collection. All anaerobic data were
collected by taking the samples from the glovebox in a tightly sealed
anaerobic cuvette. The cuvette was taken out from the box, and the
data were collected. The background was corrected prior to the
experiments, by an identical amount of solvent mixture.
EPR Data Collection. The samples (150 μL) were taken in EPR
tubes and thoroughly sealed inside the glovebox. The samples were
7.94 (s, 4H), 8.08−8.14 (dd, 4H), 8.25 (s, 2H), 8.44 (s, 2H), 8.59 (s,
2
H). 13C NMR (CDCl 100 MHz): δ 53.0, 68.1, 112.9−152.8
3
,
(
(
aromatic region). ESI-MS (positive-ion mode, CH CN): m/z 672.4
15%; [M + H] ).
3
+
FeTPC. To a solution of TPC (10 mg, 0.015 mmol) in 5 mL of dry
degassed THF was added 2,4,6-collidine (5 μL, 0.03 mmol) in a
Schlenk flask equipped with a stopper, and the solution was stirred for
brought from the box and frozen in liquid N , and the data were
2
III
collected mainly at 77 K. Only for limited samples (Fe -HS) were the
10 min in a glovebox. FeBr (13 mg, 0.6 mmol) was added, and the
2
data collected at 4 K.
flask was brought outside the glovebox and refluxed for 3 h on the
Schlenk line. After cooling to room temperature, THF was removed
in vacuo and worked up with DCM. The organic layer was washed
with a brine solution and collected. It was dried over anhydrous
Na SO and purified by column chromatography with silica gel (60−
Computational Details. All calculations were performed at the
49
IACS computer cluster using Gaussian 03 software. Both the BP86
and B3LYP functionals were tested. A mixed basis set with 6-311g*
on the iron atom and on 6-31g* on the carbon, oxygen, nitrogen, and
2
4
50,51
hydrogen atoms was used for optimization.
For the final energy
120 mesh size) using a 2% MeOH/DCM mixture as the eluent. After
and ground-state calculations, a 6-311+g* basis set on all atoms was
used. Frequency calculations were performed using the basis set used
for optimization, and no negative frequencies were found for the
structures reported. The Mulliken populations were analyzed from the
solvent evaporation, it was a purple-greenish oily residue that could
not be solidified by crashing out with any binary or ternary solvent
mixture with a wide range of polarities. Yield: 73 ± 1% (8 ± 0.5 mg).
1
λmax (DCM, nm): 414, 504, 548, 599, 648. H NMR (CDCl ): δ
3
52
1
III
single-point data using QMforge software. The relative orbital
67.26, 75.87, 85.07 (paramagnetic shift for β-pyrrolic H’s of Fe -HS
picture diagrams were drawn for the complexes, considering the
orbitals with β spin. Orbital energies were normalized with respect to
the dxy orbital. For all complexes, a spin-unrestricted scheme was
adopted, which distinguishes between α- and β-spin orbitals. The
reduction potential values of the bis(imidazole) complexes and
ferrocene were calculated in ACN. The solvent effect was corrected
complex). ESI-MS (positive-ion mode, ACN): m/z 725.7 (100%;
+
[
M] ). EPR (1 mM sample in dry THF, 77 K): an axial signal with g
values of 6.02 and 1.96 (Figure S30).
Electrochemical Measurements. Four complexes (FeTPP,
FeDEsP, FeTPC, and FeDEsC) were considered (Scheme 2). All
cyclic voltammetry (CV) data were collected under anaerobic
conditions inside a glovebox. The complexes were sparingly soluble
in ACN, and sonication of the sample helped to some extent. Hence,
the complexes were dissolved in 200 μL of THF, and 1800 μL of
ACN was added followed by the addition of 100 mM TBAP as the
supporting electrolyte. Glassy carbon was chosen as the working
electrode, aqueous Ag/AgCl in saturated KCl as the reference
electrode, and platinum as the counter electrode. Because the
complexes had different axial ligands (Scheme 2), they were
accompanied by different ligand replacement kinetics during the CV
time scale. Our goal was to understand the effect of the macrocycle;
thus, the CV data of the more stable, bis(imidazole) complexes were
recorded. CV experiments were performed in organic solvents (THF
and ACN) with ferrocene (Fc) as the internal reference, and the
potential scale was normalized with respect to the potential of the
53
using polarizability continuum model. The energy of a free electron
54
was assumed to be −4.43 eV. This was an approximation because
this value will be different for different solvents. However, when the
calculated E° of the iron porphyrins and chlorins were referenced
+
against E° of Fc /Fc in the same solvent, this systematic error will
+
cancel itself and the calculated E° with respect to Fc /Fc can be
compared to the experimental E°. For the CO complexes, both spin-
restricted and unrestricted functionals were used; however, both
produced the same frequency; hence, for all complexes, the spin-
unrestricted scheme was followed. The orbital contributions were
presented as a summation of the population in both the α and β
orbitals of the d orbitals of iron and the 2s and 2p orbitals of the
ligands. The diagram depicting the orbital overlap was drawn using
Gaussview 5.0.9 software with the isovalue of the surfaces for the
corresponding molecular orbital as 0.04.
