Solid-state NMR, Moessbauer, crystallographic, and density functional theory investigation of Fe-O2 and Fe-O2 analogue metalloporphyrins and metalloproteins

J. Am. Chem. Soc. 1999, 121, 3829-3844

3829

Solid-State NMR, Mo¨ssbauer, Crystallographic, and Density

Functional Theory Investigation of Fe-O₂and Fe-O₂Analogue

Metalloporphyrins and Metalloproteins^†

Nathalie Godbout, Lori K. Sanders, Renzo Salzmann,^‡Robert H. Havlin,^§

Mark Wojdelski, and Eric Oldfield*

Contribution from the Departments of Chemistry and Biophysics, UniVersity of Illinois at

Urbana-Champaign, 600 South Mathews AVenue, Urbana, Illinois 61801

ReceiVed September 14, 1998. ReVised Manuscript ReceiVed February 12, 1999

Abstract: We have synthesized and studied via solid-state NMR, Mo¨ssbauer spectroscopy, single-crystal X-ray

diffraction, and density functional theory the following Fe-O₂analogue metalloporphyrins: Fe(5,10,15,20-

tetraphenylporphyrinate) (nitrosobenzene)(1-methylimidazole); Fe(5,10,15,20-tetraphenylporphyrinate) (nitroso-

benzene)(pyridine); Fe(5,10,15,20-tetraphenylporphyrinate)(4-nitroso-N,N-dimethylaniline)(pyridine); Fe-

(2,3,7,8,12,13,17,18-octaethylporphyrinate) (nitrosobenzene)(1-methylimidazole) and Co(2,3,7,8,12,13,17,18-

octaethylporphyrinate)(NO). Our results show that the porphyrin rings of the two tetraphenylporphyrins

containing pyridine are ruffled while the other three compounds are planar: reasons for this are discussed.

The solid-state NMR and Mo¨ssbauer spectroscopic results are well reproduced by the DFT calculations, which

then enable the testing of various models of Fe-O₂bonding in metalloporphyrins and metalloproteins. We

find no evidence for two binding sites in oxypicket fence porphyrin, characterized by very different electric

field gradients. However, the experimental Mo¨ssbauer quadrupole splittings can be readily accounted for by

fast axial rotation of the Fe-O₂unit. Unlike oxymyoglobin, the Mo¨ssbauer quadrupole splitting in

PhNO•myoglobin does not change with temperature, due to the static nature of the Fe•PhNO subunit, as verified

2

by H NMR of Mb•[²H₅]PhNO. Rotation of O₂to a second (minority) site in oxymyoglobin can reduce the

experimental quadrupole splittings, either by simple exchange averaging, or by an electronic mechanism, without

significant changes in the Fe-O-O bond geometry, or a change in sign of the quadrupole splitting. DFT

calculations of the molecular electrostatic potentials in CO, PhNO, and O₂-metalloporphyrin complexes show

that the oxygen sites in the PhNO and O₂complexes are more electronegative than that in the CO system,

which strongly supports the idea that hydrogen bonding to O₂will be a major contributor to O₂/CO discrimination

in heme proteins.

Introduction

spectroscopy,^12-15have an important role to play, by providing

basic structural-spectroscopic correlations. To date, most

studies have focused on the binding of CO to metalloproteins

and metalloporphyrins, but the topic of O₂binding is at least

as important, albeit generally more technically demanding to

investigate due to the lower chemical stability of O₂-adducts

and their decreased spectral sensitivity. These difficulties have

The question of how O₂binds to Fe atoms in respiratory

proteins, and how CO is discriminated against binding, is a topic

of continuing interest.¹Here, both crystallographic^2-5and

spectroscopic methods, such as infrared/Raman spectroscopy,^6-8

nuclear magnetic resonance (NMR),^9-11and Mo¨ssbauer

^†This work was supported by the United States Public Health Service

(National Institutes of Health Grant HL-19481).

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11949.

^‡Swiss National Science Foundation Postdoctoral Research Fellow,

1996-1997; American Heart Association, Inc., Illinois Affiliate, Postdoc-

toral Research Fellow, 1997-1998.

^§Barry Goldwater Fellow.

Colgate Palmolive Scholar.

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Published on Web 04/08/1999

3830 J. Am. Chem. Soc., Vol. 121, No. 16, 1999

Godbout et al.

resulted in the use of a wide range of Fe-O₂analogues, such

as CoNO,^16,17FeRNO,^18,19and CoO₂^20,21complexes, for NMR,

IR, Mo¨ssbauer, and electron spin resonance (ESR) investiga-

tions. A major goal of many of these studies is to obtain a better

understanding of the geometric and electronic structures of

relatively simple metal-ligand complexes, and then to extend

these ideas to metalloproteins.

in proteins, they still need to be validated on smaller, more well

characterized model systems. Then, it should be possible to

refine both protein backbone and protein side-chain, as well as

metal-ligand binding site structures, and to probe in detail

electronic and electrostatic structures by use of quantum

chemistry. For example, once a structure is known, it now

appears to be possible to deduce charge densities (F(r)),

electrostatic potentials (Φ(r)), electric field gradients (∇•E), and

so forth from the experimental structures, which are in good

accord with the results of quantum chemical calculations.^37-39

In this article, we present results on the synthesis, structure,

solid-state NMR, Mo¨ssbauer, and quantum chemical (density

functional theory, DFT) investigations of a series of Fe-O₂-

analogue metalloporphyrins, containing the groups Fe•RNO

(HNOtO₂) and CoNO. The ability to compute the spectroscopic

observables gives some confidence in the quality of the DFT

calculations, which are then used to investigate the topic of Fe-

O₂bonding in metalloporphyrins and metalloproteins.

Quantum chemical methods^22-27should, in principle, be able

to help solve such problems, but a major difficulty is that the

resolution of both protein crystal and solution structures is often

insufficient to enable highly accurate calculations. This situation

is beginning to improve with the development of intense

synchrotron light sources for X-ray crystallography,^28,29which

together with the application of chemical shift,^30-32chemical

shift anisotropy,³³and dipolar splitting^34-36restraints in solution

NMR spectroscopy can be expected to result in protein structures

of significantly improved accuracy. But before quantum chemi-

cal methods are generally applied to investigating ligand binding

The RNO analogues of heme proteins have been known for

many years, primarily due to the fact that nitrobenzene poison-

ing⁴⁰is due to the reduction of PhNO₂to PhNHOH, which binds

(as PhNO) to metHb to form Hb•PhNO.⁴¹Indeed, PhNO binds

so strongly to Hb and Mb that it displaces even CO.⁴¹We have

synthesized and characterized a range of Fe•RNO adducts, some

of which display pronounced porphyrin ruffling, and we report

the solid-state NMR and Mo¨ssbauer spectra of these systems.

We then show that modern DFT methods enable prediction of

not only their NMR spectra but also their ⁵⁷Fe Mo¨ssbauer spectra

as well. These results validate use of the DFT method on

oxyheme analogue models, and encourage an extrapolation to

the FeO₂systems, oxypicket fence porphyrin and oxymyoglobin,

whose Mo¨ssbauer spectra have been the topic of debate for some

time.¹⁵Our results with picket fence porphyrin supports a fast

axial diffusion model for averaging of the electric field gradient,

while a more restricted motion is suggested to be the origin of

the large temperature dependence of the ⁵⁷Fe MbO₂results,

although alternative explanations are possible in the latter case.

With PhNO bound to Mb, the experimental ⁵⁷Fe Mo¨ssbauer

spectra are essentially temperature insensitive, a very different

effect to that seen with MbO₂. With the isoelectronic CoNO

complex, fast axial motion is hindered at low temperatures, plus,

we predict and find an undistorted metalloporphyrin with a

Co-N-O of ∼120°, as deduced by crystallography, NMR,

and via quantum chemical geometry optimization, essentially

the same value as that for MbO₂in the most recent structure

determinations.⁴²Since we find good agreement between the

results of calculations and experiment for the chemical shieldings

and shielding tensors, as well as for the electric field gradient

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O₂-Heme Analogue Studies

J. Am. Chem. Soc., Vol. 121, No. 16, 1999 3831

in. bore superconducting solenoid magnets, Tecmag (Houston, TX)

Aries and Libra pulse programmers, Doty Scientific (Columbia, SC) 5

mm MAS NMR probes and spin-speed controllers, and a variety of

other home-built digital and radio frequency circuitries.

tensors of the metals, which implies a good description of both

ground and excited states, we then briefly explore the question

of the electrostatic structure of the FeCO, FeRNO, and FeO₂-

metalloporphyrins, of particular interest in the context of

hydrogen-bonding and CO/O₂discrimination in heme proteins.¹

Mo1ssbauer Spectroscopy. Mo¨ssbauer spectra were recorded by

using a Ranger Scientific, Inc. (Burleson, TX) MS-900 spectrometer

equipped with a VT-900 transducer and a Kr-CO₂gas proportional

counter. The source was ⁵⁷Co in a 6 µm rhodium foil having an 8 mm

active diameter, and an initial activity of 25 mCi (Amersham Life

Sciences, Arlington Heights, IL). Low temperatures were obtained by

using a Janis Research Company, Inc. (Wilmington, MA) cryostat.

Samples were sealed in thin Delrin containers with epoxy resin.

X-ray Crystallography. Crystals were mounted with oil (Paratone-

N, Exxon) to thin glass fibers and diffraction data collected on a Bruker

(Madison, WI) SMART/CCD diffractometer using Mo KR radiation

(λ ) 0.71073 Å). Structures were solved by using SHELXTL V5.0

(Bruker) software. Space group choice was confirmed by successful

convergence of full-matrix least-squares refinements on F². Hydrogen

atoms were assigned idealized locations and given isotropic thermal

parameter 1.2 times the thermal parameter of the atom to which they

were attached. Data were corrected for Lorentz and polarization effects

and empirical absorption corrections were applied.

The color and morphology of the crystals, the crystallographic

systems, and space groups, as well as other information related to the

crystal structure determinations, are summarized in Table 1. In general,

from 7459 to 26755 data points were collected with the area detector,

and from 3761 to 11388 data points having I > 2σ(I) were used in the

refinements. The final R₁values varied from 0.039 to 0.096, and the

GOF values varied from 0.936 to 1.178, Table 1 (see also Supporting

Information). Atomic coordinates, bond lengths, angles, and thermal

parameters have also been deposited with the Cambridge Crystal-

lographic Data Centre (CCDC). Any request to the CCDC for this

material should quote the full literature citation and the reference

number.

