Electronic structure of six-coordinate iron(III)-porphyrin NO adducts: The elusive iron(III)-NO(radical) state and its influence on the properties of these complexes

Published on Web 10/23/2008

Electronic Structure of Six-Coordinate Iron(III)-Porphyrin NO

Adducts: The Elusive Iron(III)-NO(radical) State and Its

Inﬂuence on the Properties of These Complexes

V. K. K. Praneeth,^†Florian Paulat,^†Timothy C. Berto,^†Serena DeBeer George,^‡

Christian Na¨ther,^§Corinne D. Sulok,^†and Nicolai Lehnert*^,†

Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109, Stanford

Synchrotron Radiation Laboratory, SLAC, Stanford UniVersity, Stanford, California 94309, and

Institut fu¨r Anorganische Chemie, Christian-Albrechts-UniVersita¨t Kiel, Olshausenstrasse 40,

D-24098 Kiel, Germany

Received March 12, 2008; E-mail: lehnertn@umich.edu

Abstract: This paper investigates the interaction between ﬁve-coordinate ferric hemes with bound axial

imidazole ligands and nitric oxide (NO). The corresponding model complex, [Fe(TPP)(MI)(NO)](BF₄) (MI )

1-methylimidazole), is studied using vibrational spectroscopy coupled to normal coordinate analysis and

density functional theory (DFT) calculations. In particular, nuclear resonance vibrational spectroscopy is

used to identify the Fe-N(O) stretching vibration. The results reveal the usual Fe(II)-NO⁺ground state

for this complex, which is characterized by strong Fe-NO and N-O bonds, with Fe-NO and N-O force

constants of 3.92 and 15.18 mdyn/Å, respectively. This is related to two strong π back-bonds between

Fe(II) and NO⁺. The alternative ground state, low-spin Fe(III)-NO(radical) (S ) 0), is then investigated.

DFT calculations show that this state exists as a stable minimum at a surprisingly low energy of only ∼1-3

kcal/mol above the Fe(II)-NO⁺ground state. In addition, the Fe(II)-NO⁺potential energy surface (PES)

crosses the low-spin Fe(III)-NO(radical) energy surface at a very small elongation (only 0.05-0.1 Å) of

the Fe-NO bond from the equilibrium distance. This implies that ferric heme nitrosyls with the latter ground

state might exist, particularly with axial thiolate (cysteinate) coordination as observed in P450-type enzymes.

Importantly, the low-spin Fe(III)-NO(radical) state has very different properties than the Fe(II)-NO⁺state.

Speciﬁcally, the Fe-NO and N-O bonds are distinctively weaker, showing Fe-NO and N-O force constants

of only 2.26 and 13.72 mdyn/Å, respectively. The PES calculations further reveal that the thermodynamic

weakness of the Fe-NO bond in ferric heme nitrosyls is an intrinsic feature that relates to the properties

of the high-spin Fe(III)-NO(radical) (S ) 2) state that appears at low energy and is dissociative with respect

to the Fe-NO bond. Altogether, release of NO from a six-coordinate ferric heme nitrosyl requires the system

to pass through at least three different electronic states, a process that is remarkably complex and also

unprecedented for transition-metal nitrosyls. These ﬁndings have implications not only for heme nitrosyls

but also for group-8 transition-metal(III) nitrosyls in general.

Introduction

produced in vivo by the nitric oxide synthase (NOS) family of

enzymes.⁴Cardiovascular regulation by NO (produced by

endothelial (e-) NOS) is then mediated by soluble guanylate

cyclase.^3d,5The role of nitric oxide in vasodilation is exploited

by certain blood-sucking insects whose bites inject NO into their

Nitric oxide (NO) is a poisonous gas that is, however, of

great biological signiﬁcance. Therefore, in 1992 it was chosen

as the “molecule of the year” by the magazine Science.¹In the

years since then, further research has shown that NO plays a

key role in nerve-signal transduction, vasodilation, blood

clotting, and immune response by white blood cells.^2,3NO is

(3) (a) Stamler, J. S.; Singel, D. J.; Loscalzo, J. Science 1992, 258, 1898–

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Brandish, P. E.; Marletta, M. A. Proc. Natl. Acad. Sci. U.S.A. 2002,

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Luchsinger, B. P.; Rich, E. N.; Yan, Y.; Williams, E. M.; Stamler,

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^†University of Michigan.

^‡Stanford University.

^§Christian-Albrechts-Universita¨t Kiel.

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9

15288 J. AM. CHEM. SOC. 2008, 130, 15288–15303

10.1021/ja801860u CCC: $40.75

2008 American Chemical Society

Electronic Structure of Iron(III)-Porphyrin NO Adducts

A R T I C L E S

Table 1. Vibrational Properties of Ferric Heme NO Adducts in

victims using small NO-carrier proteins, the so-called nitro-

phorins (Np’s).⁶Finally, nitric oxide is an intermediate in

dissimilatory denitriﬁcation.⁷In a number of these proteins,

ferric heme NO adducts occur as intermediates of catalysis or

as enzyme-product complexes. The observed biological

Fe(III)-porphyrin NO adducts can be divided into two classes,

depending on the nature of the axial ligand coordinated trans

to NO, which is either an N-donor ligand such as histidine (His)

or a thiolate cysteinate (Cys). In cytochrome cd₁nitrite reductase

(NiR), a six-coordinate (6C) ferric heme NO complex with axial

His coordination is observed as the enzyme-product complex.⁸

The nitrophorins from Rhodnius prolixus (rNp1-rNp4) contain

ferric heme centers with axial His ligation.^6a,cThese Np proteins

reversibly bind NO and release it in the victim’s tissue. Ferric

heme NO adducts with axial Cys coordination occur at the end

of the catalytic cycle of the NOS enzymes. Recently, corre-

sponding nitrophorins containing an axial Cys ligand have also

been identiﬁed.^6cFinally, the ferric form of fungal NO reductase

(P450nor) is catalytically active and binds NO to form an

Fe(III)-porphyrin NO adduct with axial Cys coordination (the

enzyme-substrate complex).^9,10

Proteins and Model Complexes

complex

ν(N-O) [cm^-1

]

ν(Fe-NO) [cm^-1

]

refs

P450cam-NO

1806

1818

1828

1851

1868

528

520

510

530

538

537

611/600

604

594

591

596

-

9c, 41

58

9c

9c, 59

4d

13, 16a

40, 60

61

6b, 45

62

P450cam-NO + adamantone

a

SR-NO

P450nor-NO

CPO-NO

iNOS-NO

a

b

1868/1838

1903

1904

1917

1918

1921

1925

1927

[Fe(OEP)(NO)](ClO₄)

a

HRP-NO

a

NorBC-NO

rNp1-NO

a

hHO-1-NO

[Fe(OEP)(MI)(NO)](ClO₄)

14a

a

594

595

-

Hb-NO

63, 40

64, 40

14d

a

Mb-NO

[Fe(TPP)(HO-i-C₅H₁₁)(NO)](ClO₄) 1935

^aSR ) picket fence porphyrin model complex with axial thiolate

ligand; CPO chloroperoxidase; HRP horseradish peroxidase;

)

NorBC ) bacterial NOR; hHO-1 ) human heme oxygenase-1; Hb )

hemoglobin; Mb ) myoglobin. ^bData are given for the chloroform

solvate and the corresponding solvent-free complex, respectively.

In the so-called Enemark-Feltham scheme, the Fe(III)-

porphyrin NO adducts are classiﬁed as {FeNO}⁶complexes,

where the exponent “6” refers to the number of Fe d electrons

plus the unpaired electron of NO.¹¹The electronic structure of

the {FeNO}⁶heme complexes with axial N-donor coordination

has in general been described⁸as Fe(II)-NO⁺in agreement

with {RuNO}⁶systems, in which this electronic structure has

been observed previously and studied extensively.¹²Hence, the

Fe-NO interaction in these complexes is dominated by π back-

bonding from two t₂-type d orbitals (“d_π” orbitals) of low-spin

Fe(II) into the unoccupied π* orbitals of NO⁺. This bonding

description is analogous to that for the isoelectronic Fe(II)-CO

complexes. Crystal structures of Fe(III)-porphyrin NO model

complexes are known for ﬁve-coordinate (5C) [Fe(OEP)(NO)]

(ClO₄) (OEP ) octaethylporphyrin)¹³and six-coordinate (6C)

[Fe(OEP)(L)(NO)](ClO₄) (L ) neutral N-donor ligands).¹⁴

These complexes show linear Fe-N-O units and extremely

short Fe-NO bond lengths of 1.63-1.65 Å, in agreement with

the Fe(II)-NO⁺description. The N-O stretching vibrations in

these complexes are usually found in the 1900 cm^-1region

(Table 1), which also reﬂects the coordinated NO⁺. Hence, it

was believed for some time that the bonding description of

{FeNO}⁶is fully analogous to that of Fe(II)-CO.

However, recent density functional theory (DFT) studies

found that the N-O and Fe-NO stretching vibrations in

Fe(III)-porphyrin NO adducts show a direct correlation upon

porphyrin ring substitution, which is in contrast to the behavior

of Fe(II)-porphyrin CO complexes.¹⁵Therefore, further con-

tributions to the Fe-NO bond in addition to the strong π back-

bond must exist in these complexes.¹⁶Related to this, axial

thiolate coordination to {FeNO}⁶in heme proteins and model

complexes is known to induce bent Fe-N-O units ( (Fe-N-O)

) 161-165°),^17,9b,18and lower N-O stretching frequencies

(1800-1850 cm^-1). In a recent study, these differences have

been attributed to a trans effect of the thiolate on the bound

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orbital, which is responsible for the bending of the Fe-N-O

unit and the weakening of the Fe-NO and N-O bonds.¹⁹In

addition to these slight variations in the “classic” π back-bonding

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A R T I C L E S

Praneeth et al.

electronic structure model for {FeNO}⁶, it has been pointed out

by Walker^6cthat the {FeNO}⁶formalism leaves room for an

even wider variation in electronic structure: the Fe(III)-porphyrin

NO adducts could also exhibit an Fe(III)-NO(radical) electronic

structure if the low-spin Fe(III) electron conﬁguration is

and Fe(III)-NO(radical) electronic states, (b) the mechanism

of NO binding to ferric heme (i.e., the NO-to-Fe(III) electron

transfer upon coordination of NO), and (c) the surprisingly large

Fe-NO force constant in ferric hemes. It is demonstrated that

the elusive Fe(III)-NO(radical) state appears as a minimum

on the PES. The biological implications of these results are

discussed.

[d_xzd_yzd_xy¹], which requires a rufﬂed porphyrin ring; in this

electron conﬁguration, the unpaired electrons of low-spin Fe(III)

and NO are antiferromagnetically coupled. This would be

advantageous for the nitrophorins because it would prevent the

autoxidation reaction of Fe(II)-NO⁺complexes,²⁰which

ultimately leads to ferrous NO adducts and hence to deactivation

of the proteins as NO carriers. However, it is not known whether

the bent Fe-N-O unit could in fact be caused by an

Fe(III)-NO(radical) electronic structure. In order to understand

the functioning of the nitrophorins and the nature of ferric

heme-NO interactions in general, it is necessary to elucidate

the electronic structures of the Fe(III)-porphyrin NO adducts

in detail. The established Fe(II)-NO⁺and elusive Fe(III)-NO-

(radical) electronic structures lead to very different reactivities.

In addition, the formation of an Fe(II)-NO⁺complex from

ferric heme and NO requires an NO-to-Fe(III) electron transfer.

However, not much is known about the energetics and the

mechanism of complex formation in these systems.

