Just a proton: Distinguishing the two electronic states of five-coordinate high-spin iron(ll) porphyrinates with imidazole/ate coordination

Article abstract of DOI:10.1021/ja907584x

We report detailed studies on two S = 2 electronic states of high-spin iron(ll) porphyrlnates. These two states are exemplified by the five-coordinate derivatives with either neutral imidazole or anionic imidazolate as the axial ligand. The application of several physical methods all demonstrate distinctive differences between the two states. These Include characteristic molecular structure differences, Moessbauer spectra, magnetic circular dichroism spectroscopy, and Integer-spin EPR spectral distinctions. These distinctions are supported by DFT calculations. The two states are characterized by very different spatial properties of the doubly occupied orbital of the high-spin that are consonant with the physical properties.

Full text of DOI:10.1021/ja907584x

Published on Web 03/01/2010
Just a Proton: Distinguishing the Two Electronic States of
Five-Coordinate High-Spin Iron(II) Porphyrinates with
Imidazole/ate Coordination
Chuanjiang Hu,†,‡ Corinne D. Sulok,§ Florian Paulat,§ Nicolai Lehnert,*,§
Anna I. Twigg,| Michael P. Hendrich,*,| Charles E. Schulz,*,⊥ and
W. Robert Scheidt*,†
The Department of Chemistry and Biochemistry, UniVersity of Notre Dame, Notre Dame,
Indiana 46556, Department of Chemistry, UniVersity of Michigan, 930 North UniVersity AVenue,
Ann Arbor, Michigan 48109, Department of Chemistry, Carnegie Mellon UniVersity,
4400 Fifth AVenue, Pittsburgh, PennsylVania 15213, and Department of Physics, Knox College,
Galesburg, Illinois 61401
Received September 7, 2009; E-mail: scheidt.1@nd.edu; lehnertn@umich.edu;
hendrich@andrew.cmu.edu; cschulz@knox.edu
Abstract: We report detailed studies on two S ) 2 electronic states of high-spin iron(II) porphyrinates.
These two states are exemplified by the five-coordinate derivatives with either neutral imidazole or anionic
imidazolate as the axial ligand. The application of several physical methods all demonstrate distinctive
differences between the two states. These include characteristic molecular structure differences, Mo¨ssbauer
spectra, magnetic circular dichroism spectroscopy, and integer-spin EPR spectral distinctions. These
distinctions are supported by DFT calculations. The two states are characterized by very different spatial
properties of the doubly occupied orbital of the high-spin that are consonant with the physical properties.
Introduction
The wide range of physiological functions carried out by the
heme proteins with the same prosthetic group, the iron derivative
of protoporphyrin IX, has continued to fascinate and perplex.
Some aspects of heme protein function appear to be controlled
by obvious changes in the proximal axial ligand to iron. For
example, the cytochrome P-450 monooxygenases and NO
synthase (NOS) are coordinated by an anionic cysteine ligand
and the catalases are coordinated by an anionic tyrosinate.
However, the most common proximal ligand in the heme
proteins is the histidyl residue that is equivalent to imidazole,
a nominally neutral ligand. Proteins with histidine ligation still
maintain a wide variety of functions that include oxygen
transport and storage in the globins, oxygen respiration in
cytochrome c oxidase and oxygen utilization and oxidation
in the peroxidases, oxidases, cyclooxygenase, NO reduction in
bacterial NO reductase (NorBC), NO signaling in soluble
guanylate cyclase (sGC), and NO transport in nitrophorins.
Figure 1. Comparison of the hydrogen bonding network involving the
proximal histidine in heme proteins. Left: myoglobin (Mb) as an example
for the case of weak hydrogen bonding. Right: horseradish peroxidase (HRP)
constitutes the case of strong hydrogen bonding.
imidazolate ligand.3-9 The notion that a strong hydrogen bond
has a strong influence on heme protein behavior is longstanding
but has been difficult to investigate in a systematic manner.
Clearly there are significant differences in the nature of the
hydrogen bonds formed to the proximal histidine in different
A common feature in many of these systems is the appearance
of a hydrogen bond to the proximal histidine ligand as shown
in Figure 1 for deoxymyoglobin1 and horseradish peroxidase.2
The strength of these hydrogen bonds is thought to vary from
very weak proton donation to complete donation to form the
(1) Vojtechovsky, J.; Chu, K.; Berendzen, J.; Sweet, R. M.; Schlichting,
I. Biophys. J. 1999, 77, 2153.
(2) Berglund, G. I.; Carlsson, G. H.; Smith, A. T.; Szo¨ke, H.; Henriksen,
A.; Hajdu, J. Nature 2002, 417, 463.
(3) Peisach, J.; Blumberg, W. E.; Adler, A. Ann. N.Y. Acad. Sci. 1973,
206, 310.
(4) Nicholls, P. Biochim. Biophys. Acta 1962, 60, 217.
(5) Mincey, T.; Traylor, T. G. J. Am. Chem. Soc. 1979, 101, 765.
(6) Teroaka, J.; Kitagawa, T. Biochem. Biophys. Res. Commun. 1980, 93,
674.
† University of Notre Dame.
‡ Present address: College of Chemistry, Chemical Engineering and
Materials Science, Soochow University, Suzhou 215123 China.
§ University of Michigan.
(7) Morrison, M.; Schonbaum, G. R. Annu. ReV. Biochem. 1976, 45, 861.
(8) Peisach, J. Ann. N.Y. Acad. Sci. 1975, 244, 187.
(9) Valentine, J. S.; Sheridan, R. P.; Allen, L. C.; Kahn, P. Proc. Natl.
Acad. Sci. U.S.A. 1979, 76, 1009.
| Carnegie Mellon University.
⊥ Knox College.
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J. AM. CHEM. SOC. 2010, 132, 3737–3750 3737

A R T I C L E S
Hu et al.
were electronically quite distinct from the imidazolate com-
plexes. Measurements in applied magnetic field allowed the
determination of the sign of the quadrupole splitting that was
found to be negative for all imidazole derivatives (Vzz < 0) and
positive (Vzz > 0) for the imidazolate species. Moreover, the
asymmetry parameter for the imidazole species was found to
be very large, whereas that for the imidazolate derivatives was
small. The differences in the Mo¨ssbauer spectra obtained in
applied magnetic fields indicate that the doubly occupied d
orbital is different in the two classes. This change in the
d-electron configuration is clearly consistent with all observed
differing features of the two classes. These results are strikingly
similar to those observed for protein examples. Reduced HRP
was studied by Mo¨ssbauer spectroscopy and compared with
deoxymyoglobin.17,18 These Mo¨ssbauer studies, in a strong
magnetic field, showed remarkable differences between reduced
HRP and deoxymyoglobin even though both are five-coordinate
hemes with histidine as the axial ligand. Reduced HRP has a
positive quadrupole splitting constant and a rather small
asymmetry value,18 whereas deoxyMb has a negative value of
the quadrupole splitting constant and a large asymmetry
constant.19 To our knowledge, these Mo¨ssbauer measurements
in applied magnetic field are the only ones available for iron(II)
heme proteins with a proximal histidine, although deoxymyo-
globin and deoxyhemoglobin have been often measured.
Figure 2. Comparison of the coordination group geometries (averaged
values) of the high-spin iron(II) porphyrinates with 2-methylimidazole (left)
and 2-methylimidazolate (right) as the axial ligand.
heme proteins. Three distinctly different types of hydrogen
bonds are formed: the globins and a number of other proteins
form a weak hydrogen bond between histidine and a nearby
carbonyl or alcohol group, the peroxidases that are hydrogen-
bonded to a conserved aspartate and the oxidases that are
hydrogen-bonded to a conserved glutamate. The idea continues
to appeal that in the peroxidases, which have a conserved
aspartate that is hydrogen-bonded to the histidine, the histidine
shows imidazolate-like character. This strong hydrogen bond
is thought to stabilize higher oxidation states of iron and to
distinctly alter the chemical behavior of the peroxidases relative
to the globins. Nevertheless, the role of this hydrogen bond is
still one of the unresolved issues concerning the peroxidases.
As part of a systematic study of five-coordinate iron(II), we
have synthesized and characterized a number of five-coordinate
imidazole- and imidazolate-ligated iron(II) porphyrinate deri-
vatives.10-15 This work reports the effects observed on the
molecular and electronic structure when an imidazole ligand in
five-coordinate iron(II) porphyrinate derivatives of the type
[Fe(Por)(2-MeHIm)]16 is deprotonated.12 Both imidazole- and
imidazolate-ligated iron(II) porphyrinates exhibit an S ) 2
(quintet) ground state, but the structural parameters of the
coordination group are distinct with both axial and equatorial
bond distance differences and large differences in the displace-
ment of iron from the porphyrin plane. These differences are
schematically depicted in Figure 2 for the iron(II) derivatives
with imidazole and imidazolate as the axial ligand and where
averaged values for several examples of each have been given.
In addition to the structural differences, characterization by
Mo¨ssbauer spectroscopy showed that the imidazole derivatives
These results led us to conclude that there are at least two S
) 2 states of high-spin iron(II) porphyrinates with distinctly
different d-electron configurations that arise from proximal
ligand effects. The one class of high-spin iron(II) derivatives,
which we will call Class N, is represented by the coordination
of neutral nitrogen donors in small molecule systems and in
the proteins by deoxymyoglobin and deoxyhemoglobin. The
second class, which we will call Class A, is represented by the
coordination of anionic imidazolate and other anions in small
molecule systems and reduced HRP in the proteins. The
important class of cysteine-ligated hemes and heme proteins
also probably belong to this class although this has been
established only for heme derivatives.20 In this paper, we explore
a number of characterization processes that could provide ways
to distinguish between the two classes. These include integer-
spin EPR measurements, magnetic circular dichroism spectros-
copy, structure, and a reexamination of the Mo¨ssbauer fitting
analysis of spectra in applied magnetic field. We have also
explored DFT calculations and how different functionals
replicate (or fail to) the experimental results. We conclude that
the distinction of Class N and Class A electronic structures
represents a real and useful dichotomy for high-spin iron(II)
porphyrinates. We further conclude that all methods tested allow
(10) Ellison, M. K.; Schulz, C. E.; Scheidt, W. R. Inorg. Chem. 2002, 41,
2173.
(11) Hu, C.; Roth, A.; Ellison, M. K.; An, J.; Ellis, C. M.; Schulz, C. E.;
Scheidt, W. R. J. Am. Chem. Soc. 2005, 127, 5675.
(12) Hu, C.; Noll, B. C.; Schulz, C. E.; Scheidt, W. R. J. Am. Chem. Soc.
2005, 127, 15018.
(13) Hu, C.; An, J.; Noll, B. C.; Schulz, C. E.; Scheidt, W. R. Inorg. Chem.
2006, 45, 4177.
(14) Hu, C.; Noll, B. C.; Piccoli, P. M. B.; Schultz, A. J.; Schulz, C. E.;
Scheidt, W. R. J. Am. Chem. Soc. 2008, 130, 3127.
(17) Debrunner, P. In Iron Porphyrins Part 3; Lever, A. B. P., Gray, H. B.,
Eds.; VCH Publishers Inc.; New York, 1983; Chapter 2.
(15) Hu, C.; Noll, B. C.; Schulz, C. E.; Scheidt, W. R. Inorg. Chem. 2008,
47, 8884.
(18) Champion, P. M.; Chiang, R.; Mu¨nck, E.; Debrunner, P.; Hager, L. P.