Computational Aspects. All chemical shielding, electric field

gradient, and geometry optimization calculations were carried out by

using density functional theory as embodied in Gaussian-94.²³As

described in detail in several recent articles,^43,49,50it is now possible to

compute both ligand and metal shieldings, as well as ligand and metal

electric field gradients, in a variety of metalloporphyrins, with good

accord being found between theory and experiment. Typically, we have

used moderately sized, locally dense basis sets such as Wachters’ all

electronbasis(14s11p6d/8s7p4d)^51,52forirontogetherwith6-311++G(2d)

for those atoms coordinated to iron, and other atoms (e.g., O in CO)

of particular interest, while smaller (6-31G*, 3-21G*) basis sets are

used on the more distant atoms. This approach enables relatively rapid

property predictions with no perceptible increase in error over larger,

more uniform basis calculations. The gauge-including atomic orbitals

method⁵³was used for the chemical shielding calculations, with Becke’s

exchange functional and Perdew and Wang’s gradient-corrected cor-

relation functional (BPW91).⁵⁴For the metal electric field gradients,

we employed Becke’s three-parameter functional⁵⁵with a nonlocal

correlation term given by the Lee, Yang, and Parr expression,⁵⁶the

Experimental Section

Synthetic Aspects. All syntheses were carried out by using Schlenk

techniques, as described elsewhere.^18,43

Fe(5,10,15,20-tetraphenylporphyrinate)(nitrosobenzene)-

(pyridine) was prepared by the NaBH₄reduction of Fe(TPP)Cl in THF

under Ar. The Fe(TPP)(THF)₂was then converted to Fe(TPP)(py)₂with

pyridine, then 1.2 equiv of PhNO (as the dimer) added to form Fe-

(TPP)(PhNO)(py). Crystallization was from a layered toluene/heptane

system with a yield of 84%. Fe(TPP)(PhNO)(py)•0.5toluene Anal. data

Calculated (Found): C, 78.00 (77.71); H, 4.70 (4.81); N, 9.32 (9.10).

Fe(5,10,15,20-tetraphenylporphyrinate)(4-nitroso-N,N-dimethyl-

aniline)(pyridine) was prepared basically as described above except

for the addition of 1.2 equiv of 4-nitroso-N,N-dimethylaniline (NOD-

MA). Crystallization was from toluene/heptane with a yield of 80%.

Fe(TPP)(NODMA)(py)•toluene: Anal. data Calculated (Found): C,

77.64 (77.16); H, 5.19 (4.93); N, 9.90 (10.21).

Fe(5,10,15,20-tetraphenylporphyrinate)(nitrosobenzene)(1-meth-

ylimidazole) was prepared basically as described above except for the

addition of 1.2 equiv of PhNO (as the dimer) to Fe(TPP)(1-MeIm)₂.

Crystallization was from toluene/pentane with a yield of 56%. Fe(TPP)-

(PhNO)(1-MeIm): Anal. data Calculated (Found): C, 75.61 (75.69);

H, 4.58 (4.50); N, 11.42 (11.27).

Fe(2,3,7,8,12,13,17,18-octaethylporphyrinate)(nitrosobenzene)(1-

methylimidazole) was prepared basically as described above except

for the use of Fe(OEP)Cl.

Co(2,3,7,8,12,13,17,18-octaethylporphyrinate)(NO) and Co-

(5,10,15,20-tetraphenylporphyrinate)(NO) were both prepared with

use of standard literature procedures.⁴⁴[¹⁵NO] labeled OEP and TPP

and [N¹⁷O] labeled OEP derivatives were prepared from K¹⁵NO₂

(Cambridge Isotopes, Andover, MA) or by the H₂¹⁷O exchange of

KNO₂, to generate the appropriate isotopically enriched species.⁴⁵For

the OEP preparation, particular attention needs to be given to rigorous

O₂exclusion since any NO₂generated can attach to the porphyrin meso

carbons.⁴⁶All compounds had satisfactory microanalyses for C, H, and

N, and were further characterized by a strong IR band (in KBr)

corresponding to the N-O stretching frequency: Co(TPP)NO, 1697

cm^-1; Co(OEP)NO, 1675 cm^-1

.

([⁵⁷Fe]-myoglobin)(PhNO) and Fe(myoglobin)([²H₅]-PhNO). ([⁵⁷Fe]-

myoglobin)(PhNO) was prepared from met-[⁵⁷Fe] myoglobin, whose

synthesis was reported previously,⁴⁷by addition of PhNHOH, prepared

by the Zn reduction of PhNO₂.⁴⁸The phenylhydroxylamine is thought

to be oxidized by the ferric iron to PhNO,⁴¹which then binds to the

reduced Mb to give the desired product. The ([⁵⁷Fe]-myoglobin)(PhNO)

was purified over Sephadex G-25, concentrated on an Amicon

ultrafiltration apparatus, then frozen for Mo¨ssbauer spectroscopy. The

[²H₅]-nitrosobenzene adduct was prepared in basically the same manner,

except that [²H₅]-PhNO₂(Cambridge Isotope Laboratories) was used

to generate the required [phenyl-²H₅]-PhNHOH, and the product was

crystallized from concentrated (NH₄)₂SO₄(aq).

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Nuclear Magnetic Resonance Spectroscopy. Solution-state NMR

spectra were recorded by using a Varian (Palo Alto, CA) Unity Plus

500 MHz instrument. Solid-state ¹⁵N and ¹⁷O “magic-angle” sample-

spinning (MAS) NMR spectra were obtained by using 360, 500, and

600 MHz (¹H) “home-built” spectrometers, which consist of Oxford

Instruments (Oxford, UK) 8.45 T/3.5 in., 11.7 T/2.0 in. and 14.1 T/2.0

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distributed by the Molecular Science Computing Facility, Environmental

and Molecular Sciences Laboratory, which is part of the Pacific Northwest

Laboratory, P.O. Box 999, Richland, WA 99352, and is funded by the U.S.

Department of Energy under contract DE-AC06-76RLO 1830. Contact

David Feller, Karen Schuchardt, or Don Jones for further information.

(53) Ditchfield, R. Mol. Phys. 1974, 27, 789-807.

(43) Salzmann, R.; Ziegler, C. J.; Godbout, N.; McMahon, M.; Suslick,

K. S.; Oldfield, E. J. Am. Chem. Soc. 1998, 120, 11323-11334.

(44) Scheidt, W. R.; Hoard, J. L. J. Am. Chem. Soc. 1973, 95, 8281-

8288.

(45) Schenk, P. W. Handbook of PreparatiVe Inorganic Chemistry 1963,

1, 460-464.

(46) Fanning, J. C.; Mandel, F. S.; Gray, T. L. Tetrahedron 1979, 35,

1251-1255.

(54) Becke, A. D. Phys. ReV. A 1988, 38, 3098-3100. Perdew, J. P.;

Wang, Y. Phys. ReV. B 1992, 45, 13244-13249.

(47) Lee, H. C.; Gard, J. K.; Brown, T. L.; Oldfield, E. J. Am. Chem.

Soc. 1985, 107, 4087-4088.

(55) Becke, A. D. J. Chem. Phys. 1993, 98, 5648-5652.

(56) Lee, C.; Yang, W.; Parr, R. G. Phys. ReV. B 1988, 37, 785-789.

3832 J. Am. Chem. Soc., Vol. 121, No. 16, 1999

Table 1. Crystallographic Data Summary

Godbout et al.

Fe(TPP)(PhNO)(py)

Fe(TPP)(NODMA)(py) Fe(TPP)(PhNO)(1-MeIm) Fe(OEP)(PhNO)(1-MeIm)

Co(OEP)(NO)

formula

formula weight

color

crystal system

a (Å)

b (Å)

c (Å)

R (deg)

â (deg)

γ (deg)

V (Å³)

C₅₅H₃₈N₆OFe•0.5 toluene C₅₇H₄₃N₇OFe•toluene

C₅₄H₃₉N₇OFe

857.77

purple/black

triclinic

10.779(2)

17.829(3)

22.728(4)

103.220(4)

93.811(4)

97.839(4)

4190.6(13)

4

C₄₆H₅₅N₇OFe

777.82

black

C₃₆H₄₄N₅OCo

621.69

black

triclinic

10.4844(6)

10.6084(7)

14.0904(9)

79.7580(10)

89.3980(10)

80.2540(10)

1519.6(2)

2

900.83

989.97

black

purple

triclinic

11.904(11)

13.259(12)

16.452(2)

87.525(2)

70.489(2)

70.556(2)

2301.3(4)

2

orthorhombic

38.8181(13)

12.3091(4)

21.4098(7)

90

triclinic

13.67950(10)

13.9290(3)

23.1320(6)

72.4130(10)

73.5480(10)

84.640(2)

4029.43(14)

4

10239.6(11)

8

Z

D

_calc(g cm^-3

)

1.30

1.284

1.36

1.282

1.359

space group

radiation,

P1h

Iba2

P1h

Mo KRj, 0.71073 Å

Mo KRj, 0. 71073 Å

wavelength (Å)

µ (mm^-1

)

0.377

0.346

0.411

0.419

0.603

crystal size (mm)

temp (K)

0.08 × 0.19 × 0.22

0.10 × 0.24 × 0.36

0.08 × 0.24 × 0.30

0.40 × 0.16 × 0.14

0.16 × 0.19 × 0.20

198(2)

diffractometer

no. of data points

collected

Bruker SMART/CCD

13404

Bruker SMART/CCD

26755

Bruker SMART/CCD

17184

Bruker SMART/CCD

22373

Bruker SMART/CCD

7459

no. of data points:

3761

4388

3981

11388

4687

I > 2σ(I)

abs min/max

R₁^a(obsd data)

wR₂(a,b)^b

0.849/0.940

0.063

0.080 (0.010, 0.0)

0.939

0.966/0.898

0.062

0.146 (0.055, 0.0)

1.043

0.880/0.970

0.096

0.173 (0.03, 0.0)

1.178

0.338/0.291

0.059

0.135 (0.071, 0.0)

0.936

0.374/0.412

0.039

0.082 (0.034, 0.687)

1.033

GOF^c

^aR1 ) Σ(|F_o| - |F_c|)/Σ(|F_o|). ^bwR₂) [Σ[w(F_o- F_c²)²]/Σ[w(F_o)²]]^1/2where w ) 1/[σ²(F_o) + (a/P)²+ b/P] and P ) (F_o+ 2F_c²)/3. ^cGOF

2

) S ) [Σ[w(F_o- F_c²)²]/(n - p)], where n ) the number of reflections and p ) the total number of parameters refined.