2

Experimental and Computational Procedures

Syntheses. Reactions were performed by applying Schlenk

techniques using carefully puriﬁed solvents. NO gas was puriﬁed

by passing it ﬁrst through an ascarite II column (NaOH on silica

gel) and then through a cold trap at -60 to -70 °C to exclude

higher nitrogen oxide impurities. ¹⁵N¹⁸O-labeled compounds were

synthesized using ¹⁵N¹⁸O gas (Sigma-Aldrich).

Syntheses of Precursors. Tetraphenylporphyrin (H₂TPP),²²

[Fe(TPP)Cl],²³[Fe(TPP)ClO₄],²⁴and [Fe(TPP)]₂O ²⁵were synthe-

sized and puriﬁed as previously reported.

Synthesis of the Ferric NO Adduct [Fe(TPP)(MI)(NO)]-

(BF₄) (1). A sample of [Fe^III(TPP)ClO₄] (100 mg, 0.13 mmol) and

KBF₄(160 mg, 1.3 mmol) was placed in a 100 mL Schlenk ﬂask.

The ﬂask was purged three times with argon, and 20 mL of CHCl₃

was added. This heterogeneous reaction mixture was stirred at room

temperature for ∼2 h and then ﬁltered. The obtained ﬁltrate,

[Fe^III(TPP)(BF₄)] (the precursor complex), was collected in a 100

mL Schlenk ﬂask, and 1-methylimidazole (MI, 10 µL, 0.13 mmol)

was added. NO gas was then passed through the stirred solution

for 10 min, and 15 mL of n-hexane was slowly added to the reaction

mixture, which was then stored at -20 °C for 2 days. The

precipitate was ﬁltered off and obtained as a microcrystalline solid.

The IR spectrum showed ν(N-O) at 1896 cm^-1, indicative of the

formation of 1. Yield: 92 mg (86%).

Synthesis of [Fe(TPP)(NO)(NO₂)] (2). [FeTPP]₂O (0.1 g, 0.074

mmol) was dissolved in 18 mL of CHCl₃in a 100 mL Schlenk

ﬂask under an argon atmosphere. The solution was stirred for a

few minutes, and then excess nitric oxide was added. Next, 0.2

mL of air was injected into the ﬂask. The resulting solution was

allowed to stir under the NO/O₂atmosphere for 1 h. Finally, 22

mL of n-hexane was added, and the obtained precipitate was ﬁltered

off and stored under an argon atmosphere in a freezer. Yield: 86

mg (78%). IR [cm^-1]: ν(N-O), 1876; ν_as(NO₂), 1460; ν_s(NO₂),

1298.

In this study, the mechanism and energy landscape of NO

binding to ferric hemes and the possible variation in electronic

structure of ferric heme nitrosyls are investigated in detail. Since

well-characterized ferric heme NO model complexes are

surprisingly rare, we have prepared the 6C model system

[Fe(TPP)(MI)(NO)](BF₄) (1; TPP ) tetraphenylporphyrin, MI

) 1-methylimidazole) and characterized this compound using

vibrational spectroscopy coupled to isotope substitution. In

particular, nuclear resonance vibrational spectroscopy (NRVS)

was applied for the ﬁrst time to a ferric heme nitrosyl in order

to identify the Fe-NO stretching vibration of 1. The vibrational

data were then analyzed using our quantum chemistry centered

normal coordinate analysis (QCC-NCA),^21band force constants

for the Fe-NO and N-O bonds were determined. We have

also extended our QCC-NCA package to calculate NRVS

intensities directly from the NCA results. In this way, the ﬁrst

detailed analysis of the Fe-N-O vibrations in a 6C Fe(III)-

porphyrin NO model complex is provided in this paper. The

electronic structure of ferric heme nitrosyls was then evaluated

and compared to that of corresponding ferrous complexes that

we have recently investigated.^21,10bThis allowed the different

complex stabilities and Fe-NO bond strengths in these systems

to be rationalized, especially the lability of the Fe-NO bond

in the ferric case. Finally, the binding of NO to ferric hemes

was explored using potential energy surface (PES) calculations

in order to elucidate (a) the relative stabilities of Fe(II)-NO⁺

Synthesis of [⁵⁷Fe(TPP)Cl].²⁶In a Schlenk ﬂask under an argon

atmosphere, anhydrous HCl gas was bubbled slowly into a

suspension of ⁵⁷Fe powder (1.00 g, 87.8 mmol; Cambridge Isotopes)

in 50 mL of dry tetrahydrofuran (THF) at 0 °C. A white precipitate

formed during the ﬁrst hour and then dissolved after another hour,

and the THF began to thicken, indicating the decomposition of THF

to 4-chloro-1-butanol. After 5 h of reaction, argon was passed

through the solution overnight to eliminate any residual HCl. The

residual THF was distilled off, resulting in the formation of a dark-

green gel-like substance. The 4-chloro-1-butanol was removed in

vacuo to yield a green solid. This solid was washed with THF to

yield a light-green solid, which was dried in vacuo. The obtained

⁵⁷FeCl₂was used without further puriﬁcation in the metalation of

H₂TPP to yield [⁵⁷Fe(TPP)Cl].²³

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Crystal Structure Determination. Single crystals of the ferric

bis(thf)-coordinated precursor complex [Fe(TPP)(thf)₂](BF₄) (thf

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Electronic Structure of Iron(III)-Porphyrin NO Adducts

A R T I C L E S

Table 2. Crystallographic Data for [Fe^III(TPP)(thf)₂](BF₄)

chemical formula

C₅₆H₅₂BF₄FeN₄O₃

formula weight

space group

971.68

P1

j

a

b

c

10.6964(9) Å

13.4320(10) Å

18.7082(15) Å

78.173(9)°

86.379(10)°

82.788(9)°

2608.1(4) Å³

170 K

R

ꢀ

γ

V

T

Z

D_calcd

µ

λ

2

1.237 Mg/m³

0.349 mm^-1

0.71073 Å

21052

measured reﬂections

independent reﬂections

R_int

11889

0.0367

Observed reﬂections

R1 [I > 2σ(I)]^a

wR2 (all data)^b

8221

0.0475

0.1310

2

2 2

^aR1 ) ∑||F_o| - |F_c||/∑|F_o|. ^bwR2 ) {∑[w(F_o- F_c²)²]/∑[w(F_o) ]}^1/2

.

Table 3. Selected Bond Lengths and Angles for

[Fe^III(TPP)(thf)₂](BF₄)

Bond Lengths [Å]

Fe(1)-N(1)

Fe(1)-N(3)

Fe(1)-O(1)

2.026(2)

2.028(2)

2.190(2)

Fe(1)-N(2)

Fe(1)-N(4)

Fe(1)-O(2)

2.031(2)

2.137(2)

Figure 1. Top view (top) and side view (bottom) of the crystal structure

of the important precursor [Fe^III(TPP)(thf)₂](BF₄), shown with the atom-

labeling scheme. The hydrogen atoms, disordered solvent molecules, and

counteranion have been omitted for clarity.

Bond Angles [deg]

N(1)-Fe(1)-N(3)

N(1)-Fe(1)-N(2)

N(3)-Fe(1)-N(2)

N(1)-Fe(1)-N(4)

N(3)-Fe(1)-N(4)

N(2)-Fe(1)-N(4)

N(1)-Fe(1)-O(2)

N(3)-Fe(1)-O(2)

179.44(7)

N(2)-Fe(1)-O(2)

N(4)-Fe(1)-O(2)

N(1)-Fe(1)-O(1)

N(3)-Fe(1)-O(1)

N(2)-Fe(1)-O(1)

N(4)-Fe(1)-O(1)

O(2)-Fe(1)-O(1)

89.70(6)

90.81(6)

89.78(6)

89.68(6)

91.70(6)

87.78(6)

178.56(5)

89.78(6)

90.09(6)

90.04(6)

90.08(6)

179.45(7)

90.55(6)

89.99(6)

UV-vis and IR Spectroscopy. Absorption spectra were re-

corded in CH₂Cl₂solutions at room temperature using an Analytik

Jena Specord S600 UV-vis spectrometer with glass ﬁber attach-

ment. Middle-infrared (MIR) spectra were recorded on PerkinElmer

Spectrum BX and GX FT-MIR spectrometers using KBr disks. The

resolution was set to 2 cm^-1. In the far-infrared (FIR) region, a

Nicolet DX version 4.56 spectrometer was employed. Spectra were

recorded in CsI disks at a resolution of 2 cm^-1. All of the IR spectra

were recorded at room temperature.

Nuclear Resonance Vibrational Spectroscopy (NRVS). NRVS

data were obtained as described in ref 28 at beam line 3-ID-XOR

of the Advanced Photon Source (APS) at Argonne National

Laboratory. This beamline provides ∼2.5 × 10⁹photons/s in a

bandwith of ∼1 meV () 8 cm^-1) at 14.4125 keV in a 0.5 mm

(vertical) × 0.5 mm (horizontal) spot. This is achieved using a

water-cooled diamond double crystal monochromator with a 1.1

eV bandpass followed by a high-resolution monochromator consist-

ing of two asymmetrically cut Si(4 0 0) and two asymmetrically

cut Si(10 6 4) crystals.²⁹Samples were loaded into 3 × 7 × 1 mm

Lucite NRVS cells. Delayed nuclear ﬂuorescence and Fe K

ﬂuorescence were detected using a single avalanche photodiode.³⁰

Spectra were recorded from -40 to 90 meV in steps of 0.25 meV.

Each scan took ∼60 min. All of the scans were added and

normalized to the intensity of the incident beam. The ﬁnal spectra

represent averages of 6 scans. During data collection, the samples

were maintained at cryogenic temperatures using a liquid helium

cryostat. Because the temperature sensor is not in direct contact

with the sample, the temperatures for individual scans are not

exactly known. Since the anti-Stokes intensities of modes >100

cm^-1are negligible, the sample temperature was well below 50

K. Comparison of initial and ﬁnal NRVS scans conﬁrms the absence

) tetrahydrofuran) that were suitable for X-ray analysis were

obtained after two weeks from a saturated solution of the precursor

in THF layered with n-hexane. Intensity data were collected using

a STOE imaging-plate diffraction system with Mo KR radiation.

The structure was solved with direct methods using SHELXS-97,²⁷

and reﬁnement was done against F²using SHELXL-97. All non-

hydrogen atoms were reﬁned with anisotropic displacement pa-

rameters. The hydrogen atoms were placed in ideal positions and

reﬁned isotropically using a riding model. There are three crystal-

lographically independent THF molecules in the asymmetric unit.

In two cases, three carbon atoms are disordered and were reﬁned

using a split model. The third THF molecule is disordered, and no

appropriate split model could be found. Therefore, the data were

corrected for disordered solvent using the “Squeeze” option in

Platon. Selected crystallographic data are presented in Table 2.

Selected bond lengths and angles are presented in Table 3.

j

[Fe(TPP)(thf)₂](BF₄) crystallizes in the triclinic space group P1 with

all of the atoms located in general positions. The iron atom is

coordinated by the four porphyrin nitrogen atoms and an oxygen

atom from each of the two thf ligands within a slightly distorted

octahedron (Figure 1). The iron atom is located in the porphyrin

ring plane (deviation from the mean plane: 0.0044 Å).

(27) Sheldrick, G. M. SHELXS-97 and SHELXL-97: Programs for the

Solution and Reﬁnement of Crystal Structures; University of Go¨ttingen:

Go¨ttingen, Germany, 1997.

(28) (a) Sturhahn, W. J. Phys.: Condens. Matter 2004, 16, S497–S530.

(b) Petrenko, T.; DeBeer George, S.; Aliaga-Alcalde, N.; Bill, E.;

Mienert, B.; Xiao, Y.; Guo, Y.; Sturhahn, W.; Cramer, S. P.;

Wieghardt, K.; Neese, F. J. Am. Chem. Soc. 2007, 129, 11053–11060.