Biochemistry 1975, 14, 4159.
(16) The following abbreviations are used in this paper: MCD, magnetic
circular dichroism; EPR, electron paramagnetic resonance; SOC, spin-
orbit coupling; Por, generalized porphyrin dianion; OEP, dianion of
octaethylporphyrin; TPP, dianion of meso-tetraphenylporphyrin; Tpiv-
PP, dianion of picket fence porphyrin; 2-MeHIm, 2-methylimidazole;
2-MeIm-; 2-methylimidazolate: 5C, five-coordinate; hs, high-spin;
HRP, horseradish peroxidase; CuEDTA, copper salt of ethylenedi-
aminetetraacetic acid; 222 or kryptofix-222, 4,7,13,16,21,24-hexa-oxo-
1,10-diazabicyclo[8.8.8]hexacosane; Np, porphyrinato nitrogen.
(19) Kent, T. A.; Spartalian, K.; Lang, G.; Yonetani, T.; Reed, C. A.;
Collman, J. P. Biochem. Biophys. Acta 1979, 580, 245.
(20) The position of the iron(II) thiolato complexes is somewhat ambiguous.
Their structural parameters are closer to those of Class N, whereas
their Mo¨ssbauer properties have positive values of the quadrupole
splitting constant consistent with Class A species.21
(21) Schappacher, M.; Ricard, L.; Weiss, R. R.; Montiel-Montoya, R.; Bill,
E.; Trautwein, A. X. Inorg. Chem. 1989, 28, 4639.
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3738 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Five-Coordinate High-Spin Iron(II) Porphyrinates
A R T I C L E S
ethanethiol was added by syringe. The mixture was stirred at room
temperature for 3 days, then the solution was transferred into a
Schlenk flask containing sodium azide (5 mg) and Kryptofix 222
(27 mg, 0.07 mmol). The mixture was stirred for 1 h, the resulting
precipitate was filtered and dried under vacuum for 0.5 h. Then it
was redissolved in CH2Cl2 (6 mL), and filtered. X-ray quality
crystals were obtained in 8 mm × 250 mm sealed glass tubes by
liquid diffusion using hexanes as nonsolvent. There were several
different crystals formed, one of which was Na(222)[Fe(TPP)Cl],
whose structure is described herein.
Instrumental Techniques. Mo¨ssbauer measurements were per-
formed on a constant acceleration spectrometer from 4.2 to 298 K
in small field and at 4.2 K in several fields between 0.75 and 9 T
using a superconducting magnet system (Knox College). Microc-
rystalline solids for Mo¨ssbauer measurements were obtained by
liquid diffusion in Schlenk tubes using hexanes as the nonsolvent.
The solids were isolated in an inert-atmosphere box and im-
mobilized in Apiezon M grease.
MCD spectra were obtained on frozen glasses in the mixed
solvent of toluene and methylene chloride (1:1) at liquid He
temperatures (1.8-25 K). A CD spectropolarimeter (Jasco 810)
with S1 and S20 photomultiplier tubes as detectors were used where
the sample compartment was modified to accommodate an Oxford
instruments SM4-7T magnetocryostat. Spectral sample solutions
were prepared from Fe(TPP) in the presence of excess ligand (and
222 for the anionic derivatives) and concentrations were determined
by UV-vis spectroscopy at the same time as the MCD measure-
ments. The samples were frozen in metallic sample compartments
between two Infrasil quartz disks separated by 3 mm neoprene
spacers. Typical sample concentrations were in the range of 0.2-1.0
mM (University of Michigan).
X-band EPR spectra of powder and solution samples were
recorded on a Bruker 300 spectrometer equipped with an Oxford
ESR-910 liquid helium cryostat and a Bruker bimodal cavity for
generation of the microwave fields parallel and perpendicular to
the static field. The microwave frequency was calibrated with a
frequency counter, and the magnetic field was calibrated with a
NMR gaussmeter. The sample temperature was calibrated with a
carbon-glass resistor (Lake Shore CGR-1-1000) placed in an EPR
tube to mimic a sample. The powder samples were prepared in
EPR tubes fitted with graded quartz to Pyrex tops and flame-sealed.
The solution samples were 5-6 mM in chlorobenzene and frozen
in standard quartz tubes. All experimental data were collected under
nonsaturating microwave conditions with a modulation frequency
of 100 kHz. The EPR spectra and temperature dependence were
analyzed by diagonalization of the spin Hamiltonian H ) S·D·S
+ ꢀS·g·B. The simulations were generated with consideration of
all intensity factors relative to a CuEDTA spin standard which
allowed computation of simulated spectra for a specific sample
amount. The simulations therefore allow a quantitative determina-
tion of signal intensities. The Windows software package (Spin-
Count) is available for general application (www.chem.cmu.edu/
groups/hendrich/facilities/index.html) (Carnegie Mellon University).
DFT Calculations. The structures of the complexes [Fe(TPP)(2-
MeHIm)], [Fe(TPP)(2-MeIm-)]-, and [Fe(TPP)Cl]- were fully
optimized without simplifications applying the BP86 functional25
together with Ahlrichs’ TZVP basis set26 using Gaussian 03.27 Zero-
field splitting and Mo¨ssbauer parameters were calculated for both
the DFT-optimized and crystal structures (with hydrogen atoms
added in idealized positions) using the program ORCA.28 These
for the detection of electronic state distinctions (or differences)
and that they are applicable to a variety of systems.
Experimental Section
General Information. All reactions and manipulations for the
iron(II) porphyrinate derivatives were carried out under argon using
a double-manifold vacuum line, Schlenkware and cannula tech-
niques. Benzene and hexanes were distilled over sodium benzophe-
none ketyl. Chlorobenzene was washed with concentrated sulfuric
acid, then with water until the aqueous layer was neutral, dried
with MgSO4, and distilled twice over P2O5 under argon. Hexanes
were distilled over sodium benzophenone. All solvents were
degassed prior to use by three freeze/pump/thaw cycles. Ethanethiol
(Aldrich) was used as received. 2-Methylimidazole (Aldrich) was
recrystallized from toluene/methanol and dried under vacuum. All
other chemicals were used as received from Aldrich or Fisher. meso-
Tetraphenylporphyrin (H2TPP) was prepared according to Adler
et al.22 and octaethylporphyrin (H2OEP) was purchased from
Midcentury. [Fe(Por)Cl] derivatives were prepared according to a
modified Adler preparation23 and used to prepare the analogous
[Fe(Por)]2O species.24
Synthesis of [K(2-MeIm-)]. In the drybox, 2-methylimidazole
(1.52 g, 19 mmol) was dissolved in 20 mL of THF, then KH (0.72
g, 18 mmol) was added slowly. The mixture was stirred for 3 h,
then filtered by cannula, the resulting solid was dried under vacuum
at 60 °C for 6 h, collected, and stored under argon.
Synthesis of [Fe(Por)] Derivatives. [Fe(Por)]2O (0.03 mmol)
was dissolved in 10 mL of benzene, and 1 mL of ethanethiol was
added by syringe. The mixture was stirred at room temperature for
3 days, then was layered with hexanes for crystallization. Crystalline
[Fe(Por)] was collected after several days, stored under argon and
used for preparing solutions for the MCD measurements.
Synthesis of [K(222)][Fe(OEP)(2-MeIm-)]. [Fe(OEP)]2O (38
mg, 0.03 mmol) was dissolved in 10 mL of benzene, 1 mL of
ethanethiol was added by syringe, and stirred at room temperature
for 3 days. The solvent was removed under vacuum and the solid
was dried for another hour. A suspension of excess K(2-MeIm-)
(28 mg, 0.23 mmol) and Kryptofix 22216 (70 mg, 0.19 mmol) in
15 mL of chlorobenzene was added to the [Fe(II)(OEP)] solid by
cannula, then stirred for 1/2 h and cannula filtered to another
Schlenk flask. X-ray quality crystals were obtained after three weeks
by liquid diffusion using hexanes as nonsolvent in 8 mm × 250
mm sealed glass tubes.
Synthesis of [K(222)][Fe(TPP)(2-MeIm-)]. [Fe(TPP)]2O(40
mg, 0.03 mmol) was dissolved in 10 mL of benzene, 1 mL of
ethanethiol was added by syringe, and stirred at room temperature
for 3 days. The solvent was removed under vacuum, the solid was
dried for another hour, and then dissolved in 10 mL of toluene. A
suspension of excess K(2-MeIm-) (24 mg, 0.20 mmol) and
Kryptofix 222 (49 mg, 0.13 mmol) in 10 mL of toluene was added
to the above solution, the resulting precipitate was cannula filtered,
dried under vacuum for 1/2 h, then dissolved in 10 mL of
chlorobenzene. The resulting solution was cannula filtered to another
Schlenk flask. X-ray quality crystals were obtained after three weeks
by liquid diffusion using hexanes as nonsolvent in 8 mm × 250
mm sealed glass tubes.
Synthesis of [Na(222)][Fe(TPP)Cl]. Crystals of Na(222)-
[Fe(TPP)Cl] were initially prepared accidentally during the reaction
of [Fe(TPP)] with NaN3 in CH2Cl2. [Fe(TPP)]2O (40 mg, 0.03
mmol) was dissolved in 10 mL of chlorobenzene, and 1 mL of
(22) Adler, A. D.; Longo, F. R.; Finarelli, J. D.; Goldmacher, J.; Assour,
J.; Korsakoff, L. J. Org. Chem. 1967, 32, 476.
(23) (a) Adler, A. D.; Longo, F. R.; Kampus, F.; Kim, J. J. Inorg. Nucl.
Chem. 1970, 32, 2443. (b) Buchler, J. W. In Porphyrins and
Metalloporphyrins; Smith, K. M., Ed.; Elsevier Scientific Publishing:
Amsterdam, The Netherlands, 1975; Chapter 5.
(24) (a) Fleischer, E. B.; Srivastava, T. S. J. Am. Chem. Soc. 1969, 91,
2403. (b) Hoffman, A. B.; Collins, D. M.; Day, V. W.; Fleischer, E. B.;
Srivastava, T. S.; Hoard, J. L. J. Am. Chem. Soc. 1972, 94, 3620.
(25) (a) Becke, A. D. Phys. ReV. A 1988, 38, 3098. (b) Perdew, J. P. Phys.
ReV. B 1986, 33, 8822.
(26) (a) Schaefer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992, 97, 2571.
(b) Schaefer, A.; Huber, C.; Ahlrichs, R. J. Chem. Phys. 1994, 100,
5829.
(27) Frisch, M. J.; et al. Gaussian 03; Gaussian, Inc: Pittsburgh, PA, 2003.
(28) Neese, F. ORCA - an ab initio, density functional and semiempirical
program package, version 2.6 rev 40; University of Bonn: Bonn,
Germany, 2007.
9
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A R T I C L E S
Hu et al.
Figure 3. ORTEP diagram (50% probability ellipsoid) of the [Fe(TPP)Cl]-
anion illustrating the atom labeling scheme. Hydrogen atoms have been
omitted for clarity.
Table 1. Selected Distances (Å) for Iron(II) Porphyrinates with
Anionic Ligands
Figure 4. Parallel-mode X-band EPR spectra of (A) Fe(TPP)(2-MeHIm)],
powder; (B) [Fe(TPP)(2-MeHIm)]2 ·2-MeHIm, powder; K(222)[Fe(TPP)(2-
MeIm-)], 6 mM in (C) chlorobenzene and (D) powder; K(222)[Fe(OEP)(2-
MeIm-)], 6 mM in (E) chlorobenzene and (F) powder. Experimental
conditions: temperature 2 K; microwave power, 0.02 mW at 9.3(1) GHz.