2

B3LYP hybrid exchange-correlation functional. This approach has been

used by us previously to successfully predict metal ion electric field

gradients (EFGs, deduced experimentally from the ⁵⁷Fe Mo¨ssbauer

quadrupole splittings) in 14 organometallic and heme-model com-

pounds.⁴⁹The use of two different functionals may appear unusual,

but Bu¨hl has shown that (⁵⁷Fe) metal shieldings are very accurately

computed by using the B3LYP functional,⁵⁷which therefore led us to

use his approach to compute both metal shieldings as well as metal

quadrupole splittings^49,50in model heme systems.⁵⁰However, we have

previously noted that the B3LYP functional appears to overestimate

the paramagnetic shifts of ligand atoms somewhat, especially for highly

deshielded groups,⁵⁸so for accuracy and consistency, we carried out

separate calculations for ligand and metal properties.

calculations, we utilized a ruffled metallocycle, and used fixed bond

lengths and angles (since there was essentially no difference seen

between the first two sets of calculations). Selected bond lengths, bond

angles, and torsion angles (i.e., proximal base orientations) used in the

calculations are given for convenience in Table 2. We also evaluated

the charge densities F(r) and the molecular electrostatic potentials, Φ-

(r), using both BPW91 and B3LYP XC functionals. These G94 results

were displayed in Cerius2 (Molecular Simulations Inc., San Diego, CA).

We also carried out a series of semiempirical molecular mechanics

calculations (Spartan, Wavefunction, Inc., Irvine, CA; PM3 force field)

in which we deduced the barrier heights to CoNO and FeO₂rotation,

using a complete metalloporphyrin, together with all near-neighbor ethyl

or phenyl groups, as shown in Figure 11.

In most cases, the structures employed were those determined

crystallographically in this paper, but, as described previously, with

the bulky ring substituents replaced by hydrogen.^49,50In the case of

CoNO, we also carried out a partial geometry optimization of the CoNO

fragment (lanl2dz ECP and basis set/6-31G(d)/3-21G(d)/B3LYP) in

which d(Co-N), d(N-O), the four d(N_por-Co), Co-N-O, the four

N_por-Co-N_NO, and the four N_por-Co-N-O dihedral angles were

left unconstrained. For the heme-O₂calculations, we initially investi-

gated planar porphyrin macrocycles having a variety of geometries,^43,59-61

using the bond lengths and bond angles reported for oxypicketfence

porphyrin.⁶⁰We then carried out a more systematic investigation of

the ⁵⁷Fe electric field gradient using six porphyrins containing a planar

macrocycle,⁵⁹in which the orientations of the oxygen and imidazole

base were systematically varied in order to map out a wide range of

possible bonding situations, as discussed in detail in the text. In one

set of calculations, we utilized a planar metallocycle and the FeO₂bond

lengths and angles were fixed or unoptimized, while in a second set of

calculations we carried out a partial geometry optimization (d(Fe-O),

d(O-O), Fe-O-O) of the FeO₂unit at B3LYP. In a third set of

Calculations were performed on two four-processor Silicon Graphics/

Cray Research (Mountain View, CA) Origin-200 computers in this

laboratory, and on SGI Origin-2000 and Power Challenge systems at

the National Center for Supercomputing Applications, located in

Urbana, IL, using in the latter case up to 16 processors.

Results and Discussions

Structural Aspects. We show in Figures 1-5 the SHELXTL

(Bruker) structures of the five new “Fe-O₂” analogue model

systems: Fe(TPP)(PhNO)(py), Figure 1; Fe(TPP)(NODMA)-

(py), Figure 2; Fe(TPP)(PhNO)(1-MeIm), Figure 3; Fe(OEP)-

(PhNO)(1-MeIm), Figure 4; and Co(OEP)(NO), Figure 5.

Selected structural summaries for all five systems are given in

Table 3.

There are several points of interest about the structures of

these five systems. The most obvious differences occur in the

extent of the porphyrin distortions seen, as illustrated in Figures

6 and 7, where we show the deviations (in 10^-2Å) of the

indicated atoms from the porphyrin least-squares plane. In

particular, both of the pyridine-containing TPP complexes, Fe-

(TPP)(PhNO)(py) (Figures 1 and 7C) and Fe(TPP)(NODMA)-

(py) (Figures 2 and 6A), have major ring distortions, while the

two 1-methylimidazole adducts, Fe(TPP)(PhNO)(1-MeIm) (Fig-

(57) Bu¨hl, M. Chem. Phys. Lett. 1997, 267, 251-257.

(58) Havlin, R.; McMahon, M.; Srinivasan, R.; Le, H.; Oldfield, E. J.

Phys. Chem. 1997, 101, 8908-8913.

(59) Salzmann, R.; McMahon, M.; Godbout, N.; Sanders, L. K.;

Wodjelski, M.; Oldfield, E. J. Am. Chem. Soc. 1999, 121, 3818.

(60) Jameson, G. B.; Rodley, G. A.; Robinson, W. T.; Gagne, R. R.;

Reed, C. A.; Collman, J. P. Inorg. Chem. 1978, 17, 850-857.

(61) Kim, K.; Ibers, J. A. J. Am. Chem. Soc. 1991, 113, 6077-6081.

O₂-Heme Analogue Studies

J. Am. Chem. Soc., Vol. 121, No. 16, 1999 3833

Table 2. Selected Bond Lengths, Bond Angles, and Torsion Angles Used in Calculations^a

3^b

1^b

2^b

mol. 1

mol. 2

4^b

5^b

5^b,c

6^b

7^b

d(M-N_NO

)

1.818

1.252

2.113

1.468

123.9

113.0

177.4

41.0

1.859

1.251

2.096

1.436

119.7

112.3

173.7

30.6

1.803

1.258

2.094

1.454

123.8

110.1

175.4

5.2

1.808

1.270

2.091

1.448

122.7

111.7

177.8

12.2

1.800

1.254

2.029

1.444

124.8

111.3

178.7

31.8

1.845

1.152

1.840

1.167

1.833

1.15

1.854

1.195

d(N-O)

d(M-N_Im)

d(N_NO-C_Ph)

θ(M-N-O)

θ(O-N-C_Ph)

θ(N_Im-M-N_NO

123.4

119.5

135.2

119.6

)

d

φ(N_Por-M-N_base-C_base

φ(O_NO-N_NO-N_base-C_base

)

e

)

8.5

97.2

30.5

66.2

20.2

^aBond lengths are in Å, bond angles and torsion angles are in degrees. ^bThe structures are as follows: 1, Fe(TPP)(PhNO)(py); 2,

Fe(TPP)(NODMA)(py); 3, Fe(OEP)(PhNO)(1-MeIm); 4, Fe(TPP)(PhNO)(1-MeIm); 5, Co(OEP)(NO); 6, Co(TPP)(NO); 7, Co(Tp-OMePP)(NO).

From ref 17. ^cG94/DFT B3LYP partial geometry optimization (see Text for details). ^dThis angle gives the orientation of the base (imidazole or

pyridine) with respect to the N-Fe-N angle in the porphyrin ring. ^eThis angle gives the orientation of the NO ligand with respect to the base

(imidazole or pyridine).

Figure 1. X-ray structure of Fe(5,10,15,20-tetraphenylporphyrinate)-

(nitrosobenzene)(pyridine). SHELXTL (Bruker, 1998) representation

showing 35% probability ellipsoids for non-H atoms. H atoms were

omitted for clarity.

Figure 3. X-ray structure of Fe(5,10,15,20-tetraphenylporphyrinate)-

(nitrosobenzene)(1-methylimidazole) showing both molecules (A, B)

in the unit cell. SHELXTL (Bruker, 1998) representation showing 35%

probability ellipsoids for non-H atoms. H atoms were omitted for clarity.

Nitrosobenzene disorder is present in B.

Fortunately, inspection of the structures of the compounds we

have synthesized and characterized here, together with our

Figure 2. X-ray structure of Fe(5,10,15,20-tetraphenylporphyrinate)-

(4-nitroso-N,N-dimethylaniline)(pyridine). SHELXTL (Bruker, 1998)

results on the OEP/TPP py/1-MeIm CO adducts, Fe(TPP)-

(iPrNC)(1-MeIm)⁵⁹and Fe(TPP)(CCl₂),⁶²have led to a simple

logic-based model for predicting such distortions, as discussed

in detail elsewhere.⁵⁹Recapitulating briefly: for the 16 com-

pounds considered,^43,59,62there are six with highly distorted

representation showing 35% probability ellipsoids for non-H atoms. H

atoms were omitted for clarity.

ures 3 and 6B,C) and Fe(OEP)(PhNO)(1-MeIm) (Figures 4 and

7A,B), as well as the 5-coordinate OEP species, Co(OEP)(NO)

(Figures 5 and 7D), are close to planar. These results are

intriguing since they are the opposite of those seen with the

M(TPP)(CO)(base) systems discussed earlier,⁴³where we found

that it was the 1-methylimidazole adducts of M(TPP)(CO) which

were ruffled, while the pyridine complexes were planar.⁴³

(62) We have determined the high-resolution structure of the anhydrous

species Fe(TPP)(CCl₂); Ziegler, C.; Salzmann, R.; Suslick, K.; Oldfield,

E. unpublished results. The structure of an apparent hydrate was reported

by: Mansuy, D.; Lange, M.; Chottard, J. C.; Bartoli, J. F.; Chevrier, B.;

Weiss, R. Angew. Chem., Int. Ed. Engl. 1978, 17, 781-782.

3834 J. Am. Chem. Soc., Vol. 121, No. 16, 1999

Godbout et al.

The structures shown in Figures 6 and 7 also indicate that

the axial bases in all TPP systems bind with the plane of the

axial base oriented at 45° to the N1-N3 axis, i.e., along d_xy.

All four six-coordinate complexes have the metal close to the

least-squares plane of the porphyrin, while as expected¹⁶the

Co in Co(OEP)(NO) is above the plane, by about 0.15 Å (Figure

7D). The PhNO and NODMA ligands are also generally oriented

with the Fe-N-O plane at 45° to the N1-N3 axes, parallel to

the 1-MeIm plane. The same orientation is also seen with the

CoNO plane, which unlike previous studies with Co(TPP)NO

is not subject to crystallographic or rotational disorder in the

OEP case, as discussed below.

Also of interest is the observation that the phenyl rings exhibit

a wide range of torsion angles between the nitroso group and

the phenyl ring, even though the Fe-N-O angle is relatively

constant (at ∼123 ( 3°). In most cases, Table 3, the torsion

angle is ∼60-80°, that is, the nitro group is almost perpen-

dicular to the benzene ring, destroying overlap of the two

π-systems, presumably due to an otherwise unfavorable hard-

core steric repulsive interaction with the π-cloud of the

porphyrin ring. However, in the case of the 4-nitroso-N,N-

dimethylaniline, there appears to be a tendency to form the

quinonoid-like structure, 2:

Figure 4. X-ray structure of Fe(2,3,7,8,12,13,17,18-octaethylporphy-

rinate)(nitrosobenzene)(1-methylimidazole) showing both molecules (A,

B) in the unit cell. SHELXTL (Bruker, 1998) representation showing

35% probability ellipsoids for non-H atoms. H atoms were omitted for

clarity.

with a C-C-N-O torsion of ∼38° being found for Fe(TPP)-

(NODMA)(py). This correlates with an increased tilt of the

NODMA group away from the porphyrin plane, with the angle

between the phenyl and porphyrin planes increasing from ∼37°

to 54°, Table 3. The quinonoid-like structure is also reflected

in a slightly shorter C-N bond length (1.457 f 1.437 Å). In

the case of Fe(TPP)(PhNO)(1-MeIm), we were unable to obtain

a good refinement of one of the two molecules in the unit cell,

due to PhNO disorder, so the geometric parameters for this

molecule are not included in the above comparisons.