+
Fc /Fc couple.
CO Complex Preparation. Dry degassed CO was generated
RESULTS
upon the dropwise addition of deaerated concentrated H SO to
■
2
4
ammonium formate. The gas was passed through a 4 N KOH
solution, followed by a concentrated H SO solution. THF solution of
Synthesis. DEsP, TPP, and FeTPP were prepared
following the reported procedures.
44−46
2
4
One of the pyrrole
the complexes were reduced by 0.5 equiv of a Na S solution (in
2
rings of the porphyrins was saturated using a 1,3-cycloaddition
reaction with in situ prepared azomethine ylide. The reaction
was monitored after every 5 h by TLC, and sarcosine and
paraformaldehyde were added until ∼50% completion. It is
worth mentioning that ∼50% conversion was the maximum
obtained. Further the addition of sarcosine and paraformalde-
hyde led to the generation of byproducts, such as bis addition.
Hence, reactions were stopped at this stage, and substrate
porphyrin was recovered by column chromatography. TPC
was unstable because of aerobic oxidation and, hence, was
stored in a glovebox after purification. The chlorins having
pendant amine groups were moisture-sensitive; hence, after
synthesis of the porphyrin cores, further reactions were carried
out in dry solvents. Iron porphyrins were synthesized following
the usual procedures; i.e., the free-base porphyrins were
deprotonated using 2,4,6-collidine and metalated with excess
MeOH) inside a glovebox in a properly sealed, septa-attached
anaerobic vial and brought out of the box. CO gas was purged
through the samples (kept in an ice bath to reduce solution
evaporation) over a period of 5 min. The formation of the complexes
was confirmed by UV−vis absorption and FTIR spectroscopy (data
given in the Results section). The vials were perfectly sealed and used
for further investigations.
NO Complex Preparation. Dry degassed NO was generated
upon the dropwise addition of a deaerated saturated solution of
sodium nitrite to the deaerated concentrated H SO . The gas was
2
4
passed through two 4 N KOH solution bubblers, followed by one
concentrated H SO solution. The samples were prepared in the exact
2
4
same way as that described in the CO Complex Preparation section
using NO gas. NO complexes were also prepared in another way
1
5
using Ph CSNO (as well as Ph CS NO), analogous to reported
procedures.
3
3
47,48
To the reduced samples was added 1 equiv of
Ph CSNO in THF. The formation of the complexes was confirmed by
3
(
∼4 equiv) FeBr at room temperature. Metalation of the
UV−vis absorption, FTIR, and EPR spectroscopy (data given in the
Results section). The vials were perfectly sealed in septa-attached
anaerobic vials and used for further investigations.
2
chlorins (DEsC and TPC) required refluxing of the solution.
The purification procedure for the iron metalation was
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different for porphyrins and chlorins. For porphyrins, after iron
metalation, the solution was worked up with dilute HCl to
remove unreacted iron salts, but this acidic workup removed
iron from the chlorin cavity; thus, only an aqueous workup was
done for the latter. The synthetic strategy is described in detail
in Scheme 2.
X-ray Structures. ZnDEsP. Needle-shaped purple crystals
of ZnDEsP were grown from the diffusion of hexane into a
DCM solution of the complex (Figure 3). ZnDEsP crystallized
distorted square-pyramidal geometry with a τ value of 0.04.
The dimers are likely packed through C−H···O hydrogen-
bonding interactions to form a three-dimensional supra-
molecular structure (Figure S36). All of the hydrogen-bonding
dimensions are summarized in Table S1. The molecular
structure is given in Figure S33.
ZnDEsC. Purple platelike crystals were grown through the
slow diffusion of diethyl ether to the chloroform solution of
ZnDEsC. It crystallized in an orthorhombic Pnma space group
(
Figure 5). Zinc shows a five-coordinate distorted square-
Figure 3. ORTEP diagram of the ZnDEsP complex. Color code: C,
blue; Zn, brown; N, pink; O, red. Hydrogen atoms are omitted for
clarity.
Figure 5. ORTEP diagram of the ZnDEsC complex. Color code: C,
gray; Zn, pink; N, blue; O, red. Hydrogen atoms and solvent
molecules are omitted for clarity.