Simulation parameters (F, dashed line): S ) 2, D ) -8 cm-1, E/D ) 0.10,
σ(E/D) ) 0.02, gz ) 2.26.
a b
,
complex
〈Fe-Np〉
Fe-Lb
∆N4b
ref
[Fe(TPP)Cl]-
2.1161(11)
2.108(15)
2.118(13)
2.113(4)
2.11(2)
2.070(16)
2.096(4)
2.074(10)
2.114(2)
2.107(2)
2.3400(5)
2.301(2)
2.056(81)c
2.060(2)
2.002(15)
2.069(4)
2.360(2)
2.324(2)
1.937(4)
2.034(3)
0.56
0.53
0.56
0.56
0.52
0.42
0.52
0.44
0.56
0.55
twd
32
12
12
33
34
35
21
36
36
[Fe(TpivPP)Cl]-
[Fe(TPP)(2-MeIm-)]-
[Fe(OEP)(2-MeIm-)]-
[Fe(TpivPP)(2-MeIm-)]-
[Fe(TpivPP)(NO3)]-
[Fe(TPP)(SC2H5)]-
that were schematically depicted in Figure 2. These include the
larger Fe-Np and Fe out-of-plane displacements, all distinctly
different from those of the imidazole complexes. Complete
experimental details and experimental results are given in the
Supporting Information.
[Fe(TpivPP)(SC2H5)]-
[Fe(TpivPP)(OC6H5)]-
[Fe(TpivPP)(O2CCH3)]-
a Average Fe-Np distance. b Value in Å. c Large error owing to a
disordered ligand. d This work.
EPR Spectra. Figure 4 shows parallel-mode X-band EPR
spectra of imidazole- and imidazolate-ligated iron(II) porphy-
rinates. All species display signals near g ) 9 indicative of an
S ) 2 state. Solution samples of [Fe(TPP)(2-MeHIm)] and
[Fe(TPP)(2-MeHIm)]2 ·2-MeHIm did not show parallel-mode
EPR signals. The absence of a solution spectrum is probably
the result of modest intermolecular interactions in solution. The
spectrum of [Fe(TPP)(2-MeHIm)] (Figure 4A) is nearly identical
to that of the sample of [Fe(TPP)(2-MeHIm)]2 ·2-MeHIm
(Figure 4B) containing the two independent molecules: [Fe(T-
PP)(2-MeHIm)] and hydrogen-bonded [Fe(TPP)(2-MeHIm)].
This result indicates that the hydrogen-bonded [Fe(TPP)(2-
MeHIm)] molecule does not produce an EPR signal, presumably
due to large D and E/D values of this molecule. For large D
and E/D, the energy spacing between all spin levels may be
too large to observe transitions at the X-band frequency. The
temperature dependence of the EPR signal of both samples is
shown in Figure 5B, plotted as signal amplitude (base-to-trough)
times temperature. For T > 20 K, signal broadening makes
determination of the amplitude unreliable. Both samples show
the same trend in signal amplitude with temperature: the signals
originate from an excited state doublet. The theoretical curve
is the percent population of the uppermost doublet of an S ) 2
system with D ) +0.9 cm-1 and E/D ) 0.1. However, it is not
possible to quantitatively simulate this signal with these zero-
field parameters. The simulation of the signal from the ground
doublet with these parameters matches the spectrum and agrees
with the sample amount, but this doublet does not reproduce
the temperature dependence shown in Figure 5B. Thus, we
conclude that the magnetic properties of this spin system are
not adequately described by a spin Hamiltonian formalism. A
similar problem is known for the description of the EPR data
calculations were performed with BP86/TZVP, BP86/TZVP with
the larger CP(PPP) basis set29 on iron, and B3LYP/TZVP again
using CP(PPP) for iron. Here, B3LYP is Becke’s three-parameter
hybrid functional.30 The Mo¨ssbauer isomer shifts were obtained
from the calculated total electron densities at the iron nucleus using
empirical correlations established by Neese and co-workers.31
Orbitals were plotted using GaussView (University of Michigan).
Results and Analysis
X-ray Structure. The structure of the [Fe(TPP)Cl]- anion is
illustrated in the ORTEP diagram of Figure 3. The anion has
many common features with other anionic high-spin five-
coordinate iron(II) porphyrinates including the large values of
Fe-Np and the large iron out-of-plane displacements. Selected
values are compared in Table 1.32-36 The strong similarity of
[Fe(TPP)Cl]- and [Fe(TpivPP)Cl]- is to be noted. Also to be
noted are the common features with the imidazolate structures
(29) (a) Neese, F. Inorg. Chim. Acta 2002, 337, 181. (b) Sinnecker, S.;
Slep, L. D.; Bill, E.; Neese, F. Inorg. Chem. 2005, 44, 2245.
(30) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (b) Lee, C.; Yang,
W.; Parr, R. G. Phys. ReV. B 1988, 37, 785.
(31) Ro¨melt, M.; Ye, S.; Neese, F. Inorg. Chem. 2009, 48, 784.
(32) Schappacher, M.; Weiss, R. R.; Montiel-Montoya, R.; Gonser, U.;
Bill, E.; Trautwein, A. Inorg. Chim. Acta 1983, 78, L9.
(33) Mandon, D.; Ott-Woelfel, F.; Fischer, J.; Weiss, R.; Bill, E.; Trautwein,
A. X. Inorg. Chem. 1979, 101, 2442.
(34) Nasri, H.; Ellison, M. K.; Shaevitz, B.; Gupta, G. P.; Scheidt, W. R.
Inorg. Chem. 2006, 45, 5284.
(35) Caron, C.; Mitschler, A.; Riviere, G.; Ricard, L.; Schappacher, M.;
Weiss, R. J. Am. Chem. Soc. 1979, 101, 7401.
(36) Nasri, H.; Fischer, J.; Weiss, R.; Bill, E.; Trautwein, A. J. Am. Chem.
Soc. 1987, 109, 2549.
9
3740 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Five-Coordinate High-Spin Iron(II) Porphyrinates
A R T I C L E S
Figure5. TemperaturedependenceoftheEPRsignalsof(A)K(222)[Fe(OEP)(2-
MeIm-)], (B) [Fe(TPP)(2-MeHIm)] (x), and (B) [Fe(TPP)(2-MeHIm)]2 ·2-
MeHIm (+) plotted as signal amplitude times temperature. The theoretical
curves are the percent populations of an S ) 2 (E/D ) 0.1) system for (A)
the lowermost doublet with D ) -8 cm-1, and (B) the uppermost doublet
with D ) +0.9 cm-1. The D-value of [Fe(TPP)(2-MeHIm] is estimated
from the temperature dependence of the EPR signal.
of deoxymyoglobin. Deoxymyoglobin shows an EPR spectrum
similar to that of these [Fe(TPP)(2-MeHIm)] complexes, and
its characteristics are also not adequately described with a spin
Hamiltonian.37
The EPR spectra from the imidazolate-ligated complex
K(222)[Fe(TPP)(2-MeIm-)] are more complicated (Figure 4C,
D). It appears that both solution and powder samples have
multiple species present with features near g ) 14 and 9. The
temperature dependence of these signals is complicated by the
presence of multiple species, but overall suggests D < 0 with
signals originating from the ground doublet. In contrast, the EPR
spectrum of imidazolate-ligated K(222)[Fe(OEP)(2-MeIm-)]
shows a single species in the solid state (Figure 4F). A plot of
the signal times temperature is shown in Figure 5A. The
theoretical curve is percent population for the lowermost doublet
of an S ) 2 system with D ) -8 cm-1 and E/D ) 0.1. A
simulation using these parameters for the ground doublet is
overlaid on the spectrum (dashed line). This simulation is in
quantitative agreement with the spectrum and the sample
amount. The solution sample of this complex (Figure 4E) also
shows multiple species.
We can draw two conclusions from the results described here.
First, the imidazole- and imidazolate-ligated iron(II) porphyrin
complexes exhibit very different electronic properties. Based
on the determination of the sign and magnitude of the D-value,
we can conclude that the electronic structures of these complexes
must be different. Second, these complexes show a parallel-
mode EPR signal indicative of electronic properties with
significant distortion from axial symmetry. This finding cor-
relates well with crystallographic data which shows that the
Fe-NIm bond is not along the heme normal.
Mo¨ssbauer Spectra. Mo¨ssbauer spectra for both imidazole
and imidazolate-ligated derivatives have been measured in strong
applied magnetic fields and previously reported.11–15 The two
classes were found, without question, to have opposite values
for the sign of the quadrupole splitting. Quadrupole splitting
values for the imidazole-ligated derivatives were in the range
of 1.96 to 2.40 mm/s at 4.2 K with negative sign for the value,
whereas those for imidazolate were in the range of 3.60 to 3.71
mms/s and with positive signs. However in the initial fits, the
sign of the D-value was not found to be distinctly different in
the two classes. The apparent difficulty of assigning the sign of
D was commented on in our earliest work on imidazole
derivatives.11 Even so, in view of the integer-spin EPR results,
Figure 6. Electronic spectra of [Fe(TPP)(2-MeHIm)] (class N). (Top)
UV-vis spectrum measured in CH2Cl2 at room temperature. (Bottom) MCD
C-Term spectrum measured in toluene/CH2Cl2 (1:1) at 2 K. The red lines
represent a correlated fit of these data (cf. Table 3A).
the fitting of the previously obtained Mo¨ssbauer spectra have
been reexamined to determine if the Mo¨ssbauer spectra will
show sensitivity to the sign of D for the two classes. Our
conclusion remains that the sign of D is ambiguous in the fits.
Notwithstanding the ambiguity in the sign determination, good
simulations of the Mo¨ssbauer data can be obtained with the use
of the E/D and D parameters given above from the EPR
measurements. When there are large values of D, in systems
that will be EPR silent, the high-field Mo¨ssbauer data can
unambiguously determine the sign of D. However, for the
smaller D values seen in the imidazolate compounds, integer-
spin EPR is crucial for the determination of this sign.
Magnetic Circular Dichroism Spectra. Figure 6 shows the
absorption and low-temperature MCD spectra of [Fe(TPP)(2-
MeHIm)] (Class N, neutral axial ligand) together with Gaussians
obtained from a correlated fit of these data. The obtained band
positions are listed in Table 3A. These spectra show four
relatively intense features in the region of the Q and Qv bands,
plus the Soret band around 23000 cm-1, which is resolved into
three features in MCD. As shown by one of us previously, the
Soret, Q, and Qv bands of simple metalloporphyrins should give
rise to a derivative-shaped C-term signal at low-temperature in
MCD due to a low-symmetry splitting of the porphyrin Eg (π*)
LUMO of the complexes.38 This causes the Soret, Q, and Qv
excited states, which have Eu symmetry in D4h, to split into two
(37) Hendrich, M. P.; Debrunner, P. G. Biophys. J. 1989, 56, 489.
9
J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010 3741

A R T I C L E S
Hu et al.
components, and correspondingly, two bands are observed for
the Soret, Q, and Qv transitions in MCD. Importantly, due to
excited state spin-orbit coupling, the two components of these
features show opposite signs, and hence, the Soret, Q, and Qv
transitions each give rise to a derivative shaped “pseudo-A”
C-term signal in MCD at low (lq. helium) temperature.