We next briefly compare the main metal-ligand geometric

features of these Fe-O₂analogue systems with corresponding

values found in oxy-heme proteins and the FeO₂model,

oxypicket fence porphyrin.⁶⁰For comparative purposes, we show

in Table 4 selected structural parameters for oxy-heme proteins

and the model system. As can be seen from Tables 3 and 4,

there is generally good agreement in M-A-B bond angles

between the Fe•RNO and CoNO model systems and the high-

resolution X-ray structure of MbO₂, with M-A-B bond angles

of ∼120 ( 3° being found in all cases. In the case of the oxy-

heme model, the bond angle is slightly larger, but considerable

crystallographic disorder was found in this structure, so that

these values are likely less accurate. In oxyhemoglobin, the

apparent bond angle is 153-159°². While large distortions from

the ∼120° observed with the well-characterized systems might

be permitted in proteins due to various steric and/or hydrogen

bonding/electrostatic field effects, the possibility also exists that

the larger protein geometries are more difficult to determine

accurately. In the case of CoO₂complexes,^20,21it is possible

that the electronic and geometric structures are indeed different,

and do not reflect the situation found with oxy-heme proteins.

Figure 5. X-ray structure of Co(2,3,7,8,12,13,17,18-octaethylporphy-

rinate)(NO). SHELXTL (Bruker, 1998) representation showing 35%

probability ellipsoids for non-H atoms. H atoms were omitted for clarity.

porphyrin rings. These distortions correlate with the presence

of porphyrin ring phenyl groups and the presence of one (and

only one) of the following large (“distorting”) ligands: 1-MeIm,

PhNO, NODMA, or CCl₂. Thus, Fe(TPP)(PhNO)(py), Figure

1, and Fe(TPP)(NODMA)(py), Figure 2, are distorted, while

the other three systems are not, Figures 3-5. As noted before,

the presence of two of these “bulky” axial ligands results is a

net overall zero distortion.⁵⁹Whether these effects are intra- or

intermolecular steric or purely electronic is not certain, but the

very large distortions seen with the very bulky NODMA tend

to favor steric interactions.

O₂-Heme Analogue Studies

J. Am. Chem. Soc., Vol. 121, No. 16, 1999 3835

Table 3. Structural Summary for Fe Porphyrins^a

Fe(TPP)(PhNO)(1-MeIm)

Fe(TPP)(PhNO)(py) Fe(TPP)(NODMA)(py) mol.1

mol. 2^b

Fe(OEP)(PhNO)(1-MeIm)

mol. 1

mol. 2

Co(OEP)(NO)

M-N(1)

1.998(3)

1.990(3)

1.984(3)

1.988(3)

1.819(3)

2.106(3)

1.249(4)

1.472(4)

88.93(13)

178.08(13)

90.35(12)

90.87(12)

175.14(13)

89.68(12)

92.41(13)

93.82(13)

89.50(13)

91.02(13)

177.24(14)

123.9(3)

1.972(5)

2.006(4)

1.976(5)

1.970(4)

1.859(6)

2.095(5)

1.252(6)

1.437(8)

89.2(2)

178.1(2)

90.1(2)

90.3(2)

174.9(2)

90.3(2)

91.8(2)

98.2(2)

90.1(2)

86.9(2)

173.7(2)

119.8(5)

127.0(4)

112.3(6)

1.958(9) 1.977(8)

2.015(8) 2.011(8)

1.973(9) 2.005(8)

1.996(8) 2.022(8)

1.800(8) 1.802(3)/1.807(3)/1.812(3) 1.809(4)

2.029(9) 2.030(9) 2.092(4)

1.254(8) 1.267(3)/1.263(3)/1.260(3) 1.269(5)

1.444(9) 1.453(2)/1.453(2)/1.452(2) 1.448(6)

87.8(3) 90.0(4)

176.7(4) 177.6(4)

92.2(4) 89.3(4)

91.8(3) 89.0(4)

178.0(4) 178.7(4)

2.012(4)

1.999(4)

1.995(4)

2.003(4)

1.987(4)

1.995(4)

2.003(4)

2.016(4)

1.802(4)

2.094(4)

1.258(4)

1.454(6)

90.1(2)

177.6(2)

90.0(2)

89.9(2)

176.6(2)

89.8(2)

91.3(2)

87.7(2)

91.1(2)

95.6(2)

175.4(2)

123.8(3)

110.1(3)

1.996(2)

1.980(2)

1.976(2)

1.989(2)

1.844(2)

M-N(2)

M-N(3)

M-N(4)

M-N*

M-N(base)

N*-O

1.152(3)

N*-C

N(1)-M-N(2)

N(1)-M-N(3)

N(1)-M-N(4)

N(2)-M-N(3)

N(2)-M-N(4)

N(3)-M-N(4)

N*-M-N(1)

N*-M-N(2)

N*-M-N(3)

N*-M-N(4)

N*-M-N(base)

M-N*-O

90.6(2)

176.7(2)

89.3(2)

89.9(2)

177.3(2)

90.0(2)

91.1(2)

93.3(2)

91.6(2)

89.47(9)

170.98(8)

89.35(8)

90.14(8)

170.06(8)

89.48(8)

95.45(9)

94.60(9)

93.57(9)

95.34(9)

88.1(4) 91.6(4)

91.6(4) 96.5(5)/87.8(6)/89.1(7)

91.2(3) 92.1(3)/91.3(5)/89.4(8)

91.6(4) 85.7(5)/94.3(6)/93.1(7)

90.8(3) 89.1(4)/89.9(5)/91.7(8)

178.7(3) 175.4(5)/175.8(6)/176.5(9) 177.8(2)

124.8(7) 122.8(3)/123.7(3)/124.4(3) 122.8(3)

123.8(6) 123.8(3)/124.2(3)/124.3(3) 122.8(3)

111.2(8) 109.9(3)/110.6(3)/110.8(3) 111.7(4)

123.4(2)

M-N*-C

O-N*-C

torsion amplitude of

TPP-phenyl rings

angle between N(base)

ring and the porphyrin

plane

angle between (N-phenyl) 37°

plane and the

porphyrin plane

122.9(3)

113.2(3)

17°, 23°, 25°, 28°

12°(20°), 17°, 18°, 18° 21°, 13°, 4°, 2°, 13°, 11°

8°, 18°

100°

87°

54°

41°

83°

37°

81°

84°

90°

39°

66°

89°

39°

59°

35°/41°/35°

42°/21°/39°

angle between (N-phenyl) 24°

plane and the N-O axis

^aAbbreviations used: M, metal center Fe or Co as appropriate; N*, nitrosyl nitrogen of PhNO or NODMA. ^bMultiple entries exist for values

associated with the disordered PhNO group.

Figure 6. Schematic illustration showing atom displacements from the porphyrin least-squares plane (in units of 10^-2Å) for (A) Fe(TPP)(NODMA)-

(py) and (B, C) Fe(TPP)(PhNO)(1-MeIm).

In addition, of course, basic differences in atomic radii,

coordination number, and porphyrin substitutions can be

expected to lead to small but real differences in observed bond

lengths. It is of considerable interest, however, that the observed

basic geometry of a ∼120° ( 3° M-A-B bond angle is

preserved between all three diamagnetic model systems, given

the wide variety of metal and ligand types involved.

Given these similarities, it is therefore of interest to next begin

to investigate the spectroscopic properties of each of the three

different types of model system, using quantum chemical

methods to help interpret the results. Such an approach has been

used previously by us to investigate the NMR, IR, and

Mo¨ssbauer spectra of CO bound to metalloporphyrins and

metalloproteins, where we concluded that CO binds in a close-

to-linear and untilted fashion in both proteins and model

systems,^49,50,63and that electrostatic fields control the differences

in IR frequencies seen experimentally with different proteins.

Here, we investigate first the Fe•RNO and CoNO metallocycles,

to see to what extent the calculations reproduce the experimental

spectroscopic observables in systems having relatively well-

defined structures. Then, we consider the FeO₂system in both

metalloporphyrins and in metalloproteins, focusing in particular

on the use of ⁵⁷Fe Mo¨ssbauer spectroscopy as a probe of local

structure and bonding. And finally, we use the results of our

DFT calculations on the PhNO, O₂, and CO-containing metal-

loporphyrins to deduce the charge densities, F(r), and the

molecular electrostatic potentials, Φ(r), which give useful

insights into possible hydrogen bonding interactions in proteins.

Solid-State NMR and Quantum Chemical Study of

Fe•RNO and CoNO Metalloporphyrins. We have obtained

the ¹⁵N and ¹⁷O solid-state (and ¹⁷O solution state) MAS NMR

spectra of several ¹⁵N- and ¹⁷O-labeled Fe-O₂analogue

metalloporphyrins, including Fe(TPP)([¹⁵N]-PhNO)(py), Fe-

(63) McMahon, M. T.; deDios, A. C.; Godbout, N.; Salzmann, R.; Laws,

D. D.; Le, H.; Havlin, R. H.; Oldfield, E. J. Am. Chem. Soc. 1998, 120,

4784-4797.

3836 J. Am. Chem. Soc., Vol. 121, No. 16, 1999

Godbout et al.

Table 4. Selected Structural Parameters of Oxy-Heme Proteins and Model Systems^a

φ(N_Por-Fe-N_Im-C_Im)

Im orient. wrt N-Fe-N

24.8

φ(O-O-N_Im-C_Im)

O₂orient. wrt Im

68.4

PDB file no.^b

d(O-O)

d(Fe-O)

d(Fe-N_Im)

θ(Fe-O-O)

θ(O-O-Fe-N_Por)

1HHO

(â subunit)

1HHO

1.24(2)

1.87(13)

2.07(9)

159.0(12)

40

1.22(1)

1.66(8)

1.94(9)

153.0(7)

19

12.0

8.2

(R subunit)

1MBO

1.22(6)

1.22

1.29

1.83(6)

1.94

1.93

2.07(6)

2.19

2.30

115(5)

118.2

120.3

129(2)

20

41

7

2.4

1.3

2.6

23.2

43.6

6.7

2MGM

2SPN

picket^c

1.17(4)

1.75(2)

2.07(2)

48.0

20.5

117.1

^aBond lengths are in Å, bond angles and dihedral angles are in deg. Available error values are shown in parentheses. ^bThe structures given

typically refer to Brookhaven Protein Data Bank file numbers: 1HHO: B. Shaanan, hemoglobin A (oxy), human (Homo sapiens), 2.1 Å resolution

(1984); 1MBO: S. E. V. Phillips, myoglobin (oxy, pH 8.4), sperm whale (Physeter catodon), 1.6 Å resolution (1983); 2MGM: M. L. Quillin, R.