̅
in a triclinic system centrosymmetric P1 space group.
Structural analysis revealed that it is a dimer formed by the
coordination of a free carbonyl “oxygen” atom with the
neighboring zinc atom (Figure S32). Zinc shows a five-
coordinate distorted square-pyramidal geometry with a τ value
of 0.03. The dimers are packed to form a one-dimensional
chain, and the short contacts are represented in Figure S35.
FeDEsP. Greenish-purple needlelike crystals of FeDEsP were
grown from the slow evaporation of a THF solution. SCXRD
analysis revealed that it is a μ-oxo dimer ((Figure 4) and
crystallized in a monoclinic system with a centrosymmetric
pyramidal geometry with a τ value of 0.03. During crystal
packing, the nitrogen center of the azomethine unit of each
ZnDEsC moiety interacted with the zinc(II) center, making a
one-dimensional coordination polymeric chain (Figure S34).
CV. The iron-bound porphyrinoids showed quasi-reversible
iron(III/II) processes (Figure 6). The iron(III/II) reduction
potential of FeDEsP(Im)
that of FeTPP(Im) . This suggests that replacing two β-
was ∼160 mV more positive than
2
2
pyrrolic hydrogen atoms from the porphyrin ring with two
P2 /c space group. Both iron atoms show five-coordinate
1
Figure 6. Cyclic voltammograms of the complexes in THF/ACN
(
1:9) at room temperature. The working electrode was glassy carbon,
Figure 4. ORTEP diagram of the (FeDEsP) O complex. Color code:
C, black; Fe, brown; N, blue; O, red. Hydrogen atoms are omitted for
clarity.
the counter electrode was platinum, and the reference electrode was
aqueous Ag/AgCl in saturated KCl. TBAP wa used as the supporting
electrolyte (100 mM).
2
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electron-withdrawing ester groups shifted the reduction
potential positive. On the contrary, upon saturation of one
of the pyrrole rings of FeTPP, the iron(III/II) reduction
Q-band region, the 579 and 617 nm peaks disappeared and a
new peak was generated at 540 nm. For the Fe DEsP-NO
II
complex, the Soret band shifted from 431 to 417 nm, and in
the Q-band region, two new peaks were generated at 554 and
580 nm at the expense of the 536, 613, and 573 nm peaks. In
potential was lowered by ∼30 mV in FeTPC(Im) . FeDEsC-
2
(
Im) contains both electron-withdrawing groups and a
2
II
saturated pyrrole center. The iron(III/II) reduction potential
the case of the Fe TPC-NO complex, it was accompanied by a
of FeDEsC(Im) was ∼120 mV more negative with respect to
sharp change in the Soret band from 422 to 413 nm, and in the
Q-band region, the 606 nm intense peak shifted to 617 nm.
For the Fe DEsC complex, NO addition led to a small change
in the Soret band from 414 to 405 nm, and in the Q-band
region, the 594 nm peak shifted to 602 nm.
FTIR. The addition of NO to the solution of iron(II)
samples resulted in the rise of a very intense band
corresponding to N−O stretching vibration (νN−O) in the
2
FeDEsP(Im) . Thus, insertion of the electron-withdrawing
2
II
groups at the β-pyrrolic positions of porphyrin shifted the
iron(III/II) potential to a positive value, while saturation of the
same positions shifted the potential to a negative value.
CO Complexes. CO adduct formation was characterized
by UV−vis absorption and IR spectroscopy.
UV−Vis Absorption. Upon the addition of CO gas to the
II
II
−1
II
14
reduced porphyrins (i.e., Fe TPP and Fe DEsP), the color of
the solution changed from pinkish red to dark red, while for
range of 1660−1690 cm (Figure 8). For the Fe TPP- NO
14 −1
complex, the ν N−O vibration appeared at 1674 cm , and
II
15
15
the same treatment for the chlorins (i.e., Fe TPC and
upon N substitution ( NO), the N−O vibration band
II
−1 II
Fe DEsC), the light-green solution changed to intense green
shifted to 1645 cm . Similarly, for the Fe DEsP-NO complex,
II
14
−1
(
Figure S37). Upon CO addition to Fe TPP, a blue shift in the
the ν N−O vibration appeared at 1683 cm
and the
1
5
−1
Soret band occurred from 434 to 417 nm. In the Q-band
region, the 617 nm peak diminished and the 580 nm peak
corresponding N−O vibration band shifted to 1647 cm .