Additionally, since Qv is the vibronic band of Q, it should appear
about 1250 cm-1 to higher energy of Q as determined by
Gouterman.39 With this in mind, the electronic spectra of
[Fe(TPP)(2-MeHIm)] can be readily understood. At low energy,
one component of the Q-band is observed at 16315 cm-1 (band
1). The Qv band corresponds to the pseudo-A signal at 17608
and 18210 cm-1 (bands 2 and 3). This indicates a relatively
large splitting of the two components of the Q excited state of
about 600 cm-1 as determined from Qv. The MCD bands 1 and
2 correspond to the first component of the Q and Qv excited
states, respectively, as evident from (a) their energetic separation
of 1293 cm-1, which is very close to the predicted value of
∼1250 cm-1, and (b) the fact that both have positive MCD
intensity, which means that 2 must be the vibronic band of 1.
This raises one question: where is the negative, second
component of the Q-band located that corresponds to band 3
of Qv? Using the observed energy difference between bands 2
and 3 of about 600 cm-1, the energy of the negative Q
component is estimated to ∼16900 cm-1, as indicated in the
MCD spectrum in Figure 6. One circumstance that could
contribute to the absence of this feature in the MCD spectrum
is the large ∼600 cm-1 splitting of the Q state, which places
this feature just in between the positive bands 1 and 2. Since
this feature must be negative, its MCD intensity might then
cancel out against these positive features (cf. analysis of the
MCD data of [Fe(TPP)(2-MeIm-)]- below). In the case of the
Soret band, the negative component is split into two bands as
shown in Figure 6, which is not unusual, since other porphyrin
π f π* transitions appear in this region. These can specifically
mix with just one component of the Soret band, and in this
way, give rise to the experimentally observed three-band pattern
for the Soret transition. This has recently been discussed in detail
for [FeIII (TPP)Cl] by one of us.40 The main components of the
Soret feature (bands 6 and 7) are located at 22795 and 23310
cm-1, respectively, which is consistent with the observed large
energy splitting between the two components of the Qv band
mentioned above. Importantly, the molar MCD intensity of
[Fe(TPP)(2-MeHIm)] is surprisingly large, and corresponds to
“normal” low-spin ferric hemes as discussed in ref 40.
Figure 7. Electronic spectra of [Fe(TPP)(2-MeIm-)]- (class A). (Top)
UV-vis spectrum measured in CH2Cl2 at room temperature. (Bottom) MCD
C-Term spectrum measured in toluene/CH2Cl2 (1:1) at 2 K. The red lines
represent a correlated fit of these data (cf. Table 3B).
Table 2. Comparison of Observed and Calculated Structural
Parameters for High-Spin Iron(II) Porphyrinates
a b
,
complex
〈Fe-Np〉
Fe-Axb ∆N4b
φc
ref
[Fe(TPP)(2-MeHIm)]-Obsd
-Calcd
2.073 (9)
2.090 (8)
2.127 0.32 24
10
2.183 0.35 18.3 twd
[Fe(TPP)(2-MeIm-)]--Obsd 2.118 (13) 2.056 0.56 23.4 12
-Calcd
2.119 (17) 2.048 0.50
2.118 (16) 2.051 0.51 23
2.113 (19) 2.340 0.56
2.119 (19) 2.300 0.51
1
twd
twd
twd
twd
-Calcd-fixed
[Fe(TPP)Cl]--Obsd
-Calcd
-
-
a Average value. b Value in Å. c Dihedral angle between imidazole
plane and closest Fe-Np vector, value in degrees. d This work.
Figure 7 shows the electronic spectra of [Fe(TPP)(2-
MeIm-)]- (Class A, anionic axial ligand) for comparison. In
this case, the Soret, Q, and Qv features are readily identified
from the MCD spectrum via their pseudo-A C-term behavior.
Table 3B summarizes the obtained band positions from a
correlated fit of these data. Here, bands 2 and 3, observed at
16206 and 16432 cm-1 in MCD, belong to the Q transition,
and bands 4 and 5, located at 17308 and 17640 cm-1 in MCD,
are identified with Qv. The energy differences between the
corresponding features 2 and 4 (negative) and 3 and 5 (positive)
are 1102 and 1208 cm-1, which is in good agreement with the
predicted value of ∼1250 cm-1 mentioned above. The relatively
low value of 1102 cm-1 is due to the fact that the position of
band 2 is not well-defined in the fit. Interestingly, the energy
splitting between the two components of Q and Qv is only about
200-300 cm-1, which is distinctively smaller than in the case
of [Fe(TPP)(2-MeHIm)]. The intensity of the negative band 2
is surprisingly low, but this can be attributed to the fact that
this feature is close to the positive bands 1 and 3, and hence,
the intensity of band 2 is canceled out to a large extent, as
illustrated by the fit of this feature in Figure 7. This is similar
to the case of the missing negative component of the Q-band
in [Fe(TPP)(2-MeHIm)] discussed above. Due to the lability
of [Fe(TPP)(2-MeIm-)]- with the imidazolate ligand, it was
not possible to prepare MCD samples of this compound without
the presence of a slight amount of presumably a ferric impurity.
Comparison of different samples shows that bands 9 and 10
(38) (a) Praneeth, V. K. K.; Na¨ther, C.; Peters, G.; Lehnert, N. Inorg. Chem.
2006, 45, 2795. (b) Lehnert, N., Electron Paramagnetic Resonance
and Low-Temperature Magnetic Circular Dichroism Spectroscopy of
Ferrous Heme Nitrosyl. In The Smallest Biomolecules: PerspectiVes
on Heme-Diatomic Interactions; Ghosh, A., Ed.; Elsevier: Amsterdam,
2008; Chapter 6, pp 147-171.
(39) Gouterman, M. in The Porphyrins; Dolphin, D., Ed Academic: New
York, 1979, Vol. III Part A, pp1-156.
(40) Paulat, F.; Lehnert, N. Inorg. Chem. 2008, 47, 4963.
9
3742 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Five-Coordinate High-Spin Iron(II) Porphyrinates
A R T I C L E S
Table 3. Correlated Fit of the UV-Vis Absorption and MCD
Spectra Shown in Figures 6-8a
MCD
∆ꢀ(M-1cm-1T-1
UV-vis
ꢀ(M-1cm-1
)
no.
position
)
position
A. [Fe(TPP)(2-MeHIm)]
26.0
1a
1
2
3
4
5
6
7
8
15531
16315
17608
18210
19295
22031
22795
23310
24134
-
-
-
3571.4
6785.7
5357.1
5000.1
12142.9
183000.0
153000.0
6000.0
22302.3
25289.1
206.8
58.5
16315
17608
18210
19160
22131
22790
23176
23912
25048
26934
-55.5
62.9
180.4
5131.5
-1956.4
-649.4
-
8a
9
26932
-77.1
B. [Fe(TPP)(2-MeIm-)]-
1
2
3
4
5
5a
6
6a
7
8
15807
16206
16432
17308
17640
18528
19082
-
21950
22650
23288
23983
25482
26642
15.0
-68.6
118.51
-46.3
42.3
-6.0
11.6
-
-271.7
253.7
-92.9
14.9
-20.1
-39.4
15807
2716.0
4142.9
4142.9
5428.6
5428.6
-
4745.9
3048.8
92104.2
68100.6
29607.5
27830.6
34796.6
22051.0
16206
16432
17308
17491
-
19082
20321
22209
22592
23162
23612
24946
26450
9
10
11
12
C. [Fe(TPP)Cl]-
-120.2
102.3
-51.4
36.2
17.1
14.4
-762.4
807.7
1
2
3
4
5
6
7
8
9
10
16438
16587
17547
17714
19057
20864
22576
22760
23005
23652
16251
16372
17434
17614
19710
20527
22460
22595
23005
23652
4642.9
4464.3
7712.8
6000.1
2571.4
Figure 8. Electronic spectra of [Fe(TPP)Cl]- (class A). Top: UV-vis
spectrum measured in CH2Cl2 at room temperature. Bottom: MCD C-Term
spectrum measured in toluene/ CH2Cl2 (1:1) at 2 K. The red lines represent
a correlated fit of these data (cf. Table 3C).
2071.4
159000.0
144000.0
46428.5
60135.4
233.9
-98.7
shown by Michl, this can be explained by a change in the
relative HOMO (porphyrin A1u, A2u) and LUMO (porphyrin Eg)
splitting of the porphyrin ring,41 where the sequence observed
for [Fe(TPP)(2-MeHIm)] is associated with ∆(LUMO) .
∆(HOMO). [Fe(TPP)(2-MeIm-)]- (and [Fe(TPP)Cl]-) then
correspond to the ∆(HOMO) . ∆(LUMO) case.
a For the fit, the minimum number of Gaussians has been used. If a
band can be identified from the MCD spectrum, but no corresponding
band is necessary to fit the absorption spectrum, then the absorption data
is not fit with this feature. Hence, in very few cases, bands appear in the
correlated fit of the MCD spectra, but not in the fit of the absorption
spectra (or vice versa).
Finally, Figure 8 shows the absorption and MCD data of
complex [Fe(TPP)Cl]- (class A). As in the case of [Fe(TPP)(2-
MeIm-)]-, clean pseudo-A C-term signals are observed for Q,
Qv, and the Soret band, which can therefore readily be identified
from the spectra. The Q-band corresponds to bands 1 and 2
observed at 16438 and 16587 cm-1, respectively. The vibronic
Qv transition is identified with bands 3 and 4 at 17547 and 17714
cm-1. Therefore, [Fe(TPP)Cl]- shows the smallest splitting
between the two components of the Q excited state of only ∼150
cm-1. The energy difference between components 1 and 2 of
Q and Qv is 1109 (bands 1 and 3) and 1127 cm-1 (bands 2 and
4), respectively, which is in good agreement with the predicted
value of ∼1250 cm-1 (vide supra). The main components of
the Soret band are assigned to bands 7 and 8 at 22576 and 22760
cm-1, respectively, again reflecting the very small energy
splitting between the two components of the Eu excited states
as observed for Q. Band 9 could either correspond to a third
Soret component as observed for [Fe(TPP)(2-MeHIm)], or an
impurity as in the case of [Fe(TPP)(2-MeIm-)]-. The latter
explanation is the most likely, based on the observed pseudo-A
behavior of bands 9 and 10 in [Fe(TPP)Cl]-, which is very
actually belong to this impurity. Therefore, the Soret band of
[Fe(TPP)(2-MeIm-)]- is assigned to bands 7 and 8, observed
at 21950 and 22650 cm-1, respectively. Due to the strong
overlap of bands 7-9, the absolute energies of these features
are not well-defined. The most striking difference in the MCD
spectra of complexes [Fe(TPP)(2-MeHIm)] and [Fe(TPP)(2-
MeIm-)]- is the significantly lower molar MCD intensity in the
case of [Fe(TPP)(2-MeIm-)]- by one order of magnitude. This
difference relates to the electronic structures of the complexes,
and is further analyzed below (cf. DFT Sections). In addition,
the signs of the Soret, Q, and Qv MCD bands are opposite in
[Fe(TPP)(2-MeIm-)]- compared to [Fe(TPP)(2-MeHIm)], i.e.
[Fe(TPP)(2-MeHIm)] shows the sequence +∆ε/(-∆ε)/+∆ε/
-∆ε/+∆ε/-∆ε (from lower to higher energy: Q - Qv- Soret),
whereas [Fe(TPP)(2-MeIm-)]- (and [Fe(TPP)Cl]-, vide infra)
exhibits the sequence -∆ε/(+∆ε)/-∆ε/+∆ε/-∆ε/+∆ε. As
(41) (a) Michl, J. J. Am. Chem. Soc. 1978, 100, 6801 (and the other 16
articles in this issue). (b) Mack, J.; Stillman, M. J.; Kobayashi, N.