(29) Toellner, T. S. Hyperﬁne Interact. 2000, 125, 3–28.

(30) Baron, A. Q. R.; Kishimoto, S.; Morse, J.; Rigal, J.-M. J. Synchrotron

Radiat. 2006, 13, 131–142.

9

J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008 15291

A R T I C L E S

Praneeth et al.

Scheme 1

Normal Coordinate Analysis. Normal-coordinate calculations

were performed using the QCPE computer program 576 by M. R.

Peterson and D. F. McIntosh. The calculations are based on a

general valence force ﬁeld; force constants are reﬁned with a

nonlinear simplex algorithm. The simplex optimization was used

to reﬁne only selected force constants according to our QCC-NCA

scheme.^21bIn this case, a force ﬁeld from DFT calculations is used

as a starting point to generate initial force constants, and a subset

of these are ﬁt to reproduce the known experimental frequencies.

Force constants were extracted from the Gaussian output using a

modiﬁed version of the program Redong³⁶(QCPE 628). We have

now extended our QCC-NCA package to directly calculate NRVS

VDOS intensities from the normal coordinate analysis results (vide

infra). In order to simulate the vibrations of the Fe-N-O subunit

˜

of 1, the force ﬁeld calculated for model 1 was used for NCA. It

should be noted that in this case, the two Fe-N-O linear bends

show a calculated splitting of ∼8 cm^-1that is not observed

experimentally. We compensated for this in the NCA by allowing

the two corresponding Fe-N-O bending force constants to have

different values. The observed TPP-based vibrations in the NRVS

spectra of 1 could not be simulated in the present NCA because of

the use of porphine instead of TPP in the calculations. Correspond-

ing large-scale calculations on the complete complex 1 including

the full TPP ligand are currently underway to investigate the

inﬂuence of the phenyl substituents of TPP on the vibrational

properties of the complex. These studies will be the subject of a

forthcoming paper.

of spectroscopic changes due to radiation damage. MIR spectra of

the samples were also recorded before and after exposure to the

X-ray beam (Figures S4 and S5 in the Supporting Information),

showing no degradation. NRVS raw data were converted to the

vibrational density of states (VDOS) using the program Phoenix.^31,32

DFT Calculations. The structures of the model systems [Fe(P)-

(MI)(NO)]⁺(1, S ) 0), [Fe(P)(MI)]⁺(S ) ¹/₂, ⁵/₂), and [Fe(P)(MI)]

˜

(S ) 0, 2) were fully optimized at the BP86/TZVP level (i.e., using

the BP86 functional and the TZVP basis set). These calculations

used a simpliﬁed TPP ligand in which the four phenyl groups at

the meso positions of the porphyrin ring were replaced by hydrogen.

The resulting porphine ligand (P^2-) is shown in Scheme 1 along

with the applied coordinate system. No imaginary frequencies were

Results and Analysis

A. Vibrational Spectroscopy and Electronic Structure of

Fe(III)-Porphyrin NO Adducts in the Ground State.

A.1. Vibrational Properties and Assignments. The MIR spec-

trum of [Fe(TPP)(MI)(NO)](BF₄) (1) is shown in Figure 2

together with the corresponding data for the ¹⁵N¹⁸O-labeled

analogue. The N-O stretching vibration ν(N-O) was identiﬁed

as the band at 1896 cm^-1that shifts to 1816 cm^-1upon isotopic

substitution. One other isotope-sensitive band is found at 588

cm^-1, which appears at 572 cm^-1in the ¹⁵N¹⁸O compound. It

is not possible to tell a priori whether this feature belongs to

the two (degenerate) Fe-N-O linear bends, δ_lb(Fe-N-O), or

the Fe-NO stretch, ν(Fe-NO). In order to locate the other

respective feature, resonance Raman measurements on frozen

solutions of 1 at multiple excitation wavelengths were then

conducted, but no unambiguous assignment could be obtained

from these experiments. One general problem with resonance

Raman spectroscopy on heme nitrosyls is the potential photo-

decomposition of the compounds by loss of either NO or the

trans imidazole ligand. We therefore turned to nuclear resonance

vibrational spectroscopy (NRVS), which is a method that (unlike

resonance Raman) does not suffer from sample degradation.

NRVS measures the inelastic scattering that is observed upon

excitation of the ⁵⁷Fe nucleus at the 14.4125 keV nuclear

resonance (Mo¨ssbauer) line.^28a,37NRVS is ideal for the

identiﬁcation of metal-ligand stretching vibrations because

NRVS intensities are proportional to the amount of iron motion

in a normal mode.³¹Hence, metal-ligand stretching vibrations

are very intense in NRVS, and this method has recently been

successfully applied to ferrous heme nitrosyls and carbonyls.^25,38

Figure S3 in the Supporting Information shows the NRVS raw

data for [⁵⁷Fe(TPP)(MI)(NO)](BF₄) (i.e., ⁵⁷Fe-substituted 1) and

the corresponding ¹⁵N¹⁸O-labeled complex. The vibrational

density of states (VDOS) calculated from these data is shown

˜

obtained for 1. In addition, B3LYP/LANL2DZ and B3LYP/

LANL2DZ* geometry optimizations were performed on these

models and the corresponding ferrous NO adduct [Fe(P)(MI)(NO)]

(S ) ¹/₂) in order to investigate the method dependence of the

complex formation energies. B3LYP/TZVP single-point calculations

on the fully optimized B3LYP/LANL2DZ* structures were also

employed. The LANL2DZ* basis set^21aconsists of the LANL2DZ

basis set³³plus polarization functions (from TZVP³⁴) on all of the

heavy atoms. The PES calculations employed B3LYP/LANL2DZ

geometry optimizations in which the Fe-NO distances were ﬁxed

and all of the other internal coordinates were allowed to vary. The

geometry of the Fe(III)-NO(radical) S ) 0 state was also fully

optimized and showed a linear Fe-N-O unit (vide infra). However,

in the PES calculations for this state, the Fe-N-O angle was ﬁxed

at 170° for Fe-NO distances less than 2.64 Å because allowing

this angle to optimize led to severe SCF convergence problems; in

addition, the wave function frequently returned to the Fe(II)-NO⁺

state. This problem was only observed at Fe-NO bond distances

less than 2.64 Å and could be overcome by ﬁxing the Fe-N-O

angle at 170°. The error in the total energies induced this way is

only ∼0.2 kcal/mol (calculated for the fully optimized structure).

B3LYP/TZVP single-point calculations were then performed on

the B3LYP/LANL2DZ structures. Finally, to explore the effect of

the phenyl substituents on the relative energies of the two singlet

states, we fully optimized the structures of [Fe(TPP)(MI)-

(NO)]⁺(i.e., including the full TPP ligand) for both the Fe(II)-NO⁺

and Fe(III)-NO(radical) S ) 0 states using B3LYP/LANL2DZ.

The obtained energy difference between these states is very similar

to the value calculated with the porphine approximation. All of

these methods were used as implemented in Gaussian 03.³⁵Orbitals

were plotted using GaussView.

(31) Sage, J. T.; Paxson, C.; Wyllie, G. R. A.; Sturhahn, W.; Durbin, S. M.;

Champion, P. M.; Alp, E. E.; Scheidt, W. R. J. Phys.: Condens. Matter

2001, 13, 7707–7722.

(32) Sturhahn, W. Hyperﬁne Interact. 2000, 125, 149–172.

(33) (a) Hay, P. J.; Wadt, W. R. J. Chem. Phys. 1985, 82, 270–283. (b)

Wadt, W. R.; Hay, P. J. J. Chem. Phys. 1985, 82, 284–298. (c) Hay,

P. J.; Wadt, W. R. J. Chem. Phys. 1985, 82, 299–310.

(36) Allouche, A.; Pourcin, J. Spectrochim. Acta 1993, 49A, 571.

(37) Scheidt, W. R.; Durbin, S. M.; Sage, J. T. J. Inorg. Biochem. 2005,

99, 60–71.

(34) Scha¨fer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992, 97, 2571.

(35) Frisch, M. J.; et al. Gaussian 03; Gaussian, Inc.: Pittsburgh, PA, 2003.

9

15292 J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008

Electronic Structure of Iron(III)-Porphyrin NO Adducts

A R T I C L E S

Figure 2. IR spectra of [Fe(TPP)(MI)(NO)](BF₄) (1) (top) and the corresponding ¹⁵N¹⁸O-labeled complex (bottom): middle IR (left); far IR (right).

of a normal mode from NRVS, e_Fe², is proportional to the

amount of iron motion in that mode.³¹As will be shown in

section A.2, this quantity can be calculated in a straightforward

fashion for a normal mode using NCA. In the case of the linear

Fe-N-O unit in 1, where no mixing between the Fe-NO

stretch and the two (orthogonal) linear bends is possible, the

amounts of iron motion in the stretch and the two bends are

intrinsic properties of the Fe-N-O unit. Therefore, the relative

NRVS intensities of these modes should be more or less

constant³⁹and are readily available from NCA. The NCA results

presented below for 1show that the Fe-NO stretch has a

distinctively larger amount of iron motion and hence a larger

NRVS intensity than the combined linear bends. This is a

general feature of a linear Fe-X-O unit, and a similar intensity

difference between ν(Fe-CO) and δ_lb(Fe-C-O) was observed

in a recent NRVS study on [Fe(TPP)(MI)(CO)].²⁵The theoretical

NRVS intensity ratio for ν(Fe-NO) and δ_lb(Fe-N-O) from NCA

Figure 3. VDOS for [⁵⁷Fe(TPP)(MI)(NO)](BF₄) (with natural abundance

isotopes (N.A.I.) NO) and for the corresponding ¹⁵N¹⁸O-labeled complex,

calculated from the NRVS raw data using Phoenix.³²The N.A.I. NO (blue)

and ¹⁵N¹⁸O (orange) ﬁts of the isotope-sensitive feature near 580 cm^-1

yielded total (combined) integrated intensities, e_Fe², of 0.536 (N.A.I.) and

0.545 (¹⁵N¹⁸O), showing that these two features are related.

was used to ﬁt the spectrum in Figure 3.

(38) (a) Rai, B. K.; Durbin, S. M.; Prohofsky, E. W.; Sage, J. T.; Wyllie,

G. R. A.; Scheidt, W. R.; Sturhahn, W.; Alp, E. E. Biophys. J. 2002,

82, 2951–2963. (b) Leu, B. M.; Zgierski, M. Z.; Wyllie, G. R. A.;

Scheidt, W. R.; Sturhahn, W.; Alp, E. E.; Durbin, S. M.; Sage, J. T.

J. Am. Chem. Soc. 2004, 126, 4211–4227. (c) Zeng, W.; Silvernail,

N. J.; Wharton, D. C.; Georgiev, G. Y.; Leu, B. M.; Scheidt, W. R.;

Zhao, J.; Sturhahn, W.; Alp, E. E.; Sage, J. T. J. Am. Chem. Soc.

2005, 127, 11200–11201. (d) Silvernail, N. J.; Barabanschikov, A.;

Pavlik, J. W.; Noll, B. C.; Zhao, J.; Alp, E. E.; Sturhahn, W.; Sage,

J. T.; Scheidt, W. R. J. Am. Chem. Soc. 2007, 129, 2200–2201. (e)

Leu, B. M.; Silvernail, N. J.; Zgierski, M. Z.; Wyllie, G. R. A.; Ellison,

M. K.; Scheidt, W. R.; Zhao, J.; Sturhahn, W.; Alp, E. E.; Sage, J. T.

Biophys. J. 2007, 92, 3764–3783.

in Figure 3. In the complex with unlabeled NO, two intense

features that are close in energy (578 and 586 cm^-1) are

observed. The mode at higher energy corresponds to the IR

feature at 588 cm^-1, since a shift of 1-2 cm^-1to lower energy

in the NRVS data can be attributed to the ⁵⁷Fe labeling in this

case (this small energy difference is also below the resolution

of the two methods). Importantly, the integrated VDOS intensity

9

J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008 15293

A R T I C L E S

Praneeth et al.