II 14
For the Fe TPC-NO complex, the N−O vibration band
II
−1
−1
15
intensified. In the case of Fe DEsP-CO, the Soret band shifted
appeared at 1627 cm , and it shifted to 1598 cm upon N
II
14
from 431 to 418 nm, and in the Q-band region, the 613 nm
substitution. For the Fe DEsC-NO complex, the ν N−O
−
1
peak intensified at the expense of the intensities of the 536 and
vibration appeared at 1688 cm , and it shifted to 1651
II
−1
15
5
4
6
73 nm peaks. For Fe TPC-CO, the Soret band shifted from
cm upon N substitution. Therefore, the N−O stretches
II II II
22 to 414 nm, and in the Q-band region, the intense peak at
06 nm shifted to 649 nm. In the case of Fe DEsC-CO, a
(νN−O) for the Fe TPP-NO, Fe DEsP-NO, and Fe DEsC-NO
II
complexes were in the range typical for 5C ferrous heme NO
II
small change in the Soret band (from 414 to 413 nm) was
accompanied by the disappearance of the 540 and 637 nm
peaks and a new peak at 594 nm appeared in the Q-band
region.
FTIR. The addition of CO gas in the solution of iron(II)
samples was accompanied by the rise of a very intense band
complexes, while νN−O for the Fe TPC-NO complex was in
the range typical for 6C ferrous heme NO complexes. So, it is
II
likely to be a MeOH-bound six-coordinate Fe -TPC(NO)-
(MeOH) complex.
EPR. The parent iron(II) complexes were EPR-silent in
perpendicular-mode X-band EPR. Ferrous heme nitrosyls of
7
the general formula {Fe(heme)(NO)} in proteins and model
corresponding to the ν
stretch in the range of 1960−1980
C−O
1
−
1
complexes exhibited low-spin ground states of S = / total
cm (Figure 7). The C−O stretching vibrations (ν
)
2
C−O
−
1
spin. 5C ferrous heme nitrosyls exhibited characteristic EPR
spectra with average g values of about 2.10, 2.06 and 2.01,
while the g values for 6C complexes were about 2.08, 2.00, and
appeared at 1966, 1975, 1978, and 1973 cm for the
II
II
II
complexes Fe TPP-CO, Fe DEsP-CO, Fe TPC-CO, and
II
Fe DEsC-CO, respectively.
1
.98 and, hence, were markedly smaller than those for their 5C
NO Complexes. NO adduct formation was characterized
analogues. The three-line hyperfine pattern originated from the
nuclear spin I = 1 of N, and the corresponding two-line
UV−vis absorption, IR, and EPR spectroscopy.
1
4
UV−Vis Absorption. NO adduct formations were accom-
1
hyperfine pattern originated from the nuclear spin I = / of
panied by the color change akin to the CO adduct formation
2
1
5
II
N. The experimental data are shown in Figure 9. Both the
(
Figure S38). Upon the addition of NO gas to Fe TPP, there
was a blue shift in the Soret band from 434 to 414 nm. In the
experimental and simulated (Figure S40) g and A values were
in good agreement with each other (Tables S3 and S4). The
data suggested that these complexes form a six-coordinate NO
adduct at low temperature (77 K). This attributed to the lower
entropic contribution at low temperature, forming a 6C
7
55
{
FeNO} complex binding a solvent molecule (MeOH).
Density Functional Theory (DFT) Calculations. The
experimentally found iron(III/II) reduction potential values
suggested that the addition of electron-withdrawing groups
shifted the reduction potentials to positive value, while upon
saturation of one of the pyrroles of the porphyrin, the
reduction potential shifted to a negative value. The νC−O and
νN−O values obtained from FTIR experiments of the CO and
NO complexes, respectively, showed that, upon the addition of
electron-withdrawing groups or upon saturation of one of the
pyrrole rings, the C−O and N−O stretching frequencies
increased relative to the reference FeTPP complex. This
implies that perturbation on the structure of porphyrin affected
the electronic structure of the corresponding iron complexes.
Hence, DFT calculations were used to gain insight into it. The
Figure 7. FTIR data of the CO complexes drop-casted onto a KBr
window. The experimental values are tabulated below.
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Figure 8. FTIR data of the NO complexes drop-casted onto a KBr window. The experimental values are tabulated below.
Figure 9. EPR spectra of 14NO- and 15NO-bound complexes, respectively (MW frequency, 9135.3 MHz; center field, 320 mT; MW power, 1 mW;
modulation frequency, 0.25 mT).
DFT-optimized structures were compared with the crystal
structures. The average metal pyrrole bond lengths from the
experiments closely matched with the calculated values (Table
Table 1. Metal−Ligand Bond Lengths Obtained from the
Crystal Structures and DFT-Optimized Structures
ave M−Npyr bond length (Å)
crystal structure
DFT-optimized
ZnDEsP
FeDEsP
ZnDEsC
2.049
2.072
2.085
2.046
2.094
2.058
1
).