Coord. Chem. ReV. 2007, 251, 429.
9
J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010 3743

A R T I C L E S
Hu et al.
Table 4. Summary of the Experimental EPR and Mo¨ssbauer
Results for the Investigated High-Spin Ferrous Heme Complexes
similar to the MCD features of the impurity observed for
[Fe(TPP)(2-MeIm-)]-. Importantly, the molar MCD intensity
of [Fe(TPP)Cl]- is again much lower compared to [Fe(TPP)(2-
MeHIm)]. This indicates that this difference in the total molar
MCD intensity42 between Class N and Class A high-spin ferrous
hemes constitutes a general trend, and can be employed to
distinguish between the two classes of compounds. Correspond-
ingly, these two classes must differ significantly in their
electronic structures, which would then explain the apparent
difference in total MCD intensity. The experimentally deter-
mined order in MCD intensity is: [Fe(TPP)(2-MeHIm)] .
[Fe(TPP)Cl]- > [Fe(TPP)(2-MeIm-)]- as evident from Figures
6-8. The electronic structures of these complexes are compared
in detail below.
[Fe(TPP)(2-MeHIm)]
[Fe(TPP)(2-MeIm-)]-
[Fe(TpivPP)Cl]-
D (cm-1
δ (mm/s)
∆Eq(mm/s)
η
)
+0.9a
0.85
-1.95
0.8
-8b
1.00
+3.60
0.02
n/ac
1.01
4.36
n/ac
a D ) -5.0 cm-1 had initially been obtained by Lang et al. (J. Chem.
Phys. 1979, 71, 4899) from Mo¨ssbauer spectroscopy at 6T. This value
was later corrected to a positive D of about 5-30 cm-1 based on
variable-field Mo¨ssbauer measurements by Scheidt and co-workers
(J. Am. Chem. Soc. 2005, 127, 5675). However, Mo¨ssbauer fits are not
very sensitive to the value of D, and hence, the EPR-based value
presented here is much more reliable. b Determined for
[Fe(OEP)(2-MeIm-)]-. c Not available
Density Functional Theory (DFT) Calculations: Geometry
Optimizations. To determine the electronic structural differences
between the two different classes of five-coordinate, high-spin
ferrous hemes, we have fully optimized the geometries of the
complexes [Fe(TPP)(2-MeHIm)], [Fe(TPP)(2-MeIm-)]-, and
[Fe(TPP)Cl]- using BP86/TZVP without any simplifications.
Figures S1sS3 (Supporting Information) show core diagrams
of the fully optimized structures including important bond
lengths. In the case of [Fe(TPP)(2-MeHIm)], the Fe-Np and
Fe-NIm distances of 2.090 (averaged) and 2.183 Å, respectively,
are somewhat on the large side of the range of values observed
for imidazole complexes (Table 2), but are still in reasonably
good agreement with experiment. The out-of-plane displace-
ment, ∆N4, of iron of 0.35 Å and the orientation of the imidazole
relative to the porphyrin plane (dihedral angle φ ) 18°) are in
excellent agreement with the crystal structure of [Fe(TPP)(2-
MeHIm)] (exp. values: 0.32 Å and 24°, respectively). The out-
of-plane distortions of the TPP ring in both the experimental
and calculated structures are complex, and correspond to a
combination of different types of distortions43 leading to C1
symmetries of the cores. As evident from Figure S1 (Supporting
Information), ruffling is more dominant in the calculated
structure compared to the experimental result. In comparison,
the calculated Fe-Np and Fe-NIm distances of 2.119 (averaged)
and 2.048 Å, respectively, for the imidazolate complex [Fe-
(TPP)(2-MeIm-)]- are in excellent agreement with experiment
(Table 2). This is also true for the calculated out-of-plane
displacement ∆N4 of iron, which is 0.50 Å (exp. value: 0.56
Å). The largest deviation in the case of [Fe(TPP)(2-MeIm-)]-
is observed for the orientation of the imidazolate: the calculated
value for φ ) 1° is smaller compared to φ ) 23° from
experiment. We therefore recalculated the structure of [Fe(TPP)(2-
MeIm-)]- where the angle φ was fixed at 23° ([Fe(TPP)(2-
MeIm-)]- (fixed)). The structure obtained from this treatment
is only +0.7 kcal/mol higher in energy than [Fe(TPP)(2-
MeIm-)]-, and is shown in the Supporting Information, Figure
S2. Besides the change in the dihedral angle, the other geometric
parameters of [Fe(TPP)(2-MeIm-)]- (fixed) are very similar
compared to the fully optimized structure of [Fe(TPP)(2-
MeIm-)]-. More importantly, the electronic structures of
[Fe(TPP)(2-MeIm-)]- and [Fe(TPP)(2-MeIm-)]- (fixed) are
similar as discussed below. The nonplanar distortions of the
TPP core in the experimental and calculated structures of
[Fe(TPP)(2-MeIm-)]- are again complex. The experimental core
conformation of [Fe(TPP)(2-MeIm-)]- shows a clear saddling
contribution. In the case of the calculated structures, the
imidazolate complexes [Fe(TPP)(2-MeIm-)]- and [Fe(TPP)-
(2-MeIm-)]- (fixed) show pronounced contributions of the
doming distortion, but less saddling. Finally, the calculated
Fe-Np and Fe-Cl distances of 2.119 (averaged) and 2.300 Å,
respectively, for the chloride complex [Fe(TPP)Cl]- are again
in excellent agreement with experiment (Table 2). The calculated
out-of-plane displacement ∆N4 is 0.51 Å, which also compares
well with experiment (exp. value for [Fe(TPP)Cl]-: 0.56 Å).
As observed before, both the experimental and calculated
structures of [Fe(TPP)Cl]- show complex nonplanar distortions
of the porphyrin core that differ somewhat between theory and
experiment. The distortion observed in the crystal structure
corresponds mostly to ruffling and doming, whereas the
calculated structure does not predict the ruffling contribution.
In summary, the DFT calculations are able to reproduce the
geometries of the high-spin Fe(II) coordination groups well in
the three complexes, including Fesligand bond lengths, relative
orientations of imidazole and imidazolate, and, importantly, the
out-of-plane displacement of iron. The cores of the TPP ligands
show strong low-symmetry (C1) distortions, and differences are
observed in the core conformations between theory and experi-
ment. However, as a comparison of [Fe(TPP)(2-MeIm-)]- and
[Fe(TPP)(2-MeIm-)]- (fixed) shows, the effects of these dif-
ferences on the electronic structure of the iron(II) center are
very small.
Calculation of Zero Field Splitting and Mo¨ssbauer
Parameters from DFT. As described above, the EPR and
Mo¨ssbauer measurements have identified dramatic differences
in the magnitude and sign of the axial zero field splitting
parameter D and the Mo¨ssbauer quadrupole splitting ∆Eq
between high-spin ferrous heme complexes of classes N and
A, as summarized in Table 4. In particular, complexes with
neutral imidazole ligands (class N) like [Fe(TPP)(2-MeHIm)]
show negative quadrupole splittings ∆Eq and large asymmetry
parameters η, whereas complexes with axial anionic imidazolate
or chloride ligands (Class A) like [Fe(TPP)(2-MeIm-)]- and
[Fe(TPP)Cl]- exhibit a positive ∆Eq and a small η. Since ∆Eq
reflects the electron distribution of iron in the ground state, these
differences indicate that compounds of classes N and A differ
(42) It would be advantageous to actually compare molar MCD C-term
intensities here instead of the total molar MCD intensities, since the
latter also include contributions form the MCD A- and B-terms.
However, as shown in ref 40, MCD measurements at room temperature
are required to determine the absolute A- and B-term intensities. The
frozen glasses applied here do not allow these data to be measured,
since the glasses disintegrate above 150 K. Data taken at ∼100 K
still show very significant C-term contributions.
(43) (a) Shelnutt, J. A.; Song, X.-Z.; Ma, J.-G.; Jia, S.-L.; Jentzen, W.;
Medforth, C. J. Chem. Soc. ReV. 1998, 31. (b) Scheidt, W. R.
Systematics of the Stereochemistry of Porphyrins and Metallopor-
phyrins. In The Porphyrin Handbook; Kadish, K. M.; Smith, K. M.;
Guilard, R., Eds.; Academic Press: San Diego, 2000; Vol. 3, Chapter
16, pp 49-112.
9
3744 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Five-Coordinate High-Spin Iron(II) Porphyrinates
A R T I C L E S
Table 5. Summary of the DFT-Calculated EPR and Mo¨ssbauer Parameters for High-Spin Ferrous Heme Complexes Using Fully Optimized
Structures from BP86/TZVP
BP86/TZVP + CP(PPP) on Fe
B3LYP/TZVP + CP(PPP) on Fe
[Fe(TPP)(2-MeHIm)]
[Fe(TPP)(2-MeIm-)]-
[Fe(TPP)(2-MeHIm)]
[Fe(TPP)(2-MeIm-)]-
g-values
2.029
2.010
2.041
2.018
2.036
2.035
2.056
2.047
2.064
2.050
2.075
2.058
g-iso
2.043
-3.74
0.31
11826.88925
0.738
-2.213
0.761
2.032
+2.55
0.21
11826.97657
0.700
+2.213
0.391
2.058
-4.74
0.27
11815.71193
0.761
2.783
2.041
-6.21
0.16
11815.63672
0.789
+3.772
0.066
D (cm-1
E/D
)
F (o) (au-3
δ (mm/s)
∆Eq (mm/s)
η
)
0.992
Table 6. Summary of the DFT-Calculated EPR and Mo¨ssbauer
Parameters for High-Spin Ferrous Heme Complexes Based on the
Experimentally Observed Structures
electronic structural differences between class N and A high-
spin ferrous hemes are therefore analyzed based on BP86/TZVP
calculations on the crystal structures. As shown in the Support-
ing Information, the same conclusions about the electronic
ground states of these compounds are drawn from the MO
diagrams obtained for the DFT-optimized structures. This again
emphasizes that the small structural differences between the
experimental and optimized structures of the different com-
pounds do not affect the ground states of these complexes in a
significant way, as reflected by the similar ∆Eq values.
BP86/TZVP + CP(PPP) on Fe
[Fe(TPP)(2-MeHIm)]
[Fe(TPP)(2-MeIm-)]-
g-values
2.0114
2.0409
2.0739
2.0421
+3.4077
0.2917
11827.10921
0.645
2.0002
2.0296
2.0802
2.0367
-2.2767
0.3174
11827.10552
0.646
g-iso
D (cm-1
E/D
)
F (o) (au-3)]
δ (mm/s)
∆Eq (mm/s)
η
Electronic Structure from DFT. In all three complexes,
iron(II) is in the high-spin state (S ) 2), and hence, the principal
d-electron configuration of the metal is [t2]4[e]2 or [dxz, dyz, dxy]4
-2.313
0.751
+2.335
0.141
2
2
2
2
[dx - y , dz ] . In a spin unrestricted formalism, this means that
all R-d orbitals are occupied, and in addition, one of the ꢀ-d
orbitals is occupied (here, R denotes the majority spin and ꢀ
the minority spin). Correspondingly, the central questions for
the electronic structures of the complexes are (a) which one
of the three t2-type orbitals carries the ꢀ-electron, and (b) what
is the energy sequence of the remaining two unoccupied ꢀ-t2
orbitals. Because of this, the ꢀ-MO diagrams of the three
complexes are individually discussed in the following.
in their electronic ground states. DFT calculations on the
optimized structures with either BP86/TZVP (see Table S7,
Supporting Information)) or BP86/TZVP using the larger
CP(PPP) basis set for iron (see Table 5, left) reproduce this
difference in the sign of ∆Eq and the value of η very well. The
differences between these two methods are negligible.