M. Arduini, G. N. Phillips, Jr., myoglobin (oxy) mutant with initiator Met and Asp 12 replaced by Asn (Met, D122N), synthetic gene for sperm

whale myoglobin expressed in Escherichia coli, 1.9 Å resolution (1994); 2SPN, M. L. Quillin, R. M. Arduini, G. N. Phillips, Jr., myoglobin (oxy)

mutant with initiator Met, Leu 29 replaced by Phe, and Asp 122 replaced by Asn (Met, L29F, D122N), sperm whale myoglobin expressed in E.

coli, 1.7 Å resolution (1994). ^cReference 60.

Figure 7. Schematic illustration showing atom displacements from

the porphyrin least-squares plane (in units of 10^-2Å) for: (A, B) Fe-

(OEP)(PhNO)(1-MeIm), (C) Fe(TPP)(PhNO)(py) and D, Co(OEP)NO.

(TPP)([nitroso-¹⁵N]-NODMA)(py), Fe(TPP)([¹⁵N]-PhNO)(1-

MeIm), Co(OEP)(¹⁵NO), Co(OEP)(N¹⁷O), and Co(TPP)(N¹⁷O).

These compounds all have quite well characterized structures

and offer the opportunity to deduce shielding information about

both the directly attached nitroso nitrogens (in Co and Fe

systems) and the terminal oxygen atom in the Co•NO system.

We report first the solid-state NMR spectra of these systems,

and show that the DFT calculations give relatively good

shielding predictions. We then consider in the next section our

⁵⁷Fe Mo¨ssbauer results, and the corresponding EFG (Mo¨ssbauer

Figure 8. Representative NMR spectra of metalloporphyrins investi-

gated. (A) 8.45 T ¹⁵N MAS NMR spectrum of Fe(TPP)(Ph¹⁵NO)(py)

∆E_Q) calculations. Since the shielding tensors are response

properties, they are often more difficult to evaluate than are

electric field gradient tensors, which depend primarily on the

description of the ground rather than ground-and-excited states

at 298 K; (B) 11.7 T ¹⁷O MAS NMR spectrum of Co(OEP)(N¹⁷O) at

373 K; and (C) 11.7 T ¹⁷O MAS NMR spectrum of Co(TPP)(N¹⁷O) at

298 K.

potential,^37-39which can then begin to be used to describe the

details of, e.g., hydrogen bonding, in these and related systems.

We show in Figure 8 typical ¹⁵N and ¹⁷O NMR spectra: the

¹⁵N MAS NMR spectrum of Fe(TPP)([¹⁵N]-PhNO)(py) (Figure

of the molecule. Success in evaluating both shift and EFG

tensors (see below) gives increased confidence in the reliability

of the theoretical methods to predict these observables, as well

as others, such as the charge density and the electrostatic

O₂-Heme Analogue Studies

J. Am. Chem. Soc., Vol. 121, No. 16, 1999 3837

Table 5. Comparison between Experimental and Computed Chemical Shifts and Shift Tensor Elements for Fe-O₂Analogue

Metalloporphyrins^a

system

δ_i(ppm)

δ

₁₁(ppm)

δ₂₂(ppm)

δ

₃₃(ppm)

|δ₃₃- δ₁₁| (ppm)

Fe: Fe(TPP)(Ph¹⁵NO)(1-MeIm)

Fe(P)(Ph¹⁵NO)(1-MeIm)

Fe(TPP)(Ph¹⁵NO)(py)

expt

calc

expt

calc

expt

calc

expt

calc

605

628

607

644

607

603

593

643

623

1178

1220

1265

1256

1281

1170

1247

1246

1202

2070

1901

2674

2288

1959

1868

2747

1670

464

486

469

490

456

472

448

500

486

829

561

959

1247

1376

1475

779

1505

173

179

87

186

82

168

84

182

566

263

656

801

987

993

170

1200

1005

1041

1178

1070

1199

1002

1163

1064

1020

1504

1638

2018

1487

972

Fe(P)(Ph¹⁵NO)(py)

Fe(TPP)(¹⁵NO-NODMA)(py)

Fe(P)(¹⁵NO-NODMA)(py)

Fe(OEP)(Ph¹⁵NO)(1-MeIm)

Fe(P)(Ph¹⁵NO)(1-MeIm)

site 1

site 2

Co: Co(OEP)(¹⁵NO)

Co(P)(¹⁵NO)

expt

calc

expt

1155

908

Co(OEP)(N¹⁷O)

298 K

343 K

373 K

393 K

1430

1445

1441

1445

1232

1458

875

2577

470

Co(P)(N¹⁷O)

calc

expt

Co(TPP)(N¹⁷O)

298 K

^aThe theoretical chemical shieldings (σ) were calculated as described in the text and were then converted to theoretical chemical shifts (“calc”

in the Table) by using the following absolute shieldings: ¹⁵N, δ(¹⁵N, NH₃) ppm ) 244.6 - σ (ref 74) and for ¹⁷O, δ(¹⁷O, H₂O) ppm ) 306.7 -

σ (ref 75).

8A), the ¹⁷O MAS NMR spectrum of Co(OEP)(N¹⁷O) (Figure

8B), and the ¹⁷O MAS NMR spectrum of Co(TPP)(N¹⁷O)

(Figure 8C). From such spectra, we obtained the principal

components of the ¹⁵N and ¹⁷O chemical shift tensors, and/or

the isotropic chemical shifts, shown in Table 5. We then

evaluated the individual components of the ¹⁵N and ¹⁷O shielding

tensors and the corresponding isotropic shifts. These results are

also presented in Table 5, and a graphical comparison between

the experimental and theoretical shift tensor results is given in

Figure 9. There is clearly a good overall correlation between

theory and experiment, although the errors in absolute shielding

are rather more pronounced for these axial ligands (NO and

RNO) than are seen with the CO and RNC systems,^43,59

presumably due to the small n f π* separation in the NO group,

and the large overall paramagnetic shifts. Typical shielding

tensor orientations for the Fe•RNO and Co•NO systems are

shown in Figure 10. As can be seen from Figures 10A and 10B,

the orientations of the two tensors are in general similar with

the most deshielded element, σ₁₁, oriented along or close to the

NdO bond axis, while the most shielded element, σ₃₃, is

oriented perpendicular to the M-N-O plane. Having σ₁₁(δ₁₁)

oriented close to the N-O bond axis and σ₃₃(δ₃₃) more or less

perpendicular to the R-N-O (or M-N-O) plane is the

orientation seen in many other nitrosocompounds, as discussed

previously.⁶⁴

Co(TPP)(¹⁵NO), in which they observed fast axial motion of

the Co-NO fragment at 298 K, but an essentially rigid lattice

tensor at 200 K, with a phase transition at 206.7 K.⁶⁵

In the case of Co(OEP)(NO), the ¹⁷O NMR results indicate

a major increase in the barrier to axial rotation from that seen

in Co(TPP)(NO). There is essentially free rotation at room

temperature with Co(TPP)(NO), but hindered rotation or es-

sentially no motion at room temperature in the case of Co(OEP)-

(NO), as judged by ¹⁷O NMR, and via crystallography. These

results are linked to strong steric interactions between the bent

NO group and the ethyl groups in OEP, while in the TPP

analogue the phenyl groups act more as lattice spacers. This

effect can be well reproduced by carrying out a molecular

mechanics (PM3; Spartan) study of the rotational barrier using

single Co(TPP)(NO) or Co(OEP)(NO) molecules together with

additional local ethyl or phenyl groups from neighboring

molecules, as shown schematically in Figures 11A,B. In the

case of TPP, Figure 11, the barrier to rotation is very small

(1.6 kcal) and has 4-fold symmetry, making hopping from one

state to another quite facile at room temperature (and thereby

making X-ray refinements very difficult). In contrast, in the case

of Co(OEP)(NO), there are major steric interactions with

neighboring ethyl groups, with a barrier height of ∼18 kcal

mol^-1, Figure 11C, although of course in reality this will be

reduced due to ethyl librations. The presence of such fast axial

rotation enables an estimate of the Co-N-O bond angle from

the NMR data, as reported previously with the Fe-O-O

fragment in oxypicketfence porphyrin,¹¹and in Co(TPP)(NO).⁶⁵

Using a value of -388 ppm (¹/₂(1670 + 1505) - 1200) as an

estimate of the motionally averaged tensor span from the

Now, to fully describe the observed spectra, we must consider

the effects that molecular motion might have on the spectro-

scopic observablessa topic of great debate for many years in

the context of Fe-O₂interactions in heme proteins and model

systems, as described below. We find a major temperature

dependence of the ¹⁷O CSA in Co(OEP)(NO), Table 5. At 23

°C, the tensor is close to axially symmetric, with an overall

span of ∼2000 ppm, while at 120 °C (shortly before the onset

of NO loss) the tensor has narrowed considerably, to a span of

∼900 ppm, due most likely to fast axial rotation of the Co-

NO group, as found also with oxypicket fence porphyrin.^11,14

The tensor is, however, still considerably broader than the ∼470

ppm observed with Co(TPP)(NO) at 23 °C, Table 5, possibly

due to not attaining its fast motion limit. This is consistent with

the previous ¹⁵N NMR observations of Groombridge et al.⁶⁵on

1

shielding calculations, and ∆δ ) 2747 - /₂(779 - 170) )

2442 ppm as the theoretical rigid lattice limit, we obtain from

∆hδ ) ¹/₂(3 cos²θ - 1)∆δ (ref 11) a value for θ, the Co-N-O

bond angle, of about 118.5°. This value is almost identical to

the 119.5° we obtain by using a DFT quantum chemical

geometry optimization (Table 2), and the recent value of 119.6°

obtained crystallographically by Richter-Addo for Co(T(p-

OMe)PP)(NO).¹⁷All of these values are somewhat smaller than

the ∼127-135° range reported previously, for related com-

pounds.^16,65In any event, a Co-N-O bond angle of close to

120° now seems to be most general, since neither of the most

(64) Salzmann, R.; Wojdelski, M.; McMahon, M.; Havlin, R. H.;

Oldfield, E. J. Am. Chem. Soc. 1998, 120, 1349-1356.