Table 4. Geometrical and Vibrational Properties of [Fe(P*)(L)(NO)] Complexes (L ) MI, Py, etc., or Missing; P* ) Tetraphenylporphyrin- or

Octaethylporphyrin-Type Ligand)

geometrical parameters

vibrational frequencies [cm^-1

]

molecule^a

r(Fe-NO) [Å] r(N-O) [Å]

(Fe-N-O) [deg] r(Fe-L_tr) [Å] r(Fe-N_P) [Å]^bν(N-O) ν(Fe-NO)

δ(Fe-N-O)

Fe^IIIComplexes

[Fe(OEP)(NO)](ClO₄)^c,d

[Fe(OEP)(MI)(NO)](ClO₄)^c

[Fe(OEP)(Py)(NO)]Cl^e

[Fe(SP-14)(Py)(NO)]Cl^e

1.644

1.646

1.112

1.135

177

1.994

2.003

1868

1921

611

1.988

602

603

[Fe(TPP)(NO)](BF₄)

1853

1896

1816

1874

[Fe(TPP)(MI)(NO)](BF₄) (1)

[Fe(TPP)(MI)(¹⁵N¹⁸O)](BF₄)

[Fe(TPP)(NO)(NO₂)] (2)

580^f

587,^f588 (IR)

(∼569)^f

(∼575),^f572 (IR)

[Fe(P)(MI)(NO)]⁺(1) (calcd)

˜

BP86/TZVP {Fe^II-NO⁺}

1.644

1.639

1.689

1.147

1.169

1.178

180

2.018

1.976

1.967

2.022

2.023

2.021

1933

1908

1841

639

657

430

606/598

613/601

570/523

B3LYP/LANL2DZ {Fe^II-NO⁺}

B3LYP/LANL2DZ {Fe^III-NO(rad.)}

[Fe(TPP)(MI)(NO)]⁺(calcd)

B3LYP/LANL2DZ {Fe^II-NO⁺}

B3LYP/LANL2DZ {Fe^III-NO(rad.)}

1.644

1.673

1.173

1.180

173

179

1.976

1.975

2.013

2.010

Fe^IIComplexes

[Fe^II(TPP)(MI)(NO)] (3)^g

1.750

1.734

1.182

1.186

138

140

2.173

2.179

2.008

2.022

1630

1662

437

609

563(ip), 291(oop)

482(ip), 317(oop)

[Fe^II(P)(MI)(NO)] (calcd BP86/TZVP)^g

^aMI ) 1-methylimidazole; P ) porphine ligand used in the calculations. ^bValues are averages over the four Fe-N_Pbonds. ^cData taken from ref

14a,b. ^dChloroform solvate. ^eData taken from ref 43. ^fNRVS data. Natural abundance isotopes energies at 580 and 587 cm^-1are estimated from the

calculated ⁵⁷Fe shifts using NCA (experimental NRVS energies: 578 and 586 cm^-1, respectively). The energies in the ¹⁵N¹⁸O case can only be

estimated because only one band, spectroscopically centered at 571 cm^-1, is observed (see text). ^gData taken from ref 21b,e.

Table 5. Comparison of Experimental and QCC-NCA Vibrational Frequencies and of QCC-NCA and DFT-Calculated Force Constants for 1

vibrational frequencies [cm^-1

]

force constants [mdyn/Å]

DFT-calculated^b

exptl

QCC-NCA

¹⁵N¹⁸

mode

N.A.I.^a

¹⁵N¹⁸

O

⁵⁷Fe

O

QCC-NCA

Fe(II)-NO⁺

LS Fe(III)-NO(rad.)

ν(N-O)^c

ν(Fe-NO)

δ_lb(Fe-N-O)

1896

578^d

1816

1897

580

587

1815

566

573

15.178

15.62

4.82

0.46/0.47

13.72

2.26

0.31/0.37

(∼569)^d

3.922

586,^d588 (IR)

(∼575),^d572 (IR)

0.368/0.406 ^e

^aN.A.I. ) natural abundance isotopes. ^bValues for Fe(II)-NO⁺and LS Fe(III)-NO(radical) were calculated using BP86/TZVP and B3LYP/

LANL2DZ, respectively (see DFT Calculations). ^cFor free NO: ν(N-O) ) 1876 cm^-1; N-O force constant ) 15.49 mdyn/Å. For free NO⁺: ν(N-O)

) 2387 cm^-1in (NO⁺)(BF₄^-); N-O force constant ) 25.07 mdyn/Å.^{46 d}From NRVS using ⁵⁷Fe. Isotope shifts between N.A.I. iron and ⁵⁷Fe are in

the range of 1-2 cm^-1and hence of the same magnitude as the experimental error in the NRVS frequencies (which is due to the required data ﬁtting

and the relatively low resolution of NRVS). In particular, the energies in the ¹⁵N¹⁸O case had to be estimated because only one band, spectroscopically

centered at 571 cm^-1, was observed (see text). ^eDFT predicts a small splitting of the δ modes; the force constants were adjusted to ensure that the two

components are isoenergetic.

Since the 586 and 578 cm^-1features in 1 are close in energy,

their assignment is not straightforward. In the case of the ferric

NO adduct of myoglobin, δ_lb(Fe-N-O) is found at somewhat

higher energy than ν(Fe-NO),⁴⁰but this trend is reversed in

the case of the ferric cytochrome P450cam NO adduct⁴¹and

also in six-coordinate (6C) ferrous heme carbonyls.²⁵Therefore,

a better way to assign the observed modes in 1 is to consider

the fact that the 586 cm^-1feature is IR-active (observed at 588

cm^-1) whereas the mode at 578 cm^-1is not. Previous work on

[Ru(NH₃)₅(NO)]X₃(X ) halide ion, etc.), a complex containing

a linear Ru(II)-NO⁺unit similar to the Fe(II)-NO⁺unit in 1,

has shown that in this case the Ru-NO stretch is Raman active

whereas the Ru-N-O linear bends are strongly IR active.^12b

Similar observations have been made for linear dinitrogen

complexes (note that N₂is isoelectronic to NO⁺).⁴²On this

basis, the band at 588 cm^-1is assigned to the two Fe-N-O

linear bends, δ_lb(Fe-N-O), and the feature at 578 cm^-1is then

identiﬁed with ν(Fe-NO).

When ¹⁵N¹⁸O isotope labeling is used, ν(Fe-NO) and

δ_lb(Fe-N-O) merge into the band centered at 571 cm^-1. This

is evident from the fact that the total integrated VDOS intensity

of this band (0.545) is comparable to the combined intensities

of the 586 and 578 cm^-1features in the unlabeled complex

(0.536). When the ratio of integrated intensities of the

586 and 578 cm^-1bands is kept constant (e_Fe²[ν(Fe-NO)]/

e_Fe²[δ_lb(Fe-N-O)] ≈ 1.35), the 571 cm^-1feature in the

¹⁵N¹⁸O-labeled complex can be approximately resolved into two

features at 575 and 569 cm^-1

, which correspond to

(39) This assumes that mode mixing of the Fe-NO stretch and of the linear

Fe-N-O bends with porphyrin modes is reasonably small, which

should in general be a good approximation for vibrations occurring

in the 500-650 cm^-1range. Some mode mixing is of course observed,

so small variations in the relative VDOS intensity of the stretch versus

the bends are possible (see text).

δ_lb(Fe-N-O) and ν(Fe-NO), respectively. These assignments

are listed in Tables 4 and 5. In summary, the NRVS measure-

ments allow for the identiﬁcation of ν(Fe-NO) and

δ_lb(Fe-N-O) for the ferric heme NO model complex 1. The

(40) Benko, B.; Yu, N. T. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 7042–

7046.

(41) Hu, S.; Kincaid, J. R. J. Am. Chem. Soc. 1991, 113, 2843–2850.

(42) Lehnert, N.; Tuczek, F. Inorg. Chem. 1999, 38, 1659–1670.

9

15294 J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008

Electronic Structure of Iron(III)-Porphyrin NO Adducts

A R T I C L E S

spectral assignments are further substantiated by simulation of

the observed isotope shifts using NCA (vide infra).

properties of the linear Fe-N-O unit that can be estimated

2

from NCA. The integrated NRVS VDOS intensity e_Fefor a

The observed ν(N-O) frequency of 1896 cm^-1for 1 is in

good agreement with the literature value of 1921 cm^-1obtained

for [Fe(OEP)(MI)(NO)](ClO₄), as shown in Table 4. In proteins,

6C ferric heme NO adducts with trans histidine ligation also

show N-O frequencies in the 1900 cm^-1region (Table 1).

Interestingly, the Fe-NO stretch in these protein species

(590-600 cm^-1) is slightly higher in energy than in 1. In

particular, the ferric NO adduct of rNp1 exhibits ν(N-O) at

1917 cm^-1and ν(Fe-NO) at 591 cm^-1. From solution studies

of [Fe(OEP)(Py)(NO)]Cl and [Fe(SP-14)(Py)(NO)]Cl (SP-14

) strapped porphyrin) using resonance Raman spectroscopy

(407 nm excitation), ν(Fe-NO) has been assigned to bands at

602 and 603 cm^-1, respectively.⁴³The vibrational energies

predicted by DFT (BP86/TZVP) for the model system [Fe(P)-

given normal mode is readily obtained from NCA using the

atomic-displacement matrix together with the following equa-

tion:^38b

2

m_Fer_Fe

2

e_Fe

)

m r ²

∑

i

where the sum over i runs over all atoms of the molecule and

r_iis the absolute length of the mass-weighted atomic-displace-

2

ment vector for atom i from NCA. For 1, the ratio of the e_Fe

values for the stretch and the linear bends can be estimated as

follows:

e_Fe²[ν(Fe-NO)]

e_Fe²[δ_lb(Fe-N-O)]

(MI)(NO)]⁺(1) are also in good agreement with experiment

0.436

˜

)

≈ 1.35

0.173 + 0.149 0.322

(Table 4). This is especially true for the N-O stretch, which is

calculated at 1933 cm^-1. The calculated frequency of 639 cm^-1

for the Fe-NO stretch shows a larger deviation. The fact that

the Fe-NO bond strength and, correspondingly, the Fe-NO

frequency are overestimated by DFT is not uncommon¹⁶and

was also observed for ferrous heme nitrosyls.^21b

This value was used as a constraint in the ﬁtting of the NRVS

data for 1. This is necessary because the Fe-NO stretch and

the Fe-N-O linear bends are not resolved as individual peaks

in the data, and hence, the ﬁt is otherwise ambiguous. Interest-

2

ingly, the absolute values of e_Fefor both the stretch and the

As mentioned in the Introduction, detailed vibrational studies

of ferric heme NO model complexes are rare. This is most likely

due to the fact that these complexes are unstable and tend to

decompose by either loss of NO or reaction with O₂to form

the nitro-nitrosyl adduct [Fe(porphyrin)(NO)(NO₂)]. In order

to ensure that compound 1 does not correspond to [Fe(TPP)-

(NO)(NO₂)] (2), we prepared this complex and investigated its

vibrational properties. Figure S1 in the Supporting Information

shows a comparison of the IR spectra of 1 and 2, which are

clearly different. In the case of 2, ν(N-O) is located at 1874

cm^-1. The N-O stretching frequencies of the coordinated nitrite

ligand are observed as characteristic bands at 1461 cm^-1for

ν_as(NO₂) and 1299 cm^-1for ν_s(NO₂) (assignments from ref 44).

