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Copyright © 2007 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 92, Issue 11, 3764-3783, 1 June 2007

doi:10.1529/biophysj.106.093773

Biophysical Theory and Modeling

Quantitative Vibrational Dynamics of Iron in Carbonyl Porphyrins

Bogdan M. Leu^*, Nathan J. Silvernail^†, Marek Z. Zgierski^‡, Graeme R.A. Wyllie^†, Mary K. Ellison^†, W. Robert Scheidt^†, Jiyong Zhao^§, Wolfgang Sturhahn^§, E. Ercan Alp^§ and J. Timothy Sage^*, ,  

^* Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts
^† Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana
^‡ Steacie Institute for Molecular Science, National Research Council of Canada, Ottawa, Ontario, Canada
^§ Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois

Address reprint requests to J. T. Sage.

Diatomic molecules make essential contributions to the processes of life. Best known is the role of molecular oxygen, which has transformed Earth's biosphere 1 and is central to complex biomolecular networks 2. There is also growing recognition of the role of diatomic molecules in cellular signaling 3,4,5,6. Heme proteins figure prominently in interactions with these diatomic molecules 7,8,9.

The interactions between CO and heme proteins have drawn particularly intense scrutiny. In part, this stems from a favorable combination of stability and spectroscopic and photochemical properties that enable CO to function as a “stunt double” for O₂ in physical investigations. CO binding confers stability against autooxidation of the heme Fe, and spectroscopic observables, including vibrations of the FeCO group 10, provide sensitive probes of the local environment. In addition, the quantum yield for photodissociation of CO from heme proteins is higher than for other diatomic ligands 11, favoring its use for investigating the conformational response to ligand dissociation 12,13,14 and migration of CO through the protein interior 15,16,17 over a wide range of timescales.

There is increasing recognition that CO may participate directly in physiological signaling 6. CO binding to the heme-containing sensor protein NPAS2 is believed to facilitate regulation of circadian rhythms 18. CO also activates soluble guanylate cyclase by binding to its heme site, although it is less effective than NO 19,20. As a result, there is ongoing investigation of the possibility that CO can participate in physiological processes such as neurotransmission 21,22 and smooth muscle relaxation 23, where soluble guanylate cyclase is known to mediate NO signaling. CO, along with biliverdin and iron, are products of heme catabolism by the enzyme heme oxygenase 24,25,26. A bacterial heme protein CooA regulates DNA expression based on the local concentration of CO 27,28.

The affinity of CO for free heme, 2×10⁴ times that of O₂7, favors heme proteins as CO sensors. On the other hand, many heme proteins have evolved mechanisms to selectively bind O₂ in the presence of the background of endogenously produced CO. Although hydrogen bonding to the bound dioxygen is now believed to be the primary mechanism for selective O₂ binding in hemoglobins 7,29,30, discrimination against CO binding can also play an important role.

While O₂ binds end on to iron with the O-O axis ∼60° from the normal to the plane of a free porphyrin, CO binds nearly perpendicular to the plane 31. It has long been recognized that proteins can exploit this difference in geometry to sterically destabilize CO binding in favor of O₂32. Two discoveries have deemphasized the significance of steric factors in hemoglobins. For one thing, structural investigations of myoglobin with bound CO (MbCO) find much less distortion of FeCO from its energetically favorable linear geometry than was once believed 33,34,35,36,37. Moreover, quantum chemical calculations of the energetic cost of the residual distortion are significantly reduced from earlier estimates 29,38,39. The best available estimates indicate that steric distortion plays a secondary role in discriminating between CO and O₂ binding to hemoglobins 9,29,30, although questions remain about how to evaluate the energy involved in distorting the surrounding protein 35,40,41.

Although the understanding of how respiratory heme proteins avoid being poisoned by CO has advanced significantly, the means by which CO sensing proteins discriminate against false signals due to other diatomic molecules such as NO and O₂ is less well understood. Steric interactions with heme-bound diatomic ligands may also underlie their recognition by sensor proteins, for example by triggering conformational changes that regulate enzymatic activity or protein/DNA interactions 42,43,44,45. However, structural changes of the heme upon diatomic ligand binding can also drive allosteric conformational changes through the covalent link with the protein 46,47. Both factors contribute to the oxygen-binding behavior of the paradigmatic allosteric protein, hemoglobin 48,49,50.

Vibrational spectroscopy can be a sensitive probe of molecular structure and energetics, and traditional methods partially characterize heme Fe-ligand vibrations. Strong absorption in the 1900–2000cm⁻¹ frequency region, where there is minimal interference from protein or solvent vibrations, makes the bound C-O stretching vibration an effective infrared probe 51,52,53,54. Raman measurements in resonance with heme electronic transitions are primarily selective for porphyrin vibrations, but Fe-C stretching and, in some cases, FeCO bending vibrations are detectable in the resonance Raman signal from six-coordinate CO-ligated hemes 55,56,57,58,59,60.