Interestingly, our EPR results also show that [Fe(TPP)(2-
MeHIm)] and [Fe(OEP)(2-MeIm-)]- differ in the sign of the
axial zero-field splitting parameter D as shown in Table 4.
Because the value of D relates to spin-orbit coupling of the
ground state with low-lying excited states, this sign difference
in D, however, is much less diagnostic for a difference in ground
states than is ∆Eq. Using BP86 calculations on the DFT-
optimized structures, the sign change of D is again reproduced,
but in the opposite way compared to experiment: whereas
experimentally, the imidazole complex has a positive D and
for the imidazolate complex D is negative, the DFT calculations
predict exactly the opposite trend. In order to determine whether
this relates to small structural differences between the experi-
mental (crystallographic) and DFT-optimized structures, we then
recalculated the EPR and Mo¨ssbauer parameters for the observed
molecular structures. As shown in Table 6, both the trends in
∆Eq and in D are now exactly reproduced in the calculations.
In addition, the agreement for η has also distinctively improved.
This indicates that the small deviations in the porphyrin
structures as described above are significant for the calculation
of D, i.e. the excited states energies, whereas the electronic
ground state is described well in either case, and ∆Eq is
insensitive to this difference.
Figure 9 shows the frontier ꢀ-MO diagram of [Fe(TPP)(2-
MeHIm)], obtained for the observed molecular structure using
BP86/TZVP. In this case, the single ꢀ-d electron occupies the
bonding combination of the dxz orbital with one component of
the unoccupied E(g) LUMO of the porphyrin ring (cf. Figure
S4 for porphyrin orbitals, Supporting Information). As can be
seen from the contour plot in Figure 9, the ∼0.3 Å out-of-plane
displacement of iron actually allows for a weak σ backbonding
interaction between these orbitals. The resulting MO, labeled
dxz_E(g), has 65% dxz character. Importantly, the dxz orbital is
located in the plane of the imidazole ligand. The ꢀ-dyz orbital
is unoccupied, and is found 2221 cm-1 higher in energy. Again,
this orbital shows a σ backbonding interaction with the other
component of the E(g) porphyrin LUMO, and the resulting
bonding combination is labeled dyz_E(g) in Figure 9. The dyz
orbital is oriented perpendicular to the imidazole plane, and
shows an additional π bonding interaction with an occupied π
orbital of the imidazole ligand. The relatively large rhombic
splitting between the dxz and dyz orbitals is therefore mostly due
to the axial imidazole ligand, and not the splitting of the
porphyrin E(g) LUMO caused by the nonplanar distortion of the
porphyrin ring (vide supra). Finally, the dxy orbital is nonbond-
ing, and found at highest energy as shown in Figure 9. This
orbital sequence with the dxz and dyz orbitals at lower energy
than dxy is uncommon for iron porphyrin complexes, but is
consistent with the conclusions drawn earlier from Mo¨ssbauer
spectroscopy.11 The well-studied low-spin ferric hemes usually
Finally, we also tried B3LYP/TZVP calculations (using
CP(PPP) on Fe) on the DFT-optimized structures. The results,
shown in Table 5, right, do not reproduce the experimental
results, that is, no sign change for either ∆Eq or D is observed.
This method is therefore unsuitable. In the following, the
9
J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010 3745

A R T I C L E S
Hu et al.
Figure 10. Frontier ꢀ-MO diagram of [Fe(TPP)(2-MeIm-)]- calculated
with BP86/TZVP. The applied coordinate system is chosen such that x and
y are located in the porphyrin plane, and z is orthogonal to the porphyrin
ring in the direction of the axial ligand. E(g) refers to the LUMO of the
porphyrin as shown in Figure S4 (the index g is put in brackets because of
the low symmetry of the porphyrin core, Supporting Information). The
nomenclature a_b indicates that orbital a interacts with b and that a has a
larger contribution to the resulting MO.
Figure 9. Frontier ꢀ-MO diagram of [Fe(TPP)(2-MeHIm)] calculated with
BP86/TZVP. The applied coordinate system is chosen such that x and y
are located in the porphyrin plane, and z is orthogonal to the porphyrin
ring in the direction of the axial ligand. E(g) refers to the LUMO of the
porphyrin as shown in Figure S4 (the index g is put in brackets because of
the low symmetry of the porphyrin core, Supporting Information). The
nomenclature a_b indicates that orbital a interacts with b and that a has a
larger contribution to the resulting MO.
show ground states where the dxy orbital is at lower energy.44
But known exceptions of low-spin iron(III) systems where dxy
is at higher energy have π-accepting axial ligands such as
weakly basic pyridines45,46 or isocyanides47 demonstrating the
importance of axial ligation.
region of the ꢀ-MO diagram of [Fe(TPP)(2-MeIm-)]-, obtained
for the observed solid-state structure with BP86/TZVP. In this
case, the single ꢀ-d electron occupies an MO that corresponds
to d′ with a small admixture of dxz. This mixing leads to a
xy
rotation of the dxy orbital out of the xy plane. A contour plot of
the t2 type orbital obtained this way is shown in Figure 10. As
The difference in the d orbital energy sequence between
iron(II) and iron(III) can be rationalized as follows. First, in
the ferric oxidation state, the d orbitals are at low energy, and
correspondingly, the porphyrin serves mostly as a π donor
ligand. This π donation leads to an increase in the energy of
the dxz and dyz orbitals relative to dxy. On the other hand, in the
ferrous oxidation state, the d orbitals are at high energy, and
correspondingly, the porphyrin now serves as a backbonding
ligand. This interaction lowers the energy of the dxz and dyz
orbitals relative to dxy. Because of the general nature of this
effect, we believe that the electronic structure description
elaborated for [Fe(TPP)(2-MeHIm)] is in general representative
for class N high-spin ferrous porphyrin complexes.
evident from the contour plot, this rotation of the d′ orbital
xy
enables a σ backbond with one component of the E(g) LUMO
of the porphyrin. Note that the label d′ is used in the following
xy
to indicate that this orbital actually corresponds to a combination
2
2
of the dxy and dx -y orbitals (∼3:1 ratio), which leads to a slight
rotation of the dxy orbital in the xy plane to optimize bonding in
the distorted symmetry of the complex. A second combination
of d′ and the porphyrin E(g) LUMO is observed only 1365 cm-1
xy
higher in energy than the occupied ꢀ-d MO as shown in Figure
10. In summary, the d′ orbital is observed at lowest energy in
xy
[Fe(TPP)(2-MeIm-)]-, which is strikingly different from [Fe-
(TPP)(2-MeHIm)]. This relates to the very large out-of-plane
displacement of iron in [Fe(TPP)(2-MeIm-)]-, which allows
In contrast, the electronic structure of class A complexes
shows significant differences. Figure 10 shows the frontier
for the d′ orbital to undergo a backbonding interaction with
xy
the E(g) LUMO of the porphyrin as illustrated by the contour
plot of the d′ /dxz-E(g) MO in Figure 10. In contrast, dxy is strictly
xy
(44) Walker, F. A. Chem. ReV. 2004, 104, 589.
nonbonding in the case of [Fe(TPP)(2-MeHIm)]. Finally, the
dxz and dyz orbitals are observed at highest energy in the case of
[Fe(TPP)(2-MeIm-)]-, which is due to the fact that imidazolate
is a distinctively stronger donor than imidazole. This affects in
particular dyz, which interacts with an occupied π orbital of
imidazolate, as evident from the contour plot in Figure 10. This
shifts the dyz orbital to unusually high energy. In contrast, dxz
(45) Safo, M. K.; Gupta, G. P.; Watson, C. T.; Simonis, U.; Walker, F. A.;
Scheidt, W. R. J. Am. Chem. Soc. 1992, 114, 7066.
(46) Safo, M. K.; Walker, F. A.; Raitsimring, A. M.; Walters, W. P.; Dolata,
D. P.; Debrunner, P. G.; Scheidt, W. R. J. Am. Chem. Soc. 1994, 116,
7760.
(47) Walker, F. A.; Nasri, H.; Turowska-Tyrk, I.; Mohanrao, K.; Watson,
C. T.; Shokhirev, N. V.; Debrunner, P. G.; Scheidt, W. R. J. Am.
Chem. Soc. 1996, 118, 12109.
9
3746 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Five-Coordinate High-Spin Iron(II) Porphyrinates
A R T I C L E S
and |J〉 in direction w. These equations show that C-term
intensity requires two orthogonal electric dipole allowed transi-
tions, where either the excited states (mechanism 1) or the
ground and a low-lying excited state (mechanism 2) are
spin-orbit coupled in a direction orthogonal to the plane formed
by the two transition dipole moments. Previous work of one of
us has shown that for the Soret, Q, and Qv bands, mechanism
1 leads to a pseudo-A (derivative-shaped) C-term signal in the
MCD spectrum.38 This is due to the fact that metalloporphyrins
do not show strict 4-fold symmetry, leading to a small energy
splitting between the two components of the E(g) LUMO of the
porphyrin. Since the Soret, Q, and Qv bands are mostly in-plane
(x, y) polarized, the corresponding excited states can then
spin-orbit couple in the z direction (w ) z in mechanism 1).
Besides this, mechanism 2 becomes important when low-lying
(ligand field) excited states are available, for example in the
case of low-spin ferric porphyrins. As one of us has shown
recently, the two different electron configurations [dxy]2 [dxz, dyz]3
(unpaired electron located in the dxz or dyz orbital) and [dxz, dyz]4
[dxy]1 (unpaired electron located in dxy) differ significantly with
respect to SOC in z direction, and hence, can be distinguished
using low-temperature MCD spectroscopy (see Discussion in
ref 40). In a similar fashion, the MCD intensity differences
between all three complexes investigated here relate to mech-
anism 2, that is, changes in the t2-type ꢀ-d orbitals. Indeed, as
described above, all three compounds differ quite strongly in
the properties of these orbitals. In the following, we will use
the observed Soret band MCD intensity to compare the
properties of all complexes.
Figure 11. MCD C-term mechanisms (left, middle), and example for
mechanism 2 for class N five-coordinate high-spin ferrous heme complexes
like [Fe(TPP)(2-MeHIm)] (right).
mixes with an imidazolate (in-plane) σ orbital, which is a weaker
interaction, causing the energy splitting between dxz and dyz
(‘rhombicity’). The principal electronic structure descriptions
do not change when the fully optimized structures of these
compounds are considered, as evident from Figures S5sS7
(Supporting Information).
With the electronic structure descriptions of [Fe(TPP)(2-
MeHIm)] and [Fe(TPP)(2-MeIm-)]- clearly laid out, an inter-
esting question is how the properties of class A complexes
change if the anionic axial ligand is an isotropic π donor, such
that dxz and dyz experience the same level of energetic destabi-
lization. In order to evaluate this case, we consider the chloride
complex [Fe(TPP)Cl]-. Figure S7 (Supporting Information)
shows the ꢀ-MO diagram for this complex in the frontier region,
which strikingly supports our previous analysis. In this case,
the ꢀ-d electron again occupies an orbital that has predominant
dxy character (50% dxy plus 11% dxz as shown in Figure S7,
Supporting Information). Both the dxz and dyz orbitals are now
high in energy, due to chloride having equivalent 3px and 3py
donor orbitals. This supports the previous conclusion that dyz
in [Fe(TPP)(2-MeIm-)]- is high in energy because of imida-
zolate being a strong π donor.