These shift to 1401 and 1238 cm^-1, respectively, when ¹⁵N¹⁸O

is used (Figure S2 in the Supporting Information), in agreement

with this assignment. Importantly, both bands are missing in

the spectrum of 1 (Figure S1), conﬁrming that complex 1 is

not the nitro-nitrosyl complex.

linear bends are overestimated in the NCA by ∼30%. This is

due to the neglect of the phenyl rings of TPP in the NCA (and

in the DFT calculations), which reduces the effective mass of

the porphyrin ligand, and also to mode mixing of ν(Fe-NO)

and δ_lb(Fe-N-O) with porphyrin modes. A further investigation

of this point is the subject of an ongoing study.

The observed N-O stretching frequency and the correspond-

ing force constant are in good agreement with the generally

accepted Fe(II)-NO⁺description of the ferric heme NO adducts

(vide infra).^8a,11The large Fe-NO force constant of 3.92

mdyn/Å is consistent with the presence of two strong π back-

bonds between the two occupied d_πorbitals of iron and the

unoccupied π* orbitals of NO⁺. In comparison, force constants

of 15.4 and 5.0 mdyn/Å have been obtained for the N-O and

Ru-NO bonds, respectively, in the Ru(II)-NO⁺moiety of

[Ru(NH₃)₅(NO)]Br₃, which exhibits

a similar bonding

situation.^12bFor the ferric NO adduct of Np1, N-O and Fe-NO

force constants of 15.11 and 4.09 mdyn/Å, respectively, have

been estimated using a simple eight-atom model for the NCA.⁴⁵

Nevertheless, these results show that 1 serves as an excellent

model system for ferric Np1-NO.

A.2. Quantum Chemistry Centered Normal Coordinate

Analysis (QCC-NCA). A normal-coordinate analysis was carried

out in order to substantiate the vibrational assignments for 1

and obtain experimental force constants for the Fe-NO and

N-O bonds. An initial force ﬁeld was calculated for the model

A.3. Electronic Structure of [Fe(P)(MI)(NO)]⁺(1). The

˜

electronic structure of the ferric six-coordinate (6C) complex

[Fe(TPP)(MI)(NO)]⁺(i.e., the cation of 1) was analyzed in this

complex [Fe(P)(MI)(NO)]⁺(1), and selected force constants

˜

study using the model complex 1. Geometry optimization of

were then reﬁned in the ﬁtting procedure according to the QCC-

NCA approach.^21bMasses of 77 were substituted for the meso

hydrogen atoms of P^2-in the model system to better represent

the meso phenyl groups of 1. As shown in Table 5, excellent

agreement between the vibrational frequencies from the NCA

treatment and the experimental data is obtained. The corre-

sponding force constants of the N-O and Fe-NO bonds are

determined to be 15.18 and 3.92 mdyn/Å, respectively. Since

the Fe-NO stretch and the two linear bends do not mix in the

linear Fe-N-O geometry of 1, the integrated VDOS intensity,

this system was performed using BP86/TZVP and B3LYP/

LANL2DZ. As shown in Table 4, both methods give very good

agreement with the crystal structure of the 6C complex [Fe(OEP)-

(MI)(NO)](ClO₄).^14aIn particular, the very short Fe-NO bond

(<1.65 Å) and the linear Fe-N-O unit are reproduced by DFT.

For the further analysis of the electronic structure of 1, the BP86/

˜

TZVP-optimized model 1 shown in Figure 4 is used for

consistency with earlier work on ferrous heme NO adducts.²¹

In the following evaluation of the molecular orbital (MO)

˜

2

e_Fe², and thus the ratio of the iron displacements r_Feof the

diagram of 1, the coordinate system shown in Scheme 1 is

employed.

Fe-NO stretch and the two Fe-N-O linear bends are intrinsic

(43) Lipscomb, L. A.; Lee, B.-A.; Yu, N.-T. Inorg. Chem. 1993, 32, 281–

286.

(45) Maes, E. M.; Walker, F. A.; Monfort, W. R.; Czernuszewicz, R. S.

J. Am. Chem. Soc. 2001, 123, 11664–11672.

(44) Novozhilova, I. V.; Coppens, P.; Lee, J.; Richter-Addo, G. B.; Bagley,

K. A. J. Am. Chem. Soc. 2006, 128, 2093–2104.

(46) Fadini, A.; Schnepel, F.-M. Schwingungsspektroskopie; Thieme Verlag:

Stuttgart, Germany, 1985.

9

J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008 15295

A R T I C L E S

Praneeth et al.

Figure 4. Fully optimized structure of [Fe(P)(MI)(NO)]⁺(1, P ) porphine,

MI ) 1-methylimidazole) obtained using BP86/TZVP. Important structural

parameters are given in Table 4.

˜

Figure 5 shows the MO diagram obtained for complex 1.

Contour plots of important MOs are presented in Figure 6. As

evidenced by the N-O stretching frequency of ∼1900 cm^-1

(vide supra) and the linear Fe-N-O unit, and in agreement

with the literature^8a,11and recent DFT results,¹⁶the electronic

structure of complex 1 can in general be classiﬁed as

Fe(II)-NO⁺. Since iron is in the +2 oxidation state and low-

spin in these compounds (as evident from magnetic Mo¨ssbauer

6

2

6

2

measurements), it has a [d_xzd_yzd_{x -y}] ≈ [t₂] electron conﬁg-

uration. Both π* orbitals of the NO⁺ligand (isoelectronic to

N₂) are empty and hence undergo π back-bonding interactions

with the d_xzand d_yzorbitals of the metal in the applied coordinate

system. The corresponding bonding combinations, d_xz_π_x* and

d_yz_π_y* (see Figure 5 for notation), are delocalized over several

MOs (see MOs <111> and <110> in Table 6). Hence, the

strength of this back-bond is better estimated from the corre-

sponding antibonding combinations, π_x*_d_xz<123> and π_y*_d_yz

˜

<124>, which are the LUMOs of complex 1. Both have ∼68%

π* and ∼27% d orbital character, which corresponds to a very

strong π back-bonding interaction. Contour plots of these

orbitals are shown in Figure 6. The third t₂-type d orbital of

Figure 5. MO diagram of [Fe(P)(MI)(NO)]⁺(1) calculated using BP86/

TZVP. The applied coordinate system is shown in Scheme 1. The labels

A_1u, A_2u, B_2u, and E_grefer to porphine orbitals (shown in Figure S7 in the

Supporting Information).^21bThe notation “A_B” indicates that orbitals A

and B interact, with A making the larger contribution to the resulting MO.

2

˜

iron, d_{x -y}<118>, is practically nonbonding (see Table 6). In

summary, the calculated orbital coefﬁcients are also in agree-

ment with the Fe(II)-NO⁺description. However, the obtained

N-O force constant of 15.18 mdyn/Å is markedly lower than

that of free NO⁺(25.1 mdyn/Å) and is actually very close to

that of free NO.⁴⁶This is in agreement with the existence

of two very strong Fe-NO π back-bonds, which lead to the

transfer of a signiﬁcant amount of electron density from the d_xz

and d_yzorbitals of Fe to the π* orbitals of NO. The total donation

corresponds to approximately one electron. In addition, there

2

orbital (d_z_σ* in our notation) into the occupied porphyrin A_2u

<81> HOMO. This mechanism leads to the partial population of

an Fe-N-O antibonding orbital, which explains the observed trend

in the DFT calculations: a larger population of this orbital leads to

smaller values of both ν(N-O) and ν(Fe-NO) and vice versa. In

addition, this is accompanied by a bending of the Fe-N-O unit.

Experimentally, bending of the Fe-N-O unit is observed for ferric

heme nitrosyls with axial thiolate coordination. Interestingly, in a

is a very weak σ interaction between σ_nbof NO⁺and d_zof Fe.

2

The corresponding bonding combination, σ_nb_d_z<66>, has

2

51% σ_nband only 4% d_zcontributions.

A.4. The Bending of the Fe-N-O Unit. Besides the two

strong Fe-NO π bonds in ferric heme complexes, additional

contributions to the Fe-NO bond have been proposed in recent

DFT studies and related to a bending of the Fe-N-O unit.¹⁶In

5C ferric heme NO adducts, a direct correlation of the Fe-NO

and N-O stretching frequencies was observed in DFT calculations

upon core substitution of the porphyrin macrocycle.^16aThis has

been related to a small admixture of a fully Fe-N-O σ antibonding

2

recent study, the same mechanism (i.e., admixture of this d_z_σ*

orbital into the occupied porphyrin A_2u<81> HOMO) was shown

to be responsible for the direct correlation of the ν(Fe-NO) and

ν(N-O) frequencies in these systems and the observed Fe-N-O

bending.¹⁹In this case, this is mediated by a trans effect of the

thiolate. This interesting electronic effect is further substantiated

by a comparison with complex 1 investigated here: in this case,

9

15296 J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008

Electronic Structure of Iron(III)-Porphyrin NO Adducts

A R T I C L E S

Figure 6. Contour plots of important MOs of [Fe(P)(MI)(NO)]⁺(1) calculated using BP86/TZVP. The labels are explained in Figure 5 and Table 6.

˜

2

the admixture of the Fe-N-O antibonding d_z_σ* orbital into the

type orbital (antibonding combination of d_πand π*) in the 6C

case.^16bWe found no evidence for this in our studies on the 6C

complex 1, so this does not seem to be a general property of 6C

ferric heme nitrosyls.

B. Nitric Oxide Binding to Ferrous and Ferric Porphyrin

Complexes. B.1. Energetic Considerations and Method Cali-

bration. The stabilities of the ferrous and ferric NO complexes

are determined from the following complex formation reactions

using DFT calculations:

˜

A_2u<81> HOMO of the complex (see MO <122> of 1) is very

small (only 1% d_zand 0.3% σ* contributions), which is negligible

2

with respect to the total Fe-NO bond strength (see Table 6).

Correspondingly, this complex has a linear Fe-N-O unit and

larger ν(Fe-NO) and ν(N-O) frequencies than the complexes with

axial thiolate coordination. Contrary to these ﬁndings, it has also

been proposed that the nature of the N-O antibonding orbital

admixed into the porphyrin A_2u<81> HOMO changes to a π*-

9

J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008 15297

A R T I C L E S

Praneeth et al.

HS-[Fe(P)(MI)]ⁿ⁺+ NO f LS-[Fe(P)(MI)(NO)]ⁿ⁺(n ) 0, 1)

This way, reaction energies of -16 kcal/mol (ferrous) and

roughly -13 kcal/mol (ferric) were obtained; in the latter case,

the wrong ground state for the reactant is still predicted. Finally,

we used B3LYP/TZVP single-point calculations on the B3LYP/

LANL2DZ* structures. In this case, a complex formation energy

of -11.4 kcal/mol is obtained for the ferrous complex.