(65) Groombridge, C. J.; Larkworthy, L. F.; Mason, J. Inorg. Chem. 1993,

32, 379-380.

3838 J. Am. Chem. Soc., Vol. 121, No. 16, 1999

Godbout et al.

applied in the past to analyze Mo¨ssbauer spectra, only more

recently have the results of the calculations become “robust”s

that is they give the correct results for a broad range of bonding

situations without adjustable parameters,^49,71in a reliable way.

Indeed, as clearly shown by Case and co-workers, the results

of early (e.g., XR, EHT, PPP) calculations gave all sorts of EFG

and orbital energy predictionssa situation that for most systems

of interest has now been rectified by use of modern DFT

methods.^49,71

Experimentally, ⁵⁷Fe Mo¨ssbauer spectroscopy is a potentially

powerful probe of the ⁵⁷Fe center, and thereby of Fe-O₂

structure and bonding, and several studies on model systems,

such as oxypicket fence porphyrins, as well as on myoglobin

and hemoglobin, have been reported.^13-15The Mo¨ssbauer results

on a single crystal of oxymyoglobin indicate that the principal

component of the electric field gradient tensor at Fe, V_zz, defined

such that

|V_zz| g |V_yy| g |V_xx| and V_xx+ V_yy+ V_zz) 0

is oriented in the plane of the porphyrin,¹³at least at low

temperatures. In addition, the magnitude of the observed

Mo¨ssbauer quadrupole splitting, ∆E_Q, defined as:

1/2

1

2

η²

3

∆E_Q) eQV_zz1 +

(

)

where Q is the quadrupole moment of the I* ) ³/₂excited state

and η is the asymmetry parameter of the electric field gradient:

V_xx- V_yy

η )

V_zz

is temperature dependent. In oxypicket fence porphyrin, ∆E_Q

varies from about -2.1 mm s^-1at 4.2 K¹⁴to about 1.3 mm s^-1

(unsigned) at room temperature, while in oxymyoglobin, ∆E_Q

is about -2.3 mm s^-1at 4.2 K and increases to ∼|1.6| mm s^-1

at 260 K.¹⁵A variety of models have been used to explain these

results, as discussed for example by Debrunner.¹⁵In one model,

the MbO₂data have been rationalized in terms of a harmonic

model of bond angle and bond distance oscillations,^15,72while

Spartalian et al.¹⁴have proposed a 2-site jump model for

oxypicket fence porphyrin, in which there are two substates,

having QV_iiof -4.18, 1.60, 2.57 and -1.78, 0.18, and 1.60

mm s^{-1 14}

while in a third model, iterative extended Hu¨ckel

,

Figure 9. Graphs showing correlations between experimental and

theoretical chemical shifts for ¹⁵N, ¹⁷O-labeled metalloporphyrins: (A)

R¹⁵NO, slope ) 0.910, R²) 0.993; (B) Co¹⁵NO, slope ) 1.09, R²)

1.00; and (C) CoN¹⁷O, slope ) 1.233, R²) 0.992. From Table 5.

theory has been used to support a rotational diffusion model.¹²

Here, we first investigate the Fe•RNO adducts via Mo¨ssbauer

spectroscopy and quantum chemistry, to test the ability of DFT

methods to predict the observable ∆E_Qvalues in these Fe-O₂

analogue systems whose structures are accurately known. Then,

we apply the same quantum chemical methods to the problem

of the ⁵⁷Fe Mo¨ssbauer spectroscopy of oxypicket fence por-

recent crystal structures were plagued by crystallographic

disorder. The fact that the CoNO bond angle is also found to

be essentially 120° by using quantum chemical methods (Table

2) gives additional confidence in the use of such methods in

the structure refinement of FeO₂and Fe-O₂analogue metal-

loporphyrins and metalloproteins.

The Iron-57 Mo1ssbauer Problem. We next investigate the

question of the ⁵⁷Fe Mo¨ssbauer spectroscopy of FeO₂and Fe-

O₂analogue metalloporphyrins and metalloproteins. Here, there

have been major questions as to the geometry of Fe-O₂bonding

(bent versus symmetric, the degree of bend of the Fe-O-O

unit), as well as many discussions of electronic structure (see,

e.g., refs 66-70). While quantum chemical methods have been

(67) Olafson, B. D.; Goddard, W. A., III Proc. Natl. Acad. Sci. U.S.A.

1977, 74, 1315-1319.

(68) Case, D. A.; Huynh, B. H.; Karplus, M. J. Am. Chem. Soc. 1979,

101, 4433-4453.

(69) Newton, J. E.; Hall, M. B. Inorg. Chem. 1984, 23, 4627-4632.

(70) Dedieu, A.; Rohmer, M.-M.; Veillard, H.; Veillard, A. NouV. J.

Chim. 1979, 3, 653-667.

(71) Grodzicki, M.; Flint, H.; Winkler, H.; Walker, F. A.; Trautwein,

A. X. J. Phys. Chem. 1997, 101, 4202-4207.

(72) Wise, W. W.; Debrunner, P. G. Bull. Am. Phys. Soc. 1984, 29, 365.

(73) W. Arnold, N. Godbout, and E. Oldfield, unpublished results.

(74) Jameson, C. J.; Jameson, A. K.; Oppusunggu, D.; Willie, S.; Burrell,

M.; Mason, J. J. Chem. Phys. 1981, 74, 81-88.

(66) Pauling, L. Nature 1964, 203, 182-183. Weiss, J. J. Nature 1964,

203, 183.

(75) Wasylishen, R. E.; Mooibroek, S.; Macdonald, J. B. J. Chem. Phys.

1984, 81, 1057-1059.

O₂-Heme Analogue Studies

J. Am. Chem. Soc., Vol. 121, No. 16, 1999 3839

Figure 10. Orientations of the principal components of the ¹⁵N shielding tensors for two metalloporphyrins: (A) Fe(TPP)(Ph¹⁵NO)(1-MeIm) and

(B) Co(OEP)(¹⁵NO).

Figure 11. Barriers to NO ligand rotation in CoNO porphyrins. (A, B) Co(TPP)(NO) and Co(OEP)(NO) molecules and lattice fragments used to

evaluate the barriers to rotation and (C) rotational barriers. Co(TPP)(NO) is shown as a solid line; Co(OEP)(NO) is shown as a dashed line. The

barriers were evaluated by using molecular mechanics (Spartan 4.1) allowing only Co-NO rotation.

phyrin and oxymyoglobin, testing some of the ideas put forth

previously, which then leads to a final investigation of electro-

statics in these and related systems.

We show in Figure 12A a representative ⁵⁷Fe Mo¨ssbauer

spectrum of Fe(TPP)(PhNO)(1-MeIm) at 298 K, together with

a spectral simulation, in addition to ⁵⁷Fe Mo¨ssbauer spectra of

3840 J. Am. Chem. Soc., Vol. 121, No. 16, 1999

Godbout et al.

a cytochrome c model,⁴⁹as have Walker et al. for the PMe₃

and TMP adducts noted above.⁷¹We therefore next consider

the FeO₂systems, oxypicket fence porphyrin and oxymyoglobin.

In these O₂containing systems there is more uncertainty in the

geometries to be used, since oxypicket fence porphyrin has

crystallographic disorder,⁶⁰and in proteins the FeO₂geometries

and porphyrin ruffles are less certain, although disorder is clearly

less of a problem than with the picket fence system.⁴²

We first carried out an exploratory series of calculations using

both a planar (C₂-cap) and a less planar (picket fence) porphyrin

geometry, using d(Fe-O) ) 1.75 Å, d(O-O) ) 1.24 Å, and

Fe-O-O ) 131°,⁶⁰together with several basis sets, func-

tionals, and FeO₂/1-MeIm/porphyrin geometries. The results of

these initial calculations are shown in Table 6 and indicate ∆E_Q

values ranging from -1.7 to -2.4 mm s^-1, V_zzis in the

porphyrin plane (Figure 13), and η is small (∼0.1-0.3). This

is all in good accord with experiment, and most importantly,

the calculations are essentially identical in form to previous

successful EFG (∆E_Q) calculations for other (CO, RNC,

cytochrome c, RNO, PMe₃) metalloporphyrin systems, indicat-

ing reliability. The results shown in Table 6 also indicate that

there is a significant effect of porphyrin geometry on the

computed ∆E_Qvalues, although the effects of rotating the O₂

ligand by 90°, 180°, and 270° are quite small (only 0.07 mm

s^-1). On the basis of these results, there seems to be little support

for the existence of a second site having ∆E_Q∼ -0.9 mm s^-1

as being the origin of the temperature-dependent Mo¨ssbauer

results.¹⁴We then extended our calculations to cover the six

major imidazole/FeO₂/porphyrin relative orientations shown

schematically in Figure 14, since these relative orientations

might be significant in causing EFG changes at Fe. We also

carried out geometry optimizations of the FeO₂fragment (d(Fe-

O), d(O-O), Fe-O-O). The optimized structural parameters

are shown in Table 7, together with their relative energies. The

results of the unoptimized (d(Fe-O) ) 1.75 Å, d(O-O) ) 1.24

Å, Fe-O-O ) 131°) and optimized EFG calculations are

shown in Table 8, for an essentially undistorted metallopor-

phyrin. These results do show a sizable range in ∆E_Q(from

-1.53 to -2.62 mm s^-1), but all values are far larger than the

tensor required in the early model¹⁴to describe the temperature

dependence of the ⁵⁷Fe Mo¨ssbauer ∆E_Qin picket fence

porphyrin.¹⁴Interestingly, there is only a small change in ∆E_Q

on geometry optimization, Table 8. We then evaluated the ∆E_Q

values for six ruffled porphyrins, Table 8, with results in general

accord with those obtained using the planar model, except that

Figure 12. Representative ⁵⁷Fe Mo¨ssbauer spectra: (A) Fe(TPP)-

(NODMA)(py), at 298 K; (B) [⁵⁷Fe] Mb•PhNO at 50 K and (C) [⁵⁷Fe]-

Mb•PhNO at 200 K.