On the basis of these results, the two important general
differences between the electronic structures of class N and class
A five-coordinate high-spin ferrous heme complexes are: (a)
dxy is low in energy in class A compounds due to the large out-
of-plane displacement of iron, which allows for a backbonding
interaction with the porphyrin E(g) LUMO, and (b) dyz and dxz
are high in energy due to the presence of strong anionic (π)
donor ligands. As a consequence, the ꢀ-d electron occupies the
dxz orbital in class N compounds, and the dxy orbital in class A
complexes. This difference in electronic structure is directly
reflected by the molar MCD intensities of these complexes, as
further evaluated below.
Assuming that state |K〉 in mechanism 2 is a low-lying ligand
field excited state, and keeping in mind that the Soret band is
x, y polarized, the C-term intensity for this feature is predomi-
nantly generated by SOC in the z direction. The corresponding
reduced SOC matrix element between the ground state |A〉 and
the low-lying excited state |K〉 is defined as (cf. mechanism 2
in Figure 11):
jKA
Lz ) Im〈K|HzSOC|A〉
(1)
where HzSOC ) ΣAꢁ(rA)lA, z (the integrals over the spin functions
have already been taken into account in the derivation).48 Here,
the sum runs over all atoms A of the molecule, ꢁ(rA) is the radial
SOC operator of atom A, and lA, z is the angular momentum
operator lz on center A. Using the LCAO approximation for
molecular orbitals, that is, |K〉 ) Σrcrφr and |A〉 ) Σscsφs, and
neglecting two- and three-center integrals, the total SOC
contribution becomes:
Electronic Structure and MCD Intensity. With the electronic
structure descriptions of all three complexes in hand, we can
now analyze the observed drastic difference in low-temperature
MCD intensities between these complexes. In general, MCD
C-term intensity is due to two different mechanisms, which
require spin-orbit coupling between two energetically close
excited states (mechanism 1), or the ground state and a low-
lying excited state (mechanism 2). Figure 11 shows illustrations
of these mechanisms, together with mathematical expressions
Im[
ꢂA
〈c φA|lA,z|csφsA〉]
∑ ∑
r
r
Im〈K|HzSOC|A〉
A
r,s
)
(2)
∆
∆
KA
KA
where ꢂA is the one-electron spin-orbit coupling constant of
atom A. Importantly, the value of this integral is dominated by
the contributions of iron d orbitals for two reasons: (a) the SOC
48
constant ꢂ3d for iron(II) is about 400 cm-1
,
which is much
for the resulting MCD C-term intensities as derived in ref 48.
larger than SOC constants for light elements like C and N, and
(b) since lA, z|pAz 〉 ) 0, the integrals in eq 2 should all vanish for
pure porphyrin π or π* MOs. Although the d orbitals in the
complexes all show quite large admixtures of the porphyrin E(g)
LUMO π* orbitals (vide supra), these contributions can therefore
be neglected for the SOC matrix elements in eq 2, which
become:
AJ
b
Here, Du is the transition dipole moment for the electronic
transition between states |A〉 and |J〉 in direction u (u, V, w )
-1
x, y, z), ∆ is the energy difference between states |K〉 and |J〉,
KJ
and LKwJ is the reduced SOC matrix element between states |K〉
j
(48) Neese, F.; Solomon, E. I. Inorg. Chem. 1999, 38, 1847.
9
J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010 3747

A R T I C L E S
Hu et al.
ꢂA
〈c φA|lA,z|csφsA〉]
Im[ꢂFe
〈cdφd|l |cdφd〉]
can be seen in Figure 2, the iron displacement from the four
nitrogen atom plane and the Fe-Np bond distances are both
significantly larger in the imidazolate than in the imidazole
derivatives. Simply put, in the imidazolate derivatives the size
of the iron(II) atom appears to be larger than it is in the
imidazole species. The structures for a number of iron(II) species
with an axial anionic ligand are similar to those of the
imidazolate derivatives as well as that of anionic [Fe(TPP)Cl]-
reported herein. The strong similarities between members of
each group strongly suggested that the origin of these structural
differences are the consequence of differing electronic structures.
In exploring this possible dichotomy in this paper, we
distinguish between two classes of iron(II) porphyrinate deriva-
tives, which we call Class N, those species with neutral, mostly
nitrogen donors, and Class A, those species with an anionic
axial ligand. Clearly, the charge on the axial ligand has a strong
effect on the electronic structure. Because the d-orbitals are all
at relatively high energy in the ferrous state, the π-donating
character of the anionic ligands leads to the raising of the dxz
and dyz orbital energies and concomitant relative lowering of
the energy of the dxy orbital which then becomes the doubly
occupied orbital. This effect holds whether the axial ligand has
two equivalent π-donor orbitals (px and py), as in the case of
atomic anions (halides, oxyanions), or anisotropic (in-plane and
out-of-plane) π donor orbitals, as in the case of imidazolate,
hydroxide, or thiolate ligands. Keeping in mind that protonation/
deprotonation in the imidazole case occurs at the distant ε-N
atom, formally sp2 hybridized, the strong effect of the proton-
ation state of this N-atom on the electronic structure of the
complex is certainly surprising. In contrast, the corresponding
change in electronic structures in the cases of water versus
hydroxide, or thiol versus thiolate complexes is somewhat less
surprising, since in these cases the deprotonation occurs directly
at the coordinating donor atom, thereby strongly increasing its
π-donor abilities. This increase in π donation also strengthens
the Fes(anionic ligand) bonds as evident from the shorter Fe-N
bond distance in imidazolate versus imidazole complexes. This
basis for the electronic structure differences suggests that a five-
coordinate aqua species would be a class N whereas a hydroxo
species would be class A. Indeed, de Gracia et al.50 describe
solution-based Raman and UV-vis spectroscopy for five-
coordinate high-spin ferrous aqua and hydroxo complexes,
which they conclude have the same electronic structures as the
analogous imidazole and imidazolate species, respectively. We
believe that these class N and A groupings are broadly applicable
and we expect additional data will continue and strengthen the
classification. Classification of six-coordinate high-spin ferrous
species is somewhat limited. We note that the only well-
characterized six-coordinate high-spin iron(II) species are those
of bis(tetrahydrofuran) coordination51,52 that are clearly shown
to be members of class N by Mo¨ssbauer characterization.51
As we have outlined in Results and Analysis, we have
explored several physical approaches to determine whether the
methods provide clear-cut distinctions between the two classes
of high-spin iron(II) porphyrinates. In every case, within the
Im[
∑ ∑
∑
r
r
r
r
z
s s
A
r,s
r,s
)
∆
∆
KA
KA
(3)
where the φd now refer to the principal d orbitals of iron.
Equation 3 can be used to obtain qualitative insight into the
relative MCD C-term intensities for the three complexes of
interest. A quantitative evaluation is prohibited by the fact that
the intensity contribution from mechanism 1 is not exactly
known, but should at least be quite similar for the three
complexes of interest.
For [Fe(TPP)(2-MeHIm)], the four lowest lying excited states
(using the nomenclature from Figure 9) give rise to the four
contributions:
Im[ꢂFe〈dyz-E(g)|lz|dxz-E(g)〉] Im[ꢂFe〈dxy|lz|dxz-E(g)〉] ) 0
Im[ꢂFe〈E(g)-dxz|lz|dxz-E(g)〉] ) 0 Im[ꢂFe〈E(g)-dyz|lz|dxz-E(g)〉]
Since SOC has to be active in the z direction, the matrix
elements 〈dxy|lz|dxz〉 and 〈dxz|lz|dxz〉 vanish. Importantly, this means
that dxy cannot contribute to MCD intensity for x, y polarized
transitions. In addition, Im〈dyz|lz|dxz〉 ) 1.49 Using the ground
state molecular orbital coefficients, and the orbital energies as
a rough approximation for the corresponding excited state
energies from Figure 9, one can estimate that the contribution
from 〈dyz_E(g)|lz|dxz_E(g)〉 will be dominating. (From TD-DFT:
dyz_E(g) excited state ) +1543 cm-1.) The same type of analysis
can be applied to [Fe(TPP)(2-MeIm-)]-, which allows us to
approximate the C-term intensity ratio between complexes
[Fe(TPP)(2-MeHIm)] and [Fe(TPP)(2-MeIm-)]-. In the case
of [Fe(TPP)(2-MeIm-)]-, the dominating contribution to MCD
intensity comes from the integral 〈dyz_E(g)|lz|d′ /dxz_E(g)〉. (From
xy
TD-DFT: dyz_E(g) excited state ) +5741 cm-1.) Considering
the main d-orbitals of complexes [Fe(TPP)(2-MeHIm)] and
[Fe(TPP)(2-MeIm-)]-, the MCD C-term ratio between com-
plexes [Fe(TPP)(2-MeHIm)] and [Fe(TPP)(2-MeIm-)]- is roughly
estimated to be ∼6:1. (Using TD-DFT excited state energies,
the ratio becomes 7:1.) This number is somewhat low compared
to experiment, which could be due to the fact that (a) the orbital
energies are only crude approximations of the corresponding
excited state energies, and more importantly, (b) the occupied
ꢀ-d orbital of [Fe(TPP)(2-MeIm-)]- still shows a distinct dxz
admixture, which might not be the case experimentally.
Discussion
This study began with the idea that there are at least two
distinctly different electronic states of high-spin iron(II) por-
phyrinates. This dichotomy was initially seen from the clear
structural differences between five-coordinate species with a
neutral nitrogen donor versus species with an anionic axial
ligand. These differences are schematically depicted in Figure
2 for the iron(II) derivatives with imidazole and imidazolate as
the axial ligand. The values shown for imidazolate are the
averages for the structures reported earlier12 and those for the
imidazole averages are drawn from previously defined struc-
tures.10,11,13–15 Although both types of species are high-spin (S
) 2), the coordination geometry differences are striking. As
(50) de Gracia, A. G.; Bordes, L.; Desbois, A. J. Am. Chem. Soc. 2005,
127, 17634.
(51) Reed, C. A.; Mashiko, T.; Scheidt, W. R.; Spartalian, K.; Lang, G.
J. Am. Chem. Soc. 1980, 102, 2302.
(52) Hu, C.; Noll, B. C.; Scheidt, W. R. Acta Crystallogr., Sect. E 2005,
61, m830–m831.
(49) Lever, A. B. P.; Solomon, E. I. Ligand Field Theory and the Properties
of Transition Metal Complexes. In Inorganic Electronic Structure and
Spectroscopy, Volume 1; Solomon, E. I., Lever, A. B. P., Eds.; Wiley:
New York, 1997.
(53) The best choice of mean plane comparison is the displacement of iron
from the mean plane of the central four nitrogen atoms as this
minimizes effects from differing core conformations.
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3748 J. AM. CHEM. SOC. VOL. 132, NO. 11, 2010

Five-Coordinate High-Spin Iron(II) Porphyrinates
A R T I C L E S
necessary limitations of a small number of examples, the result
is in the affirmative, and we briefly summarize the strengths
and limitations of each approach.
several attempts are needed to obtain a glass of sufficient quality
for measurement.