Importantly, this method ﬁnally predicted the correct HS ground

state for ferric [Fe(P)(MI)]⁺, with a calculated HS/LS energy

difference of 5.6 kcal/mol. Because of this stabilization of the

HS state of the reactant, the magnitude of the reaction energy

from eq 1 decreased dramatically, and a ∆ε value of only -3.9

kcal/mol was obtained. In summary, these calculations show

that the formation of nitrosyl complexes is energetically more

favorable for ferrous compared to ferric hemes by ∼7.5 kcal/

mol. This number is equivalent to a factor of 3 × 10⁵difference

in the NO binding constant. This is in excellent agreement with

the experimentally determined trend in dissociation rate con-

stants: for ferric hemes, dissociation rate constants range from

0.65 to 40 s^-1, leading to a relatively small binding constant

for NO (K_eq) 10³-10⁵M^-1). In contrast, for Fe(II) systems,

(1)

The calculated complex formation energies for both low-spin

(LS) and high-spin (HS) reactants [Fe(P)(MI)]ⁿ⁺are listed in

Table 7. From experiment, it is known that both ferrous and

ferric ﬁve-coordinate (5C) hemes with axial imidazoles are

high-spin.^47-49As will be shown in the following analysis, the

prediction of the correct ground state for the reactant turned

out to be a major challenge for DFT. Importantly, the calculated

reaction energies, ∆ε, exhibit a large degree of method

dependence, as shown in Table 7. Using BP86/TZVP gave

unrealistically large energies (|∆ε| > 30 kcal/mol), and the 5C

reactants are predicted to be low-spin for both ferrous and ferric

heme. Hence, as has been noted before, total energy calculations

with gradient-corrected functionals are problematic.⁵⁰Therefore,

we instead focused on using the B3LYP functional. Using

B3LYP/LANL2DZ* calculations on the fully optimized struc-

tures from BP86/TZVP leads to much more realistic reaction

energies. For the ferrous case, eq 1 is exothermic, with ∆ε )

-16.7 kcal/mol. In this case as well as in all of the other B3LYP

calculations, the correct high-spin ground state for the ferrous

reactant is predicted. In the ferric case, this approach leads to

degenerate HS and LS states for [Fe(P)(MI)]⁺and ∆ε ≈ -12.5

kcal/mol. In order to further improve on these numbers, we next

applied full geometry optimizations using B3LYP/LANL2DZ*.

the dissociation rate constants are on the order of 10^-4s^-1

,

leading to very large binding constants for NO (K_eq) 10¹¹-10¹²

M^-1).⁵¹The calculated complex formation energies therefore

show that the Fe-NO bond is thermodynamically stronger in

Table 6. Charge Contributions of Important MOs of [Fe(P)(MI)(NO)]⁺(1) Calculated Using BP86/TZVP

˜

Fe

d

N

O

MO no.

label

energy [hartree]

s

p

s

p

N_MI(s + p)

∑MI

2

<125>

<124>

<123>

d_z_σ_nb+ Im(σ)

π_y*_d_yz

-0.23526

-0.25401

-0.25465

56

27

4

0

3

42

1

0

2

26

9

0

11

1

0

π_x*_d_xz(LUMO)

2

<122>

<121>

<120>

<119>

<118>

<117>

<116>

<114>

<112>

<111>

<110>

<109>

<108>

<107>

<106>

<102>

<73>

A_2u<81> + d_z_σ* (HOMO)

-0.29701

-0.30203

-0.32744

-0.32773

-0.33166

-0.33288

-0.34318

-0.36820

-0.37193

-0.39427

-0.40249

-0.40746

-0.41055

-0.41102

-0.43033

-0.44589

-0.61229

-0.61271

-0.63694

1

0

10

86

0

6

14

2

55

36

1

0

4

0.2

0

0.3

0

1

0

0.1

0

2

3

0

9

0

2

0

4

0

13

4

0

1

47

48

26

2

0

0.5

0

1

2

18

5

1

5

0

5

0

36

0

2

0

1

0

41

2

52

13

1

14

0

21

0

67

0

7

A_1u<79>

E_g<75/76> + d_yz_π_y*

E_g<75/76> + d_xz_π_x*

2

d_{x -y}

0

B_2u<74> + Im(π)

0.2

2

0

6

5

1

0

1

41

43

14

2

A_2u<72> + d_{x -y}

Im(π) + d_yz_π_y*

E_g<70/71>

d_xz_π_x*

d_yz_π_y* + Im(π)

E_u<77/78> + π_x*

B_1u

E_u<77/78> + Im(π)_d_yz

B_1g<80>_d_xy

19

13

2

3

4

2

Im(σ)_d_z+ σ_nb

π_x^b

π_y^b

<72>

2

<66>

σ_nb_d_z+ Im(σ)

2

Table 7. Reaction Energies for [Fe(P)(MI)]ⁿ⁺+ NO f [Fe(P)(MI)(NO)]ⁿ⁺at 298.15 K

∆ε(BP86) [kcal/mol]

∆ε(B3LYP) [kcal/mol]^b

complex

method

HS educt

LS educt

HS educt

LS educt

[Fe^II(P)(MI)] + NO

BP86/TZVP full opt.

-44.1

-53.4

-37.9

-41.2

-16.7^a

-12.6^a

-22.3^a

-12.3^a

[Fe^III(P)(MI)]⁺+ NO

[Fe^II(P)(MI)] + NO

B3LYP/LANL2DZ full opt.

Structure of NO adduct incorrectly described

[Fe^III(P)(MI)]⁺+ NO

-8.9

-9.2

[Fe^II(P)(MI)] + NO

B3LYP/LANL2DZ* full opt.

B3LYP/LANL2DZ*//B3LYP/TZVP

-16.1

-13.3

-21.7

-12.8

[Fe^III(P)(MI)]⁺+ NO

[Fe^II(P)(MI)] + NO

-11.4

-3.9

-18.8

-9.5

[Fe^III(P)(MI)]⁺+ NO

^aFrom single-point B3LYP/LANL2DZ* calculations on the fully optimized BP86/TZVP structures. ^bBold numbers indicate the lower complex

formation energy.

9

15298 J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008

Electronic Structure of Iron(III)-Porphyrin NO Adducts

A R T I C L E S

ferrous than in ferric heme NO adducts. In contrast, the Fe-NO

force constant is distinctively smaller in ferrous heme NO

adducts and has been determined to have the value 2.38 mdyn/Å

in the directly related six-coordinate complex [Fe^II(TPP)-

(MI)(NO)] (3).^21eThis again raises the question of why the

Fe-NO force constant is so much larger in the ferric case and

how this relates to the complex stabilities and thermodynamic

Fe-NO bond strengths.

B.2. Potential Energy Surface (PES) Calculations for the

Interaction of NO with Ferric Heme. In order to obtain detailed

insight into the mechanism of NO binding to ﬁve-coordinate

(5C) ferric heme, we next performed PES calculations using

model system 1 on (a) the closed-shell Fe(II)-NO⁺electronic

˜

state and (b) the Fe(III)-NO(radical) electronic state, where

the unpaired electrons of Fe(III) and NO are antiferromagneti-

cally coupled. Since the 5C reactant [Fe(P)(MI)]⁺can either

be high-spin or low-spin, we considered both of these spin states

in the calculations; therefore, in case (b), the total spin is either

S ) 0 [low-spin Fe(III)] or S ) 2 [high-spin Fe(III)]. Geometry

optimizations were performed on these three different electronic

structures, varying the Fe-NO distance in a systematic way

between 1.5 and 5 Å. Because this endeavor requires a very

large number of calculations, we employed the B3LYP/

LANL2DZ method for these partial optimizations, since this

method generates a fully optimized structure for model system

Figure 7. Potential energy surfaces for (a) closed-shell Fe(II)-NO⁺(blue),

(b) LS Fe(III) antiferromagnetically coupled (AFC) to NO (open shell S )

0, red), and (c) HS Fe(III) AFC to NO (S ) 2, black). Surfaces (a) and (b)

were obtained using single-point B3LYP/TZVP calculations on the B3LYP/

LANL2DZ structures (see Figure S6 in the Supporting Information). Surface

(c) was obtained by shifting the corresponding B3LYP/LANL2DZ surface

to lower energy to reproduce the calculated NO dissociation energy of 3.9

kcal/mol obtained with B3LYP/TZVP (Table 7). All of the calculations

˜

1 that compares well with experiment and the BP86/TZVP

were performed using model complex [Fe(P)(MI)(NO)]⁺(1).

˜

structure (see Table 4). Figure S6 in the Supporting Information

shows the PES obtained from these calculations. Importantly,

B3LYP/LANL2DZ predicts that the Fe(III)-NO(radical) (S )

0) state is in fact the ground state of the complex, being 1.0

kcal/mol lower in energy than the Fe(II)-NO⁺state. Since this

is not in agreement with experiment, we then performed B3LYP/

TZVP single-point calculations on the B3LYP/LANL2DZ

structures for the two S ) 0 states, because this method has

been shown to give better energies, as described in section B.1.

The S ) 2 surface was not recalculated but instead was shifted

in energy to reproduce the calculated NO dissociation energy

of ∼4 kcal/mol obtained from the B3LYP/TZVP treatment

(Table 7), which is in better agreement with experiment. Figure

7 shows the three PES obtained this way.

In order to investigate whether the porphine approximation

has a signiﬁcant effect on the calculated energies of the S ) 0

states, we also fully optimized the structure of [Fe(TPP)-

(MI)(NO)]⁺without any simpliﬁcation (i.e., applying the

complete TPP ligand). Importantly, the observed energy dif-

ference between the Fe(III)-NO(radical) S ) 0 state and the

Fe(II)-NO⁺state in this case is 1.3 kcal/mol, which compares

well with the value of 1.0 kcal/mol obtained with the porphine

approximation. Structural parameters are listed in Table 4. As

observed in previous work,^21bthe phenyl substituents have only

a small effect on the properties of the central Fe-N-O unit in

heme nitrosyls.

As shown in Figure 7, the B3LYP/TZVP calculations predict

the Fe(II)-NO⁺state to have the lowest energy, in agreement

with experiment (vide supra). The corresponding PES for this

state (shown in blue in Figure 7) is unusually steep for a

metal-ligand bond, which is a result of the fact that a

dissociation of the complex into Fe(II) and NO⁺is energetically

very unfavorable. The equilibrium Fe-NO distance for this state

is predicted to be <1.65 Å (Table 4), which is in excellent

agreement with experiment (vide supra). The Fe-N-O unit in

this state is linear. The PES for the S ) 0 state in which low-

spin Fe(III) is antiferromagnetically coupled to NO (the LS

Fe(III)-NO(radical) state) is shown in red in Figure 7. To our

great surprise, this state is energetically very close to the

Fe(II)-NO⁺ground state (predicted to be ∼1.0 kcal/mol higher

in energy). In this case, a stable minimum is obtained. Calculated

spin densities are +0.84 on Fe and -0.73 on NO, which

resembles the antiferromagnetic coupling between LS Fe(III)

and NO. The two unpaired electrons are roughly located in the

d_yzand π_y* orbitals (see below). Therefore, because of the small

energetic separation between the two S ) 0 states, the

Fe(II)-NO⁺PES crosses the LS Fe(III)-NO(radical) surface

at an elongation of the Fe-NO bond of only ∼0.05 Å from

the equilibrium distance. In addition, the crossing from the

Fe(II)-NO⁺state to the LS Fe(III)-NO(radical) state does not

produce a bending of the Fe-N-O unit: full geometry

optimization of the LS Fe(III)-NO(radical) state leads to an

Fe-NO bond length of 1.69 Å and a linear Fe-N-O unit. The

electronic structure of this state is illustrated in Scheme 2. In

this case, a strong polarization of the Fe-NO back-bond is

observed, where the R-spin interaction almost disappears. MO

coefﬁcients for the corresponding antibonding combinations

(47) Walker, F. A.; Simonis, U. In Encyclopedia of Inorganic Chemistry,

2nd ed; King, R. B., Ed.; Wiley: Chichester, U.K., 2005; Vol. IV, pp

2390-2521.

(48) Scheidt, W. R.; Geiger, D. K.; Lee, Y. J.; Reed, C. A.; Lang, G. J. Am.

Chem. Soc. 1985, 107, 5693–5699.

(49) Momenteau, M.; Loock, B.; Tetreau, C.; Lavalette, D.; Croisy, A.;

Schaeffer, C.; Huel, C.; Lhoste, J.-M. J. Chem. Soc., Perkin Trans. 2

1987, 249–257.

(50) (a) Ghosh, A.; Persson, B. J.; Taylor, P. R. J. Biol. Inorg. Chem. 2003,

8, 507–511. (b) Ghosh, A.; Vangberg, T.; Gonzalez, E.; Taylor, P. J.