CO Complexes. The Fe -CO complexes were optimized
II
using the BP86 and B3LYP functionals. The calculated C−O
stretching frequencies are given in Table 2.
The frequencies obtained from the BP86 functional are in
II
NO Complexes. The Fe -NO complexes were optimized
using the BP86 and B3LYP functionals. The calculated C−O
stretching frequencies are given in Table 3.
better agreement with the experimental values (Figure 7).
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Table 2. DFT-Calculated C−O Stretching Frequencies
positive than that of FeTPP-Im (Table 4). The iron(III/II)
2
reduction potential depended on two factors: (a) the electron
affinity of the iron(II) center and/or (b) the stability of
iron(II). The electron affinity of iron(III) was judged from the
natural population analysis (NPA) and natural bond orbital
(NBO) analysis, which gave the natural charge over the
νC−O (cm−1)
II
II
II
II
Fe TPP-CO Fe DEsP-CO Fe TPC-CO Fe DEsC-CO
expt
uBP86
uB3LYP
1966
1980
2090
1975
1985
2097
1978
1981
2094
1973
1983
2096
56
atoms. Both analyses suggested that the natural charge over
III
iron was slightly higher in the case of Fe DEsP-Im relative to
2
III
Table 3. DFT-Calculated N−O Stretching Frequencies
Fe TPP-Im (Table 5). The inductive withdrawing (−I) effect
2
νN−O (cm−1)
Table 5. Theoretically Calculated Natural Charge over Iron
in the Corresponding Bis(imidazole) Complexes Using the
uBP86 Functional in Both the NPA and NBO Methods
II
II
II
II
Fe TPP-NO Fe DEsP-NO Fe TPC-NO Fe DEsC-NO
(
5C)
(5C)
(6C)
(5C)
expt
uBP86
uB3LYP
1674
1707
1768
1683
1726
1810
1627
1704
1820
1688
1713
1782
natural charge over iron
NPA
NBO
FeTPP-Im2
FeDEsP-Im2
FeTPC-Im2
FeDEsC-Im2
0.49
0.52
0.49
0.50
0.19
0.21
0.19
0.20
The calculated νC−O and νN−O frequencies obtained from the
BP86 functional were in better agreement with the
experimental values (Figures 7 and 8); hence, further analysis
was carried out with the BP86 functional.
Iron(III/II) Reduction Potential. The reduction potential
values of the bis(imidazole) complexes and ferrocene were
calculated using the BP86 functional (Table 4). The calculated
E° iron(III/II) reduction potential of the iron porphyrins and
chlorins were referenced against the calculated E° iron(III/II)
of the ester groups was responsible for making the iron center
III
III
in Fe DEsP-Im more electropositive than that of Fe TPP-
2
III
Im . As a result, Fe DEsP-Im had greater electron affinity
2
2
III
over Fe TPP-Im .
2
The electron-withdrawing ester groups exerted the −I effect,
making the ligand (pyrrolic nitrogen atoms of the porphyrin) a
weak σ donor. This should result in a relatively weaker
+
of Fc /Fc. In ACN, the iron(III/II) reduction potential of
FeTPP-Im was found to be −0.71 V, while for FeDEsP-Im , it
2
2
was −0.52 V. However, in FeDEsP, two meso-phenyl groups
2
2
interaction between the porphyrin and d
orbital of iron.
x −y
were substituted by hydrogen. Hence, iron diphenylporphyrin,
The relative molecular orbital diagram of the corresponding
FeDPP(Im) , was considered for a comparison by DFT. The
2
bis(imidazole) complexes (to minimize the axial ligand effect)
calculated redox potential was −0.70 V, which closely
2
2
did not show any significant change in the energy of the d
x −y
resembled the value obtained from FeTPP-Im ; hence, the
orbital (Figure S41). This implies that the ester groups may
not have a prominent effect in σ bonding; they should be
involved mainly in π interaction. In fact, the Mulliken
2
difference in the redox potential between FeTPP and FeDEsP
was solely due to the electron-withdrawing groups. For
56
II
FeTPC-Im , the potential was −0.87 V, which was negatively
population analysis of reduced Fe DEsP-Im indicates that
2
2
shifted from that of FeTPP-Im . For FeDEsC-Im , the
there was greater mixing between the iron(II) and π* orbitals
2
2
potential was found to be at −0.78 V, which was shifted to
of the DEsP macrocycle (DEsP−π*), relative to TPP−π* (of
II
lower values relative to that of FeDEsP-Im . Therefore, the
Fe TPP-Im ), which was responsible for greater charge
2
2
trend obtained from the calculated E° values was in good
agreement with that from the experimental E° values.
delocalization in the reduced [iron(II)] porphyrin, making
FeDEsP-Im easy to be reduced compared to FeTPP-Im
2
2
(
Figure 10 and Table 6).