[⁵⁷Fe]Mb•PhNO at 50 and 200 K, Figures 12B and 12C,

respectively. The observed quadrupole splittings for both the

protein and model compounds (Table 6) are quite large and are

similar in magnitude to those observed previously for Fe(TPP)-

(iPrNO)(nPrNH₂) by Mansuy et al.^18,19in their pioneering

studies of nitrosoalkane-containing metalloporphyrins, in which

the sign of ∆E_Qwas determined to be negative. We then carried

out ⁵⁷Fe EFG calculations using DFT (G94/DFT/B3LYP/Fe

Wachters’/6-311++G(2d)/6-31G*/3-21G*), with the results

shown in Table 6. As can be seen from Table 6, the ∆E_Qvalues

computed from the known crystal structures vary from -0.98

to -1.4 mm s^-1, to be compared with the experimental range

of from -1.3 to -1.5 mm s^-1, about a 0.2 mm s^-1errors

about the same error range as that reported previously for 14

organometallic and metalloporphyrin model compounds.⁴⁹Also

of interest is the observation that the orientation of the principal

component of the EFG tensor, V_zz, is in the porphyrin plane,

Figure 13, just as it is with oxymyoglobin,¹³a result that might

be anticipated based on the isoelectronic nature of Fe•O₂and

Fe•RNO (Fe•HNO) systems. This gives strong support for the

use of DFT calculations to predict the EFGs in metalloporphy-

rins, and indeed in work described elsewhere we have found

good accord for ∆E_Qfor Fe(TPP)(CO)(1-MeIm), Fe(TPP)(CO)-

(py), Fe(TPP)(py)₂, Fe(TMP)(1-MeIm)₂, Fe(OEP)(PMe₃)₂, and

the range of ∆E_Qvaried from -1.86 to -2.65 mm s^-1

.

These results demonstrate that DFT methods give both the

correct magnitude for ∆E_Qin oxyheme models, and the correct

sign, orientation (Figure 13), and η values, but they also point

to the difficulty of accurately describing the oxyheme geometry

by using just ∆E_Q. On the basis of the latest high-resolution

crystal structures of MbO₂,⁴²it appears that the proximal

histidine (our 1-MeIm axial base) is oriented along an N1-N3

vector, thus the models shown in Figures 14D-F would be

closest to experiment, with E also being in good agreement with

the Fe-O-O orientation seen in two oxymyoglobins, Table 4.

The energies of B, C, and E in Figure 14 (Table 7) are also the

lowest of the series, which when combined with the X-ray data

suggests conformer E as a likely candidate for accurate EFG

calculations. The results we obtain are ∆E_Q) -1.84, -1.82,

and -2.45 mm s^-1for the planar/unoptimized, planar/optimized,

and ruffled/unoptimized structures, and at present it is not

possible to get more accurate results, although this may be

possible in the future by carrying out very large scale geometry

O₂-Heme Analogue Studies

J. Am. Chem. Soc., Vol. 121, No. 16, 1999 3841

Table 6. Computed ⁵⁷Fe Electric Field Gradient Tensor Elements and Mo¨ssbauer Quadrupole Splittings for RNO, O₂-Metalloporphyrins, and

Experimental Data for Mb•PhNO

electric field gradient (au)

∆E_Q(mm s^-1

expt

)

V₁₁

V₂₂

V₃₃

calc

(PhNO)(1-MeIm)(TPP)Fe^a

(PhNO)(py)(TPP)Fe^a

-0.3738

-0.6162

-0.7498

-0.2216

-0.1433

0.0187

0.5954

0.7595

0.7311

-0.975

-1.31

1.39

-1.31^b

-1.42^b

1.51^b

(p-Me₂NC₆H₄NO)(py)(TPP)Fe^c

PhNO•Mb

50 K

100 K

150 K

200 K

-1.758^b

-1.752^b

-1.747^b

(O₂)(1-MeIm)(TPP)Fe (C₂-cap)^d

(O₂)(1-MeIm)(TPP)Fe (C₂-cap)^e

(O₂)(1-MeIm)(TPP)Fe (C₂-cap) BP86^e,f

(O₂)(1-MeIm)(TPP)Fe (C₂-cap) BPW91^g

(O₂)(1-MeIm)(TPP)Fe (picket) xray^e,h

(O₂)(1-MeIm)(TPP)Fe (picket) xray+90^d,h,j

(O₂)(1-MeIm)(TPP)Fe (picket) xray+180^d,h,j

(O₂)(1-MeIm)(TPP)Fe (picket) xray+270^d,h,j

(O₂)(1-MeIm)(TPP)Fe (picket) xray d(O-O) ) 1.24 Å ^d,h,k

^aG94/B3LYP/Fe Wachters/6-311++G(2d) NO and N coordinated to Fe/6-31G* CR/3-21G* others. ^bThis work. ^cG94/B3LYP/Fe Wachters/

6-311++G(2d) N coordinated to Fe/6-31G* CR/3-21G* others. ^dG94/B3LYP/Fe Wachters/6-311++G(2d) O₂and N coordinated to Fe/6-31G*

CR/3-21G* others. ^eG94/B3LYP/Fe Wachters/6-311G(2d) O₂and N coordinated to Fe/6-31G* CR/3-21G* others. ^fBP86 exchange-correlation

functional (ref 23). ^gG94/BPW91 (ref 54)/Fe Wachters/6-311++G(2d) O₂/6-31G* others. ^hX-ray structure from ref 60. ⁱExperimental data from

ref 14 and ref 15. ^jX-ray + angle value means the Fe-O-O fragment was rotated with respect to the porphyrin normal by the amount indicated.

^kd(O-O) was set at 1.24 Å.

-0.8430

-0.8081

-0.8337

-0.8397

-0.7128

-0.7007

-0.7116

-0.7022

-0.7910

-0.6480

-0.6392

-0.6223

-0.6174

-0.3731

-0.3471

-0.3720

-0.3442

-0.4132

1.4911

1.4472

1.4560

1.4527

1.0859

1.0478

1.0837

1.0464

1.2043

-2.42

-2.35

-2.37

-2.36

-1.79

-1.73

-1.78

-2.1, -2.25, -2.31ⁱ

}

-1.73

-1.98

Figure 13. Orientations of the principal components of the electric field gradient tensor for ⁵⁷Fe in RNO, O₂model systems: (A) Fe(P)(PhNO)-

(1-MeIm) and (B) Fe(P)(O₂)(1-MeIm). Note that V₃₃(i.e., V_zz) is in the porphyrin plane in both cases.

Table 7. Optimized Structural Parameters for Oxy-Heme Model Systems^a

d(Fe-O) (Å)

d(O-O) (Å)

Fe-O-O (deg)

energy (au)

∆E (kcal/mol)

A

B

C

D

E

F

1.789

1.781

1.787

1.780

1.791

1.269

1.268

1.262

1.270

1.268

1.261

121.2

120.2

125.8

121.4

120.1

126.0

-2667.60946695

-2667.61541650

-2667.61490064

-2667.60968027

-2667.61554189

-2667.61020635

3.81

0.078

0.40

3.68

0.00

3.35

^aG94/DFT B3LYP partial geometry optimization carried out by using a planar porphyrin, as described in the text, for the six molecules whose

structures are shown in Figure 14. For comparison, the (unoptimized) picket fence porphyrin values are the following: d(Fe-O) ) 1.750 Å;

d(O-O) ) 1.240 Å; Fe-O-O ) 131°. The basis sets used are the following: Fe Wachters/6-31G* C,N,O and 3-21G* H.

optimizations on protein fragments. In any case, all of the ∆E_Q

calculations yield a value of ≈-2 mm s^-1, in very good

agreement with the low-temperature Mo¨ssbauer experiments.

Since we were unable to reproduce the very small ∆E_Q

suggested previously¹⁴we therefore need an alternative explana-

tion for the temperature dependence of the ⁵⁷Fe Mo¨ssbauer

quadrupole splitting. Fortunately, there are two additional pieces

of information we can use. First, previous NMR measurements

on oxypicket fence porphyrin have revealed very fast axial

rotation of O₂in the FeO₂subunit,¹¹and second, in oxymyo-

3842 J. Am. Chem. Soc., Vol. 121, No. 16, 1999

Godbout et al.

Table 8. DFT Computed ⁵⁷Fe Electric Field Gradient Tensor Elements and Mo¨ssbauer Quadrupole Splittings for Oxy-Heme Models^a

electric field gradient (au)^b,c

quadrupole splitting

structure

V₁₁

V₂₂

V₃₃

∆E_Q(mm/s)

planar, unoptimized

A

B

C

D

E

F

-0.8417

-0.7042

-0.5898

-0.8202

-0.6958

-0.8268

-0.7734

-0.4332

-0.3449

-0.7395

-0.4398

-0.6883

1.6150

1.1374

0.9346

1.5597

1.1356

1.5152

-2.62

-1.86

-1.53

-2.53

-1.86

-2.46

optimized

A

B

C

D

E

F

-0.8337

-0.7512

-0.7762

-0.8227

-0.7391

-0.8482

-0.7902

-0.3704

-0.3487

-0.7925

-0.3819

-0.7124

1.6238

1.1216

1.1249

1.6152

1.1210

1.5606

-2.63

-1.85

-1.86

-2.62

-1.85

-2.53

ruffled, unoptimized

A

B

C

D

E

F

-0.7749

-0.7470

-0.7429

-0.7678

-0.7428

-0.8692

-0.7450

-0.3983

-0.3852

-0.7441

-0.4046

-0.7678

1.5199

1.1453

1.1280

1.5119

1.1474

1.6370

-2.46

-1.88

-1.86

-2.45

-2.65

^aThe basic geometric structures used, A-F, are shown in Figure 14. ^bLocally dense basis set scheme: Fe Wachters’, 6-311++G(2d) for O₂and

5N coordinated to Fe, 6-31G* for the others except H are 3-21G*. ^cThe models were built by using either a planar porphyrin or a ruffled porphyrin

(the latter based on the structure seen in Fe(TPP)(CO)(1-methylimidazole), ref 43). For the unoptimized geometries we used d(O-O) ) 1.24 Å,

d(Fe-O) ) 1.75 Å and Fe-O-O ) 131°. The optimized values are given in Table 7.

less frequent, and would only mix-in a small contribution from

one of the rotameric states, consistent with the well-defined

electron density for the FeO₂unit in MbO₂,⁴²and very large

barriers to axial rotation as deduced from molecular mechanics

simulations (data not shown).

Given that the EFG principal components have been calcu-

lated, we can predict the resultant motionally averaged EFG,

basically as done by Spartalian et al.¹⁴For axial rotation, the

in-plane tensor components (V_zz, V_xx; Figure 13) simply ex-

change and are averaged. For example, using mean V_zz, V_xx

tensor values (from Table 6) of -1.88, 0.67 mm s^-1the average

becomes (-1.88 + 0.67)/2 ) -0.61 mm s^-1. As a result, the

principal component is now perpendicular to the porphyrin

plane, and has a value of 1.21 mm s^-1, which is very close to

the 1.288 mm s^-1(unsigned) value observed experimentally at

high temperature with the picket fence system.¹⁴The DFT

method therefore predicts not only the sign, magnitude, and

orientation of the ⁵⁷Fe EFG (or V_zz) at low temperatures but

also the correct magnitude for V_zzat high T, where motional

averaging is known to be present - without use of any

adjustable parameters. Given that DFT methods now correctly

predict the EFGs in almost 20 compounds, including ∼10

metalloporphyrins, and that we find no evidence for a second

site having a very small ∆E_Qseven when more than a dozen

possible geometries are investigatedswe conclude that this

simple motional model is correct, at least for the model system.