Porphyrins Phthalocyanines 2001, 5, 345–356. (c) Swart, M.; Groen-

hof, A. R.; Ehlers, A. W.; Lammertsma, K. J. Phys. Chem. A. 2004,

108, 5479–5483. (d) Chang, C. H.; Boone, A. J.; Bartlett, R. J.;

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(51) (a) Antonini, E.; Brunori, M.; Wyman, J.; Noble, R. W. J. Biol. Chem.

1966, 241, 3236–3238. (b) Traylor, T. G.; Sharma, V. S. Biochemistry

1992, 31, 2847–2849. (c) Sharma, V. S.; Traylor, T. G.; Gardiner, R.;

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9575.

9

J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008 15299

A R T I C L E S

Praneeth et al.

Scheme 2

away from the iron, the LS Fe(III)-NO(radical) PES crosses

the HS Fe(III)-NO(radical) PES (shown in black in Figure 7),

which corresponds to the electronic state where high-spin Fe(III)

is antiferromagnetically coupled to the unpaired electron of NO.

Hence, this process is identical with a spin crossover of the

Fe(III) center from low-spin to high-spin. The HS Fe(III)-

NO(radical) state corresponds to the product state that is

obtained after complete dissociation of NO, because 5C ferric

heme is known to be high-spin. The crossing of these surfaces

is roughly predicted to occur at ∼1.9 Å and hence, still at an

Fe-NO distance below 2 Å. These two states cannot directly

interact by spin-orbit coupling, so the intersystem crossing must

be mediated by intermediate S ) 1 states, but we have not

investigated this point further. In summary, the major ﬁnding

of these PES calculations on 6C ferric heme NO adducts is

that upon an elongation of the Fe-NO bond of only ∼0.25 Å,

the complex passes through at least three different electronic

states (plus at least one intermediate S ) 1 state). The observed

complexity of this energy landscape is unprecedented, even for

transition-metal nitrosyls.

show a d orbital contribution of only 8%, compared to 27% for

the Fe(II)-NO⁺state. As mentioned above, the unpaired

electrons in the LS Fe(III)-NO(radical) case are primarily

located in R-d_yzand ꢀ-π_y*. The latter orbital is strongly mixed

with ꢀ-d_yz, leading to a corresponding antibonding combination

that consists of 47% π_y* and 44% ꢀ-d_yz. This strong covalency

quenches part of the spin density, leading to a distribution of

roughly +0.5 on Fe and -0.5 on NO. The remaining backbond

between ꢀ-d_xzand ꢀ-π_x* is comparable to that of the

Fe(II)-NO⁺state (see Scheme 2), and hence shows a strong

contribution to the total spin density, resulting in the calculated

values listed above. In summary, although one of the ꢀ-spin

back-bonds is strengthened, this is not sufﬁcient to compensate

for the loss of R-spin back-bonding. This explains the distinc-

tively weaker Fe-NO bond in the LS Fe(III)-NO(radical) case,

for which ν(Fe-NO) is predicted to be only 430 cm^-1(Table

4) with an Fe-NO force constant of 2.26 mdyn/Å (Table 5).

For ν(N-O), a shift of ∼70 cm^-1to lower energy is predicted

because of the formal electron transfer from iron to the π*

antibonding orbitals of NO in this case. The observation that

the Fe-N-O unit is not bent in the LS Fe(III)-NO(radical)

Discussion

In this work, the new six-coordinate (6C) Fe(III)-porphyrin

NO model complex [Fe(TPP)(MI)(NO)](BF₄) (1) was prepared

from [Fe(TPP)(BF₄)] by careful reaction with puriﬁed NO. The

structure of the bis(thf)-coordinated precursor has been deter-

mined. Complex 1 was used to explore the spectroscopic

properties and electronic structures of 6C ferric heme nitrosyl

model complexes using IR and, for the ﬁrst time, nuclear

resonance vibrational spectroscopy (NRVS) along with normal

coordinate analysis (NCA) and DFT calculations. For complex

1, N-O and Fe-NO stretching frequencies of 1896 and 580

cm^-1(578 cm^-1measured with ⁵⁷Fe), respectively, are assigned

using isotope labeling. From NCA, force constants of 15.18 and

3.92 mdyn/Å for the N-O and Fe-NO bonds, respectively,

are determined for complex 1. The electronic structure of this

complex is in agreement with the widely accepted Fe(II)-NO⁺

description for ferric nitrosyls, as reﬂected by the N-O

stretching frequency of ∼1900 cm^-1and the linear Fe-N-O

unit observed in crystal structures of closely related model

complexes.^8a,11The Fe-NO interaction in ferric heme is

dominated by strong π back-bonding between two d_πorbitals

of the metal and two empty π* orbitals of NO⁺. This leads to

a net transfer of one electron from the metal to the ligand and

explains the decrease of the N-O stretching frequency from

2390 cm^-1in free NO⁺to ∼1900 cm^-1in complex 1. In

contrast, the Fe-NO σ interaction in ferric heme is weak, as a

result of the generally poor donor abilities of the σ_nborbital in

diatomics with multiple bonds. In accord with this strong π/weak

σ bonding description, the Fe-N-O unit is linear and short to

maximize the π interactions. Recently, it was proposed that the

ferric heme NO adducts in nitrophorins might have an electronic

structure that corresponds to Fe(III)-NO(radical) in order to

prevent autoxidation.^53,6cHowever, the experimental data avail-

able for ferric heme NO adducts with axial N-donor coordination

in proteins and model complexes show that these species have

2

state is due to the fact that the coupling of the E state from

[d_xz,d_yz]³on iron with the ²E state from [π_x,π_y]³on NO leads to

nondegenerate states, even in an effective C_4Vsymmetry. The

resulting electronic state is therefore not Jahn-Teller-active.

In conclusion, a crossing from the Fe(II)-NO⁺ground state to

the LS Fe(III)-NO(radical) state is induced by an elongation

of the Fe-NO bond and leads to a distinct weakening of the

Fe-NO and N-O bonds. The closeness in energy of these states

implies that there might be some LS Fe(III)-NO(radical)

admixture into the Fe(II)-NO⁺ground state. This mixing could

be dynamically mediated by the Fe-NO stretch, which means

that the Fe(II)-NO⁺ground state and the LS Fe(III)-NO(radical)

state would become vibronically coupled. In C_4Vsymmetry,

coupling between the ¹A₁ground state of Fe(II)-NO⁺with the

¹A₁component of LS Fe(III)-NO(radical) could indeed be

induced by ν(Fe-NO).⁵²

On the basis of these ﬁndings, the dissociation of NO from

the ferric iron center could be envisioned as follows: Upon a

small elongation of the Fe-NO bond to ∼1.70 Å (see Figure

7), the critical electron transfer that leads from the Fe(II)-NO⁺

state to the LS Fe(III)-NO(radical) electronic structure, which

is a symmetry-allowed process, takes place. If NO moves farther

(53) In the case of the nitrophorins, it has been proposed that the rufﬂed

2

conformation of heme plays a major role in stabilizing a [d_xzd_yzd_xy¹]-

type ground state. This could lead to the formation of a corresponding

ferric heme nitrosyl in the LS Fe(III)-NO(radical) state where the

unpaired electron is localized in the d_xyorbital (see the Introduction).

This is different from the LS Fe(III)-NO(radical) form invoked here,

where the unpaired electron is localized in the d_yzorbital.

(52) It should be noted that the symmetry of 6C ferric heme nitrosyls is

lower than C_4V, which further diminishes symmetry-related restrictions.

C_4Vis the maximum possible symmetry of a corresponding 5C

[Fe(porphyrin)(NO)]⁺complex.

9

15300 J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008

Electronic Structure of Iron(III)-Porphyrin NO Adducts

A R T I C L E S

the assumed Fe(II)-NO⁺ground state (on the basis of Fe-NO

bond distances and Fe-NO and N-O stretching frequencies).

For example, the N-O stretching frequency of 1917 cm^-1for

ferric rNp1-NO is very close to the values obtained for model

complex 1 and corresponding enzymatic species (see Table 1).

Hence, this indicates that the electronic structures of ferric

nitrophorin NO adducts do not differ from those of the other

ferric nitrosyl species. In summary, a very detailed picture of

the electronic properties of the Fe(II)-NO⁺electronic ground

state of these complexes has now emerged.

Scheme 3. Ground State PES, Taking into Account the Relative

Error of ∼2 kcal/mol in the DFT Energies

The quantitative description provided here for the Fe-NO

bond in the ferric heme complex 1 makes possible a detailed

comparison with the corresponding ferrous complex [Fe(TPP)-

(MI)(NO)] (3) studied previously.^21a,bInterestingly, the one-

electron reduction of the Fe(II)-NO⁺complex 1 is mostly

ligand-centered, resulting in an Fe(II)-NO(radical) electronic

structure description of 3. This is evident from the N-O

stretching frequency, which is reduced from 1896 cm^-1in 1 to

1630 cm^-1in 3. The Fe-NO bond in the ferrous complex

corresponds to a σ bond mediated by the singly occupied π*

corresponding to ∆G° in the range from -15 to -16 kcal/mol).

This dramatic increase in the Fe-NO bond strength in going

from the ferric to the ferrous case is not in agreement with the

difference in MO bonding descriptions elaborated above. In

addition, this trend is also not in agreement with the observed

trend in force constants: 1 has a much larger Fe-NO force

constant (3.92 mdyn/Å) than 3 (2.38 mdyn/Å). Therefore, we

still lack a fundamental understanding of the Fe-NO interaction

in the ferric heme complexes. This becomes evident from a

detailed evaluation of the potential energy surfaces (PES) of

the different electronic states of 1.

2

orbital of NO and the d_zorbital of iron along with a π back-

bond between the unoccupied π* orbitals of NO and the d_π

orbitals of the metal.^21bThe strong σ bond explains both the

large stability of the Fe-NO bond in this case and the observed

trans effect of the NO ligand on axially coordinated N-donor

ligands, which induces long Fe(II)-N_imidazolebond lengths

compared to the corresponding bis(imidazole)-ligated

Fe(II)-porphyrins.⁵⁴In contrast, the σ bond is clearly much

weaker in 1 because it is mediated by the low-lying orbital σ_nb.

Hence, this difference in σ bonding explains the very short

Fe-NO bond in 1 and the lack of a signiﬁcant trans effect of

NO in ferric heme NO adducts. In fact, all Fe(II)-NO⁺

complexes with trans N-donor ligands exhibit Fe-N_imidazolebond

lengths comparable to those of the corresponding [Fe^III(por-

phyrin)(imidazole)₂]⁺complexes.^14aOn the other hand, the

overall π back-bond is stronger in 1.⁵⁵When these contributions

are summed up, one would expect a somewhat stronger Fe-NO

bond in ferrous hemes because of the stronger σ bond. In

contrast to this prediction, the actual experimental difference

in Fe-NO bond strengths in these systems is much more

dramatic: experimental complex formation constants (K_eq) of

ferrous compared to ferric heme nitrosyls differ in general by

4-5 orders of magnitude (∼6 orders of magnitude in the case

of the nitrophorins⁵⁶). This trend is nicely reproduced by the

calculated free energies of complex formation, ∆G°, given in

Table 7. In the case of the 6C ferric complex 1, the obtained

formation energy is only about -4 kcal/mol. The magnitude of

this value is at the low end of the experimentally determined

values, which range from -4 to -7 kcal/mol (K_eq) 10³-10⁵

M^-1) for ferric heme nitrosyls. In comparison, the Fe-NO bond

is predicted to be thermodynamically stronger by 7.5 kcal/mol

for the corresponding 6C ferrous complex, which is again in

In order to obtain detailed insight into the Fe-NO interaction

in 6C ferric heme nitrosyls, we calculated the PES for three

key electronic states, as shown in Figure 7: (a) Fe(II)-NO⁺

(S ) 0, blue curve), which is the established ground state as

described above; (b) the open-shell singlet state low-spin (LS)

Fe(III)-NO(radical), where LS Fe(III) is antiferromagnetically

coupled to NO (S ) 0, red curve); and (c) the corresponding

state high-spin (HS) Fe(III)-NO(radical) (S ) 2, black curve),

which corresponds to the product state upon dissociation of NO.