V. DFT Approaches. DFT calculations on the BP86/TZVP
level are able to provide good ground state descriptions that
are in overall agreement with experiment if based on known
experimental structures. As shown in this paper, and as noticed
before,54,40 DFT is challenged when it comes to the prediction
of the correct out-of-plane distortion of the porphyrin ring, which
affects the accuracy of ground state property (EPR, zero-field
splitting, and Mo¨ssbauer parameters) predictions. Hence, when
only DFT-optimized structures are used, the confidence level
of the DFT results is lower. The properties that show the least
amount of variation between the experimental and optimized
structures are (a) the nature of the MO that carries the single
ꢀ-electron, and (b) the calculated sign of the Mo¨ssbauer
quadrupole tensor. These two properties show good agreement
with experiment even if the somewhat flawed DFT structures
are used for the calculations. Hence, high-level DFT calculations
can significantly help predict ground states for high-spin ferrous
hemes in new complexes or proteins, particularly when cor-
related with spectroscopic studies.
The initial recognition of two possible types of high-spin
iron(II) electronic structures led to this multimethod study to
examine various possible experimental methods for discrimina-
tion. We have outlined several methods that we show can be
used successfully to make distinctions between the two classes
of high-spin d6 porphyrinates. The two classes are distinguished
by the difference in character of the one doubly occupied d
orbital. Although there may be some minor differences within
each class, in the class N complexes the doubly occupied orbital
is effectively perpendicular to the porphyrin plane. Moreover,
this orbital is in the plane of the ligated imidazole as shown by
the DFT calculations reported herein (dxz in the coordinate
system applied here). In class A species, on the other hand, the
doubly occupied orbital has principally dxy character. Some
variation occurs as the π bonding character of the axial ligand
changes and there is some symmetry lowering in imidazolate
vs chloride, but the doubly occupied orbital can be considered
effectively parallel to the heme plane. We emphasize that we
believe that the distinction of the two groups of high-spin iron(II)
porphyrinates is generally applicable to all high-spin iron(II)
systems.55
I. X-ray Analysis. Assigning electronic states from the
molecular structure of course requires obtaining an X-ray quality
crystal and getting a reasonably precise and accurate structure.
With those requirements accomplished, the results needed for
assignment to the two classes are fairly unambiguous, based
on the coordination group parameters and especially the values
of Fe-Np and the displacement of the iron from the mean
planes.53 To our knowledge, only the group of high-spin thiolate-
ligated hemes have structural parameters intermediate to those
of class N and class A. Cataloguing their behavior with other
physical methods is still not complete, but their position as
members of class A seems certain based on the available
Mo¨ssbauer data.21 Finally and unfortunately, the X-ray method
is not easily applicable to the assignment of the electronic
structure of the heme centers of proteins.
II. Mo¨ssbauer Spectroscopy. The measurement and fitting
of Mo¨ssbauer spectra obtained in high field leads to the
unequivocal assignment of the sign of ∆Eq. The sign of ∆Eq
for all members of class N is negative, whereas for all members
of class A it is positive. For small molecule systems, obtaining
enough material for an adequate signal-to-noise ratio is relatively
straightforward but may present problems for some heme protein
samples. The need for obtaining Mo¨ssbauer spectra in high
applied magnetic field (and concomitant low temperature) in
order to unequivocally determine the sign of the quadrupole
splitting constant and somewhat larger sample sizes are the
major limitation for the use of this method. The use of 57Fe
-labeled proteins would clearly aid in making good measure-
ments on proteins.
III. EPR Spectroscopy. As described earlier the integer-spin
EPR spectra clearly show that the class N examples have a
positive sign for D, whereas those for class A have a negative
value of D. Establishing the experimental requirements for
integer-spin EPR spectra can be challenging, but the major
limitation is the magnitude of D, which if too large will render
a sample EPR silent. Although it seems that most samples are
within the values needed for the X-band range, measurements
at higher frequencies can alleviate this issue. As is well-known,
the sensitivity of EPR spectroscopy means that relatively small
samples of a heme protein are required for good measurements.
IV. MCD Spectroscopy. The difference in the electronic
structures of class N and class A high-spin ferrous hemes is
clearly reflected by the absolute molar C-term intensities of the
signals, as demonstrated here for the first time. Note that MCD
also distinguishes between high-spin and low-spin ferric por-
phyrins in a similar way.40 For practical purposes, comparison
of the C-term intensities of the very intense Soret bands is best,
which also allows for the usage of quite dilute samples (<100
µM). On the other hand, the observed energies of the transitions,
and the overall appearance of the spectra do not show much
variation in the case of high-spin ferrous hemes, and hence,
cannot be used to distinguish between the two classes. The
advantage of MCD spectroscopy is the small amount of sample
needed in order to determine the C-term intensity of the Soret
band. With sample volumes in the range of 2-3 mL, the total
amount of protein required is <0.2 µmol. For the measurement,
frozen glasses are needed, which are usually easily achieved
for proteins by addition of glycerol. Finding a combination of
organic, noncoordinating solvents that glass upon freezing for
the small molecule studies is a bigger challenge, and sometimes
Does this recognition of electronic state differences provide
new insight into the oft-asked question of the role of the
proximal ligand in heme proteins? We believe so. The differ-
ences must clearly reflect differences in the function of the heme
protein; a tenet of many bioinorganic metalloprotein studies is
that electronic structure contributes to function. This clearly is
seen in the proteins that can be definitively assigned to one of
the two classes. Deoxyhemoglobin and -myoglobin, members
of class N, are reversible O2 binders. The O2 complex has
(partial) ferric-superoxide character;56 the location of the single
ꢀ-electron in a t2g-type orbital that is orthogonal to the heme-
plane and overlaps with the incoming O2 is certainly advanta-
geous for O2 binding and electron transfer. In addition, imidazole
is a weaker ligand than imidazolate, which helps to stabilize
(54) Lehnert, N.; Silvernail, N. J.; Sage, J. T.; Scheidt, W. R.; Sturhahn,
W.; Zhao, J. ms. in preparation.
(55) We note that the one known group of six-coordinate high-spin iron(II)
species are those with two neutral tetrahydrofuran ligands.51,52
Mo¨ssbauer characterization clearly places these in the class N group.51
(56) (a) Weiss, J. J. Nature 1964, 203, 183. (b) Reed, C. A.; Momenteau,
M. M. Chem. ReV. 1994, 94, 659.
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A R T I C L E S
Hu et al.
the O2 complex by avoiding a σ-trans interaction between the
axial ligands. In this sense, the Fe-N(His) bond strength could
be used to fine-tune the properties of the Fe-O2 bond, and this
could be mediated by the strength of the hydrogen bond to the
His imidazole ring. This is yet another indication of how well
fine-tuned the active sites of Hb and Mb are for O2 binding and
transport. On the other hand, HRP and potentially cytochrome
P450, members of class A, are involved in catalytic oxidations
and do not reversibly bind O2. In this case, the link to function
is likely due to the fact that stronger donating, anionic proximal
ligands promote heterolytic O-O bond cleavage, leading to
formation of high-valent ferryl intermediates. This has to be
avoided in Mb and Hb, so in these cases, the weaker imidazole
donor is preferred over imidazolate as in HRP.
Thus to the extent that data are currently available for proteins,
they also follow the classification. Now that the possible
significance of electronic state differences are known, we expect
that further heme protein classification will be made. Currently
the best data are from Mo¨ssbauer, but as noted in this paper,
MCD may become the most generally useful method for
categorizing. From the class N and A classification from
spectroscopy it might therefore be possible to predict the likely
function of a newly discovered heme protein, i.e. whether it
might serve as O2 binder or sensor, or as an O2 activating
peroxidase, hydroxylase, etc.
Another interpretation of the biological importance of the
electronic structure differences has been provided by theoretical
investigations of Jensen and Ryde, who have studied the effects
of hydrogen bonding to imidazole on the properties of histidine-
ligated heme proteins.57 They have noted that hydrogen bonding
leads to large changes in the relative energies of the high-spin
state and the intermediate-spin state. In the presence of weak,
or no, hydrogen bonding, the ferrous state shows very small
differences between the two spin states. Indeed, the difficulty
of predicting the correct ground state for deoxymyoglobin
models is demonstrated by the number of DFT calculations that
failed to correctly predict the high-spin ground state.58 Jensen
and Ryde also showed that in the presence of strong hydrogen
bonding to histidine, it is conversely the ferric state that shows
the small difference between the intermediate- and high-spin
states.57 They further suggest that it is this difference that leads
to the differences in function. In a subsequent paper,59 they
suggest that the closeness of varying spin states in the deoxy-
derivatives is essential for facile O2 binding and that this is the
solution to the spin-forbidden nature of the reaction and to the
differing nature of HRP. We do note, however, that Jensen and
Ryde did not suggest differences in the ground state descriptions
for the different hydrogen-bonded states, which may simply be
a reflection of the difficulty of the calculations.
Where else might the issue of the two electronic states in
iron(II) be of biological significance? Although we are unaware
of any definitive data on the issue, we suspect that proteins like
the gas-sensing heme proteins, such as FixL or soluble guanyl
cyclase, will found to be members of class N whereas heme
proteins involved in O2 activation will be found to be members
of class A. The differences between the class N and A species
are not simply the strength of the ligand field. Although it is
known that imidazole is a weaker field ligand that imidazolate,60
imidazole is clearly a stronger field ligand than chloride, a class
A ligand like imidazolate. Thus, the distinction is not simply a
ligand field parameter. Further developments in the significance
of this dichotomy will wait characterization of more heme
proteins in one or the other cases.
Acknowledgment. We thank the NIH for support under Grant
GM-38401 (WRS) and Grant GM-77387 (MPH), and the Dow
Corning Corporation (NL).
Supporting Information Available: Figures S1sS3, compar-
ing DFT-calculated and observed structural parameters for
[Fe(TPP)(2-MeHIm)]-, [Fe(TPP)(2-MeIm-)]-, and [Fe-
(TPP)Cl]-; Figure S4, MO diagram for free porphine; Figures
S5sS7 giving MO diagrams for the fully optimized structures
of [Fe(TPP)(2-MeHIm)]-, [Fe(TPP)(2-MeIm-)]-, and [Fe-
(TPP)Cl]-; details of the structure determination for K(222)-
[Fe(TPP)Cl], complete ref 27; Tables S1sS6 giving complete
details for the structure of K(222)[Fe(TPP)Cl]. Crystallographic
data is available as a CIF file. This material is available free of
charge via the Internet at http://pubs.acs.org.
(57) Jensen, K. P.; Ryde, U. Mol. Phys. 2003, 101, 2003.
(58) (a) Rovira, C.; Kunc, K.; Hutter, J.; Ballone, P.; Parrinello, M. J. Phys.
Chem. A 1997, 101, 8914. (b) Kozlowski, P. M.; Spiro, T. G.; Zgierski,
M. Z. J. Phys. Chem. B 2000, 104, 10659. (c) Ugalde, J. M.; Dunietz,
B.; Dreuw, A.; Head-Gordon, M.; Boyd, R. J. J. Phys. Chem. A 2004,
108, 4653.
JA907584X
(59) Jensen, K. P.; Ryde, U. J. Biol. Chem. 2004, 279, 14561.
(60) Landrum, J. T.; Hatano, K.; Scheidt, W. R.; Reed, C. A. J. Am. Chem.
Soc. 1980, 102, 6729.
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