DISCUSSION
II
−1
■
The νC−O value for Fe TPP-CO was at 1966 cm , which
−
1
II
The addition of electron-withdrawing groups and/or saturation
of one of the pyrrole rings of the porphyrin resulted in changes
in the electronic structures of porphyrins, which is reflected in
the iron(III/II) reduction potential as well as in their
corresponding CO and NO vibrations of the adducts. DFT
calculations reasonably reproduced the trends in the
experimental data and were used to further analyze the
ground-state wave functions. The effects of the electron-
withdrawing group and saturation are discussed separately, and
finally the combined effect is discussed.
increased to 1975 cm for Fe DEsP-CO. The corresponding
NO complexes showed similar trends. The ν value for
Fe TPP-NO was at 1974 cm , which increased to 1983 cm
for Fe DEsP-NO. Both indicated a lowering in back-bonding
from Fe d /d to the π* orbitals of CO/NO. The Mulliken
population analysis suggested that in FeDEsP less mixing
occurred between Fe d /d and the π* orbitals of CO/NO
relative to the corresponding CO/NO complexes of FeTPP
(Tables 7 and 8). On the contrary, the mixing between Fe dxz/
d and the porphyrin−π* orbitals increased in both CO/NO
complexes of FeDEsP compared to Fe TPP-CO/NO.
N−O
II
−1
−1
II
xz yz
xz yz
yz
II
Effect of the Electron-Withdrawing Group. The
iron(III/II) reduction potential of FeDEsP-Im was more
Previously, Ryan et al. had reported the reduction of N−O
2
Table 4. Experimentally and Theoretically Calculated Reduction Potential Values of the Bis(imidazole) Complexes
E°(FeIII/II) vs Fc /Fc (V)
+
FeTPP(Im)2
FeDEsP(Im)2
FeDPP(Im)2
FeTPC(Im)2
FeDEsC(Im)2
experimental
theoretical
−0.58
−0.71
−0.42
−0.52
−0.61
−0.87
−0.54
−0.78
−0.70
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II
II
Figure 10. Orbital overlap between the iron and π* orbitals of the porphyrin macrocycle for the (A) Fe TPP-Im and (B) Fe DEsP-Im
2
2
complexes.
Table 6. Summation of Mulliken Population of the Iron(II)
Scheme 3. Effect of Back-Bonding in the X−O (X = C, N)
Orbitals (dxz and d ) in the π* Orbitals (α + β) of the
Stretching Frequency: Competitive Back-Bonding between
yz
Porphyrin Macrocycle
Macrocycle−π* and Axial Ligand π*
iron orbital content
[P−π*]
iron orbital content
[P−π*]
II
II
Fe TPP-Im
33.02
34.24
Fe TPC-Im
30.78
30.36
2
2
II
II
Fe DEsP-Im2
Fe DEsC-Im
2
Table 7. Orbital Contributions of the Respective d Orbitals
(α + β) of Iron in Macrocycle−π* and CO−π* Orbitals
contribution of iron
CO−π1*ab
CO−π2*ab
[P−π*]
and C18 positions of heme b were replaced by two electron-
II
withdrawing groups: hydroxyfarnesyl and a formyl group,
Fe TPP-CO
26.58
24.48
22.00
27.64
27.58
24.80
25.68
24.28
11.36
11.62
12.32
11.04
9
II
respectively (Figure 1A). Because of the electron-withdrawing
Fe DEsP-CO
II
groups attached with the β-pyrroles of the heme increased the
Fe TPC−CO
II
iron(III/II) reduction potential, it can reduce O to water at a
2
44
Fe DEsC−CO
very low overpotential. In fact, the iron(III/II) potential of
CcO is ∼350 mV, which is much higher than that of Hb/Mb
Table 8. Orbital Contributions of the Respective d Orbitals
(
−200 mV). A significant part of this difference may be derived
(α + β) of Iron in NO−π* and Macrocycle−π* Orbitals
23
from the -I effect of the formyl and hydroxyfarnesyl groups.
A
contribution of iron
weakly bound CO or NO adduct may be an alternative defense
mechanism against the inhibitions by them.