These results show that the elusiVe LS Fe(III)-NO(radical) state

does in fact exist as an energy minimum and that this state is

surprisingly close in energy to the Fe(II)-NO⁺ground state

(with a DFT energy separation of only ∼1 kcal/mol). It is likely

that the calculated energy difference between these states is

underestimated by a few kilocalories per mole, at least for the

case of axial N-donor coordination. This is evident from the

vibrational properties, which do not show any indication of the

presence of the LS Fe(III)-NO(radical) state. From the

calculated complex formation energies, a realistic error of 1-2

kcal/mol is estimated for the B3LYP/TZVP method. This would

shift the crossing point of the two S ) 0 surfaces to about

1.70-1.76 Å. In addition, the two states could be mixed, so

the shape of the ground-state PES around the crossing point is

clearly not well-deﬁned by the DFT treatment. Scheme 3 shows

the ground-state PES with corrected energies as an illustration,

including key energy parameters. When the small error in

relative energies from DFT is taken into account, it is estimated

that an Fe-NO bond elongation of only 0.05-0.1 Å from the

equilibrium position leads to a change in ground state, i.e. the

critical iron(II)-to-NO⁺electron transfer occurs at an Fe-NO

distance of only 1.70-1.75 Å. Importantly, the properties of

the Fe(II)-NO⁺and LS Fe(III)-NO(radical) states are very

different. From the calculations, the LS Fe(III)-NO(radical)

state has a weaker (longer) Fe-NO bond and hence, a lower

Fe-NO stretching frequency. Because of the electron transfer

from Fe(II) into a π* orbital of NO⁺, the N-O bond is also

weaker, shifting ν(N-O) to lower frequency. Hence, both the

reasonable agreement with experiment (K_eq) 10¹¹-10¹²M^-1

,

(54) Wyllie, G. R. A.; Schulz, C. A.; Scheidt, W. R. Inorg. Chem. 2003,

42, 5722–5734.

(55) Interestingly, this actually relates not to the observed π*_d mixing

but only to the fact that the number of back-bonds is reduced from

2 in 1 to 1.5 in 3. This is evident from the d orbital contributions

to the corresponding π*_d antibonding orbitals, which are 27%

for 1 and 25% (average) for 3 for the π_y*_d_yzorbital (R/ꢀ<126>

in ref 21b). This indicates that the linear Fe-N-O unit and the

shorter Fe-NO bond alone do not facilitate the π back-bond in 1

as compared to 3.

(56) Berry, R. E.; Ding, X. D.; Shokhireva, T. K.; Weichsel, A.; Montfort,

W. R.; Walker, F. A. J. Biol. Inorg. Chem. 2004, 9, 135–144.

9

J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008 15301

A R T I C L E S

Praneeth et al.

Fe-NO and N-O bonds become weaker in going from

Fe(II)-NO⁺to LS Fe(III)-NO(radical), which corresponds to

a direct correlation of these bond strengths.

On the basis of these properties of ferric heme nitrosyls, the

occurrence of ferric instead of ferrous hemes in the NO delivery

proteins (nitrophorins) and enzyme-product complexes of

cytochrome cd₁NiR and NOS is not arbitrary. This is actually

related to the weakness of the Fe-NO bond in the ferric

systems, which enables loss of NO. The corresponding rate

constants k_offare about 4-8 orders of magnitude larger in ferric

than in ferrous hemes, as discussed above.^2g,57Hence, formation

of ferrous heme NO adducts has often been called a “dead end”

for protein activity and would certainly impose a rather dramatic

limit on the dissociation of NO from the NiR or NOS

enzyme-product complexes or the nitrophorins.

Finally, it is interesting to compare the properties of ferric

heme nitrosyls to those of the corresponding Ru(III)-NO

adducts. In the latter case, the electronic structure also corre-

sponds to Ru(II)-NO⁺, but no lability of the bound NO is

observed. On the contrary, these compounds are some of the

most stable transition-metal nitrosyls known. What is the reason

for this substantial difference? Since ruthenium is a second-

row transition metal, its tendency to form high-spin complexes

is greatly reduced. Hence, 5C Ru(III) products obtained upon

dissociation of NO from the corresponding nitrosyl complexes

in general have low-spin ground states, and therefore, no spin

crossover is observed upon loss of NO. In view of Figure 7,

this would mean that the HS metal(III)-NO(radical) energy

surface (black) disappears (i.e., shifts to much higher energy)

for Ru(III). Independently of how the energies of the S ) 0

states play out, the removal of the HS metal(III)-NO(radical)

surface from the picture automatically leads to a much more

stable complex. This is another demonstration of how signiﬁcant

these results are for the detailed understanding of the electronic

structures, thermodynamic stabilities, spectroscopic properties,

and reactivities of group 8 metal(III) nitrosyls.

These results are very important, as they imply that a small

perturbation of the Fe-N-O unit (due to interaction of the

bound NO with a protein side chain, for example) might be

able to induce a change of the ground state from Fe(II)-NO⁺

to LS Fe(III)-NO(radical). This should be evident from the

spectroscopic properties of the complex. However, no clear

experimental proof for the existence of a ferric heme nitrosyl

with the LS Fe(III)-NO(radical) ground state is available to

date. Nevertheless, this ﬁnding offers an attractive explanation

for the observed weaker Fe-NO and N-O bond strengths in

ferric heme nitrosyls with axial thiolate coordination, which

could be attributed to a change in ground state to LS

Fe(III)-NO(radical). However, this would not explain the

observed bending of the Fe-N-O unit; our results demonstrate

that a bent Fe-N-O unit is not an intrinsic property of the LS

Fe(III)-NO(radical) state. On the other hand, recent DFT results

show that all of these properties of thiolate-coordinated ferric

heme nitrosyls can also be explained within the Fe(II)-NO⁺

ground-state model.¹⁹This point needs further investigation.

Upon a further elongation of the Fe-NO bond, the LS

Fe(III)-NO(radical) crosses the HS Fe(III)-NO(radical) PES

at a distance of ∼1.9 Å (see Scheme 3). This transition

corresponds to a spin crossover of Fe(III) and is related to the

fact that the ﬁve-coordinate (5C) ferric heme product is actually

high-spin. Importantly, the HS Fe(III)-NO(radical) PES is

dissociatiVe for the Fe-NO bond. This generates a small kinetic

barrier for the dissociation of NO, which, however, is <1 kcal/

mol and hence negligible. More importantly, this dramatically

lowers the thermodynamic stability of the ferric Fe-NO bond,

from about -10 kcal/mol on the LS surfaces to only about -4

kcal/mol (Scheme 3). Therefore, the properties of the HS

Fe(III)-NO(radical) PES determine the thermodynamic weak-

ness of the Fe-NO bond and the large dissociation rate constant

of NO in ferric heme nitrosyls. This means that once the system

has entered the HS Fe(III)-NO(radical) electronic state, the

dissociative nature of this PES will actually drive the NO away

from the metal center, allowing the NO to diffuse off and

inhibiting its retention. This explains the surprisingly large rate

constants for NO dissociation measured experimentally for ferric

hemes. On the other hand, the large Fe-NO force constant and

short Fe-NO distance in the ground state relate to the properties

of the Fe(II)-NO⁺surface, which is very steep as a result of

the fact that the dissociation of the complex into Fe(II) and NO⁺

is energetically very unfavorable. For this reason, the experi-

mentally derived Fe-NO force constant is not a measure of

the stability of the Fe-NO bond in this case. These quantities

are actually completely unrelated because they depend on the

properties of different electronic states. Because of this, complex

1 has a large Fe-NO force constant but actually a quite weak

Fe-NO bond, in agreement with the small NO binding constant

and the observed rapid loss of NO from the iron center in

solution. Altogether, dissociation of NO from the ferric iron

center requires the system to pass through at least three different

electronic states (plus an S ) 1 intermediate state). The observed

complexity of this energy landscape is unprecedented, even for

transition-metal nitrosyls. The “simple” Fe(II)-NO⁺description

of the electronic structure of these complexes, and the analogies

normally drawn to Fe(II)-heme CO complexes, do not in any

way implicate such complexity as determined here.

Conclusions

In this study, we have characterized the Fe(II)-NO⁺ground

state of six-coordinate ferric heme nitrosyls in detail using the

model complex [Fe(TPP)(MI)(NO)](BF₄) as an example. Through

theuseofDFTcalculations,theelusivelow-spinFe(III)-NO(radical)

(S ) 0) state has then been investigated. Surprisingly, this state

is located just a few kilocalories per mole above the ground

state. It is characterized by properties very different from those

of Fe(II)-NO⁺; in particular, the Fe-NO and N-O bond

strengths are greatly reduced as a result of the reduction of π

back-bonding. Upon a small (0.05-0.1 Å) elongation of the

Fe-NO bond from the equilibrium position, the Fe(II)-NO⁺

ground-state potential energy surface (PES) crosses the low-

spin Fe(III)-NO(radical) (S ) 0) energy surface. Importantly,

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9

15302 J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008

Electronic Structure of Iron(III)-Porphyrin NO Adducts

A R T I C L E S

Center for Research Resources, Biomedical Technology Program,

and by the Department of Energy, Ofﬁce of Biological and

Environmental Research. This work was supported by the Deutsche

Forschungsgemeinschaft (DFG) (Grant LE 1393/1-2). F.P. ac-

knowledges the Fonds der Chemischen Industrie (FCI) for a

Chemiefonds fellowship.

this shows that the low-spin Fe(III)-NO(radical) (S ) 0) state

could become the ground state of ferric heme nitrosyls upon

small steric or electronic perturbations that may occur in protein

active sites. This is especially true for axial thiolate (cysteinate)

coordination, because thiolates tend to stabilize Fe(III). The

observed thermodynamic weakness of the Fe-NO bond in ferric

heme nitrosyls is due to the properties of the high-spin

Fe(III)-NO(radical) (S ) 2) state, which is dissociative with

respect to the Fe-NO bond. These PES calculations reveal an

unexpectedly complex energy landscape for binding of NO to

ferric heme.

Supporting Information Available: Complete ref 35, vibra-

tional spectra of [Fe(TPP)(NO)(NO₂)] (including the ¹⁵N¹⁸O-

labeled compound), NRVS raw data, IR spectra of NRVS

samples, the PES obtained using B3LYP/LANL2DZ, MO

diagrams for porphine dianion (P^2–) and NO, Cartesian coor-

dinates of DFT-optimized structures, and crystallographic data

for [Fe(TPP)(thf)₂](BF₄) in CIF format. This material is available

free of charge via the Internet at http://pubs.acs.org.

Acknowledgment. The Advanced Photon Source is supported

by the DOE, Basic Energy Sciences, Ofﬁce of Science, under

Contract DE-AC02-06CH11357. S.D.G. acknowledges SSRL for

funding. SSRL operations are funded by the Department of Energy,

Ofﬁce of Basic Energy Sciences. The Structural Molecular Biology

program is supported by the National Institutes of Health, National

JA801860U

9

J. AM. CHEM. SOC. VOL. 130, NO. 46, 2008 15303

Article Doi

Electronic structure of six-coordinate iron(III)-porphyrin NO adducts: The elusive iron(III)-NO(radical) state and its influence on the properties of these complexes

DOI: 10.1021/ja801860u

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Article abstract of DOI:10.1021/ja801860u

Full text of DOI:10.1021/ja801860u

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