Figure 14. Schematic illustration showing the six orientations of O₂

(solid line) and 1-methylimidazole (dashed line) with respect to the

porphyrin macrocycle in Fe(P)(O₂)(1-MeIm) used for DFT calculations.

In proteins, the situation is more complex, at least for O₂.

We have investigated the variable-temperature ⁵⁷Fe Mo¨ssbauer

spectra of Mb•PhNO, and we show these results, together with

representative points from those reported previously by Parak

for MbO₂, in Figure 15. As can be seen, the ⁵⁷Fe ∆E_Qis

essentially temperature independent with the PhNO complex,

but is highly temperature sensitive in the case of MbO₂. Since

the two species are isoelectronic, these results tend to rule out

any “unusual” electronic effects as contributing to the ⁵⁷Fe EFG,

and in fact the simplest model to explain the differences between

the two sets of EFGs would be one in which in Mb•PhNO the

whole ligand is rigid, while in MbO₂, the ligand becomes

globin, the O₂molecule is crystallographically well-defined.⁴²

A quite reasonable general model for both model systems and

proteins would therefore appear to be one in which in the model

compound there is fast and complete axial rotation, or equiva-

lently jumps between the low-energy conformerssjust as found

with CoNO in Co(TPP)(NO), and Co(OEP)(NO) at high

temperature, and indeed as suggested as one possible model

previously by Loew.¹²But in the proteins, the jumps would be

O₂-Heme Analogue Studies

J. Am. Chem. Soc., Vol. 121, No. 16, 1999 3843

2

as shown in Figure 15B the H NMR spectrum of Mb•[²H₅]-

PhNO is characteristic of a rigid-lattice ²H NMR powder pattern,

even at 298 K. In the case of the ¹⁷O NMR of Mb¹⁷O₂and

Hb¹⁷O₂, so far we have only been able to obtain ¹⁷O spectra at

77 K (due to strong H₂¹⁷O overlaps and sensitivity problems at

high temperatures), and the spectra are essentially the same as

those obtained with oxypicket fence porphyrin at low temper-

ature.¹¹Nevertheless, a small (10-20%) contribution from a

second FeO₂orientation having essentially the same principal

components, but rotated by 90° in the heme plane, will clearly

cause a major decrease in ∆E_Q, while maintaining the same

basic Fe-O-O geometry. Thus, the same basic motional model

appears to be able to explain both the model compound and

metalloprotein results, using the DFT computed EFG tensor

values and orientations. Unfortunately, however, the results of

the calculations shown in Table 8 also indicate the possibility

that both alternate O₂-substates and porphyrin ruffling could

contribute to decreasing ∆E_Qin the proteins.

Electrostatics. With the availability of the results of accurate

high-level DFT calculations, it becomes of great interest to

investigate some of the other properties which then become

available, and here, we focus on the electrostatic potential, Φ(r),

mapped onto a charge density isosurface, F(r). Obtaining a better

understanding of the molecular electrostatic potential Φ(r) is

of interest because it can be expected to lead to more detailed,

quantitative descriptions of possible hydrogen bonding to CO

and O₂, which is thought by many to be of importance in CO/

O₂discrimination in hemoglobin and myoglobin.¹While the

topic of charge distribution in FeO₂and FeCO in proteins has

been the subject of considerable debate over the years,⁶⁶most

studies have estimated what are essentially local, atomic-based

charges, such as Mulliken populations or molecular electrostatic

potential derived charges, which necessarily involve use of some

model to deduce how to localize the charge. While this approach

may be necessary in, for example, molecular mechanics

methods, it would clearly be desirable to have a better

understanding of the full potential surface, Φ(r), since this can

in favorable cases be readily related to experiment. For example,

Flaig and co-workers^37,38have recently deduced the charge

density, F(r), as well as the electrostatic potential, Φ(r), for

several amino acids, from high-resolution X-ray crystallographic

data, and have shown that F(r) and Φ(r), as well as the

Figure 15. Graph showing Mo¨ssbauer and NMR results on heme

proteins: (A) The effect of temperature on the Mo¨ssbauer quadrupole

splitting, ∆E_Q, for MbO₂(b) and Mb•PhNO (9). The MbO₂results

are based on the values given in ref 15. (B) 8.45 T ²H NMR spectrum

of Mb•[²H₅]-PhNO crystals at 298 K.

mobile, or at least partially so, on increasing temperature. There

is strong support for this idea in the case of Mb•PhNO, where

Figure 16. Electrostatic potential surfaces, Φ(r), mapped onto a 0.017 au charge density surface, F(r), for (A) Fe(P)(CO)(1-MeIm), (B) Fe(P)-

(PhNO)(1-MeIm), and (C) Fe(P)(O₂)(1-MeIm). The dark blue color in the case of O₂and PhNO indicates a large negative electrostatic potential.

The electrostatic potential on CO is -0.06 au on the carbonyl oxygen (in A) but -0.086 au in the oxy complex (in C), and -0.10 au in the PhNO

adduct, (in B). The electrostatic potential surfaces are plotted for values between -0.06 and 0.17 au.

3844 J. Am. Chem. Soc., Vol. 121, No. 16, 1999

Godbout et al.

2

Laplacian of the charge density at bond critical points, ∇ F(r),³⁹

are in excellent agreement with the results of high-level quantum

chemical calculations. We have obtained similar results for

asparagine and a small peptide,⁷³which gives some confidence

in the ability of modern DFT calculations to reproduce these

electrostatic properties.

model systems. In proteins, the rotational barriers are higher,

but only a small contribution from a second rotamer (having

essentially the same EFG principal components) can have a

major effect on ∆E_Q. However, these results alone are insuf-

ficient to rule out significant contributions from porphyrin

ruffling and other geometric changes, in the case of the heme

proteins. Finally, the results of molecular electrostatic potential

calculations show that the electrostatic potentials for CO and

O₂are quite different, with the O₂complex being much more

negative (at oxygen) than in CO, consistent with a strong

hydrogen-bonding propensity in proteins, with the PhNO results

being similar to those seen with O₂. Overall, the ability of DFT

methods to successfully predict the chemical shifts and shift

tensors, as well as the ⁵⁷Fe electric field gradient tensors (the

Mo¨ssbauer quadrupole splittings, signs, and orientations) gives

confidence in the future use of these methods to probe in more

detail structure and bonding in both model systems, and in

metalloproteins themselves.

We therefore evaluated F(r) and Φ(r) for three CO, PhNO,

and O₂-containing metalloporphyrin models, and we show in

Figure 16 Φ(r), F(r) results in which the molecular electrostatic

potential is mapped onto a charge density isosurface, for CO,

PhNO, and O₂-containing metalloporphyrins. These results show

that there is a clear distinction between Φ(r), the electrostatic

potential, for bound CO and O₂, with the bound O₂exhibiting

a more negative potential, -0.086 vs -0.06 au. Also of interest

is the observation that the electrostatic potential for the terminal

oxygen in the PhNO adduct of -0.10 au is similar to that

observed in the O₂adduct, consistent with the isoelectronic

nature of the two systems. These electrostatic potential results

were essentially independent of the functional used. Also of

note is the observation that the electrostatic potential for oxygen

in the bound CO is essentially indistinguishable from that of

the adjacent porphyrin atomssan observation that would argue

against any specific, strong hydrogen bonding to COsin contrast

to the situation found with the O₂adduct, where quite clearly

the oxygen is highly electronegative (as with PhNO) and able

to selectively interact with H-bond donors, such as a proximal

histidine or glutamine.

Acknowledgment. We are grateful to B. J. Moreno, W. D.

Arnold, S. R. Wilson, T. Prussak-Wieckowska, P. Debrunner,

and C. E. Schulz for experimental assistance and T. Martinez

for helpful comments. This work was supported in part by use

of the SGI/Cray Origin 2000 and Power Challenge clusters at

the National Center for Supercomputing Applications (funded

in part by the US National Science Foundation, grant CHE-

97002ON). The X-ray crystallographic facilities were supported

by the National Science Foundation (grant CHE 95-03145).

Solution NMR spectra were obtained in the Varian-Oxford

Instrument Center for Excellence in NMR Laboratory. Funding

for this instrumentation was provided in part from the W. M.

Keck Foundation, the National Institutes of Health (grant RR

10444), and the National Science Foundation (grant CHE 96-

10502). High resolution mass spectra were obtained in the Mass

Spectrometry Laboratory, School of Chemical Sciences, Uni-

versity of Illinois, which is supported in part by a grant from

the National Institute of General Medical Sciences (grant GM-

27029). The 70-VSE mass spectrometer was purchased in part

with a grant from the Division of Research Resources, National

Institutes of Health (grant RR-04648).

Conclusions

The results we have obtained above are of interest for several

reasons. First, the synthesis and structural characterization of a

range of Fe•PhNO, CoNO metalloporphyrins is of interest

because it helps the development of a model for metallopor-

phyrin distortion.⁴³In addition, there are many structural

similarities with respect to axial ligand orientation between

FeO₂, FeRNO, and CoNO, reflecting their similar electronic

structures, and validating their use in testing theoretical methods

for predicting spectroscopic observables for Fe-O₂and Fe-

O₂analogue systems. Second, we have obtained solid-state

NMR and as appropriate Mo¨ssbauer spectra on these FeO₂-

analogue systems, and used DFT methods to predict the

experimental results with good success, validating the calcula-

tions. Third, we have used the same DFT methods to investigate

the Fe-O₂interaction in a metalloporphyrin and in oxymyo-

globin (and oxyhemoglobin). The magnitude, sign, and orienta-

tion of the ⁵⁷Fe Mo¨ssbauer EFG tensor is correctly predicted

for all low-temperature data, and a motional averaging model

in which the FeO₂rotates (as found for FeO₂and CoNO

metalloporphyrins by NMR) gives a ∆E_Qof 1.21 mm s^-1, versus

the 1.288 mm s^-1(unsigned) value seen experimentally with

Supporting Information Available: Tables of crystal data,

structure solution and refinement, atomic coordinates, aniso-

tropic displacement parameters, hydrogen coordinates, bond

lengths and angles, and torsion angles (PDF). X-ray crystal-

lographic files, in CIF format, are also available through the

Internet. This material is available free of charge via the Internet

at http://pubs.acs.org.

JA9832820

Article Doi

Solid-state NMR, Moessbauer, crystallographic, and density functional theory investigation of Fe-O2 and Fe-O2 analogue metalloporphyrins and metalloproteins

DOI: 10.1021/ja9832820

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Article abstract of DOI:10.1021/ja9832820

Full text of DOI:10.1021/ja9832820

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