ΝΟ−πv*
[P−π*]
II
Effect of Saturation of One of the Pyrrole Rings. The
Fe TPP-NO
67.81
53.95
44.96
46.59
19.68
27.61
23.23
24.45
II
reduction potential of FeTPC-Im was negative relative to
Fe DEsP-NO
2
II
FeTPP-Im2 (Table 4). The relative energy level diagram
suggested that, upon saturation of one of the pyrrole rings of
Fe TPC-NO
II
Fe DEsC-NO
2
the porphyrin, the energy of the d orbital decreased by 0.26
z
2
2
eV and that of the d
orbital increased by 0.15 eV (Figure
x −y
7
stretching frequency in [FeNO] species with octaethylpor-
phyrin because the pyrrole rings were oxidized to pyrrolidones
in contrast to observations here, suggesting a different effect of
S42). Weaker interaction with the axial imidazole lowered the
2
2
2
energy of the dz orbital. The d
orbital became more
x −y
destabilized because of the stronger σ donation from of the
pyrrolic nitrogen atoms. Hence, saturation exerted a +I effect
to make the pyrrole nitrogen atom a better σ donor. The
Mulliken population analysis indicated a relatively lower
mixing between the iron(II) and π* orbitals of the chlorin
32
pyrrole oxidation on back-bonding from iron to NO.
Therefore, a competitive back-bonding between macro-
cycle−π* and the axial ligand π* with the Fe d /d orbitals
xz yz
was operative here (Scheme 3). The extent of back-bonding
was responsible for tuning of the reduction potential and its
reactivity. Hence, iron porphyrins with electron-withdrawing
groups attached with the β-pyrroles exerted a relatively positive
reduction potential and made the CO and NO adducts weaker.
The effect that arose from the presence of the electron-
withdrawing groups can be extended to O reactivity. The O
macrocycle (TPC−π*), in comparison with porphyrin
II
(TPP−π*; Table 6). In Fe TPC-Im , back-bonding with the
2
macrocycle−π* orbitals was weaker because the planarity of
the TPC macrocycle was compromised because of the weak
hydrogen-bonding interaction between the pendant amine and
II
axial imidazole (Figure S31). Therefore, in Fe TPC-Im , less
2
2
2
binding site in CcO is heme a , while the O carrier protein
charge delocalization from iron(II) to the macrocycle π*
3
2
II
hemoglobin/myoglobin contain heme b. The difference
between heme a and heme b was that in heme a the C
3
orbital occurred relative to Fe TPP-Im , and hence the former
2
was relatively difficult to reduce, which was reflected in the
3
3
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Scheme 4. Plausible Mechanistic Pathway of Nitrite Reduction Catalyzed by CcNiR and Cd NiR
1
reduction potential values. The ν
value for five-coordinate
enzymes follow similar mechanistic pathways until the
formation of a {Fe(NO)} intermediate (Scheme 4). However,
C−O
II
−1
−1
6
Fe TPP-CO was at 1966 cm , which shifted to 1978 cm for
II
Fe TPC-CO (Figure 7), which was due to less back-bonding
in the case of Cd NiR, it takes one electron from cytochrome c
1
to the π* orbitals of CO because of competitive back-bonding
and NO is released, while in CcNiR, the reaction cycle
(Table 7).
continues until the formation of an ammonium ion. The fast
II
7
−1
The Fe TPC formed a six-coordinate {FeNO} complex at
NO dissociation (koff ∼ 6−35 s ) from ferrous cd nitrite
1
59
room temperature where the sixth ligand was likely the solvent
reductase supports the argument. Iron porphyrin possessing
both electron-withdrawing groups and a saturated center forms
7
MeOH. Lehnert et al. showed that the NO in {FeNO}
7
complexes has a much stronger trans effect than that in the
weakly bound NO adduct {FeDEsC-NO} relative to the
corresponding {FeNO}6 complexes because of the much
situation where iron porphyrin is having only electron-
5
7,58
II
stronger σ bonding in the former.
Fe TPC formed a six-
7
7
withdrawing groups, {FeDEsP-NO} . Hence, in the {FeNO}
intermediate of heme d in Cd NiR, the NO adduct should be
II
coordinate Fe TPC(NO)(N-methylimidazole) complex with
1
1
only 1 equiv of N-methylimidazole, while in the case of
so weakly bound (due to competitive back-bonding) that NO
can be released prior to a further reduction to ammonium.
II
Fe TPP, excess N-methylimidazole was required to form the
II
six-coordinate Fe TPP(NO)(N-methylimidazole) complex.
−
1
The N−O stretching vibration appeared at 1627 cm ,
which shifted to 1598 cm− upon N substitution (Figure
1
15
CONCLUSIONS
■
S19). The N−O stretching vibrations clearly indicated that
In this Article, we have discussed how perturbation of iron
porphyrinoids can alter the electronic structure, by introducing
electron-withdrawing groups to the β-pyrrolic positions and by
saturating the same. The electron-withdrawing group (−I
effect) and saturation (+I effect) have little effect on the σ
bonding between macrocycles and the iron. Instead, they affect
the π bonding by lowering the energy of the π* orbitals of the
porphyrin ring. Therefore, resulting in a competitive back-
bonding scenario between Fe dπ and (a) the NO/CO π* and
II
back-bonding to NO−π* was significantly low in Fe TPC-
(
NO)(N-methylimidazole), relative to its FeTPP analogue.
While the strength of the σ bond drove the trans effect, as
pointed out by Lehnert et al., the Mulliken population analysis
II
indicated that back-bonding from Fe dπ to NO π* was lower
II
as well in Fe TPC(NO), likely because of the synergistic
II
effect, relative to Fe TPP(NO) (Table 8), which was reflected
in their relative NO frequencies; i.e., the NO vibration acted as
a spectator to the dominantly σ-bonding effect. Therefore,
saturating the 3 and 4 positions of one of the pyrrole rings of
the porphyrin weakened both σ bonds and exerted lower back-
bonding to the axial ligand (CO and NO) relative to the fully
unsaturated FeTPP.
FeDEsC is a system in which both the electron-withdrawing
group and saturated pyrrole center is present together. These
two substitutions have opposite effects on the redox potential:
the electron-withdrawing group makes it positive, while
(
b) the vacant porphyrin−π* orbitals. Electron-withdrawing
groups significantly increase the iron(III/II) reduction
potential, which is also relevant to heme a in CcO. The
3
electron-withdrawing group and saturation together form a
very weakly bound ferrous nitrosyl complex, which is
hypothesized as the reason why heme d in Cd NiR releases
1
1
7
NO from the {FeNO} intermediate.
ASSOCIATED CONTENT
saturation reduces it. As a result, FeDEsC-Im showed a
■
2
redox potential that was higher than that of FeTPP-Im2
*
S
Supporting Information
(because of its electron-withdrawing effects) but lower than
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b02063.
FeDEsP-Im (because of the saturation). It was observed that
2
both the electron-withdrawing and saturation effects led to
lower back-bonding to the CO/NO π* orbitals. Therefore, the
II
Additional NMR, mass, UV−vis, and FTIR data and
coordination of the DFT-optimized structures (PDF)
higher C−O and N−O stretching frequencies of Fe DEsC-CO
II
and Fe DEsC-NO, respectively, stemmed from a consequence
of competitive back-bonding (Scheme 3).
FeDEsC can be structurally correlated with heme d in
Accession Codes
1
Cd NiR, which contains two electron-withdrawing formyl
groups and two saturated pyrrole carbon atoms (Figure 1C).
CCDC 1854375−1854377 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing 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.
1
11
Cd NiR, a nitrite reducing enzyme, terminates the reduction
1
cycle at NO, in strict contrast to CcNiR, a multi-c-heme-
containing enzyme, which reduces nitrite to an ammonium ion
10
without releasing NO (Figure 1B). Both nitrite-reducing
K
DOI: 10.1021/acs.inorgchem.8b02063
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(
16) Crane, B. R.; Getzoff, E. D. The relationship between structure
AUTHOR INFORMATION
■
and function for the sulfite reductases. Curr. Opin. Struct. Biol. 1996, 6
6), 744−756.
17) Crane, B. R.; Siegel, L. M.; Getzoff, E. D. Sulfite Reductase
Corresponding Author
E-mail: icad@iacs.res.in.
(
*
(
ORCID
Structure at 1.6 Å: Evolution and Catalysis for Reduction of Inorganic
Anions. Science 1995, 270 (5233), 59.
Abhishek Dey: 0000-0002-9166-3349
Notes
The authors declare no competing financial interest.
(
18) Allen, J. W.; Ferguson, S. J.; Fulop, V. Cytochrome cd1 Nitrite
̈
̈
Reductase. In Encyclopedia of Inorganic and Bioinorganic Chemistry;
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ACKNOWLEDGMENTS
O’Brian, M. R.; Warren, M. J. Prokaryotic Heme Biosynthesis:
Multiple Pathways to a Common Essential Product. Microbiol. Mol.
Biol. Rev. 2017, 81 (1), XXX.
■
This research is funded by Council of Scientific and Industrial
Research (CSIR) Grant 01(2874)/17/EMR-II. S.A. acknowl-
edges CSIR-SRF, and P.S. acknowledges UGC-JRF.
(
20) Murshudov, G. N.; Grebenko, A. I.; Barynin, V.; Dauter, Z.;
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