These vibrations have been heavily exploited to monitor FeCO structure, environment, and energetics in heme proteins. Both Fe-C and C-O frequencies are well-calibrated electrostatic probes 10,61,62, and polarized IR measurements can infer distortion of the FeCO unit by determining the orientation of the C-O infrared transition dipole 33,34,35. Investigations of the FeCO bending vibration 55,56,57,63,64 have motivated quantum chemical calculations of the energetics of FeCO distortion 29,38,39, although the relevant lowest frequency distortion mode, predicted to lie below 100cm⁻¹, has not yet been observed experimentally.

Unfortunately, attempts to identify other Fe-ligand vibrations for CO-ligated heme proteins have been inconclusive. In particular, the vibration of the covalent Fe-histidine link with the protein only contributes strongly to the Raman signal after dissociation of CO 13,14,65, and Fe coordination to the porphyrin is usually probed only indirectly through its influence on high frequency porphyrin vibrations 66. A more comprehensive vibrational picture would identify additional vibrational probes for the Fe nearest-neighbor bonds and address unresolved questions surrounding previously observed vibrations. In particular, a fuller characterization of the FeCO bending vibrations would quantify the asymmetry of the quasi-degenerate bending vibrations, confirm that the observed features are vibrational fundamentals 63,64,67,68,69, and provide an experimental test of quantum chemical predictions for the FeCO energetics 29,38,39.

Nuclear resonance vibrational spectroscopy (NRVS) provides direct experimental access to all Fe vibrations. NRVS exploits characteristic properties of synchrotron radiation sources to reveal the complete vibrational spectrum of a probe nucleus 70,71,72,73,74 and provides a comprehensive picture of the vibrational dynamics of ⁵⁷Fe in many materials 75,76,77, including proteins 78,79,80,81,82,83,84 and model compounds that mimic protein active sites 85,86,87,88,89,90,91,92,93. NRVS yields quantitative values for vibrational amplitudes and directions, as well as frequencies, and thus provides a rigorous test of vibrational predictions. This information is proving particularly valuable for evaluating quantum chemical calculations based on density functional theory (DFT), which provide detailed predictions of many molecular ground state properties, including the optimized structure and a comprehensive description of vibrational dynamics 38,39,60,90,93,94,95,96,97,98.

Here, we use NRVS measurements and DFT calculations to characterize the vibrational dynamics of ferrous carbonyl porphyrins, including [Fe(TPP)(1-MeIm)(CO)] (Fig. 1), that mimic the active site of six-coordinate CO-ligated heme proteins. Fe-ligand vibrations dominate the observed NRVS signal, in contrast to other vibrational techniques. We compare the resulting quantitative information on the amplitude and direction of the Fe motion, as well as mode frequencies, directly with vibrational predictions from DFT calculations. This comparison provides a rigorous and successful test of the predictions, which describe vibrational modes observed in the experimental spectra. Vibrations involving all six covalent bonds to the iron contribute prominently to the NRVS signal, including the imidazole and pyrrole nitrogens as well as FeCO vibrations previously observed using resonance Raman spectroscopy. Lower frequency modes involving heme doming or reorientation of the diatomic CO ligand are likely to control the reactivity of heme proteins. Vibrational frequencies identified here can be used both to probe the Fe coordination structure and to provide quantitative insight into molecular energetics. In particular, experimental confirmation of predicted behavior for FeCO bending vibrations supports quantum chemical estimates of the cost of FeCO distortion.

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Figure 1

Structure resulting from geometric optimization [Fe(TPP)(1-MeIm)(CO)], showing the 1-MeIm and CO ligands below and above the plane of the porphyrin. Hydrogen atoms are omitted for clarity. (Cyan, iron; green, carbon; blue, nitrogen; and red, oxygen.) Subsequent figures use the same color scheme. Molecular structures and vibrational mode depictions (Figure 8 and Figure 9 and Figure 10 and Figure 11) were generated with the program MOLEKEL 152.

⁵⁷Fe-enriched porphyrinates were prepared using a small-scale metallation procedure described by Landergren and Baltzer 99. The synthesis of [Fe(TPP)(1-MeIm)(CO)] 88, [Fe(TPP)1,2-Me₂Im)(CO)] 100, [Fe(OEP)] 90, [Fe(TPP)(2-MeHIm)] 101, and [Fe(OEP)(2-MeHIm)] 102, was described previously. [Fe(OEP)(1-MeIm)(CO)] and [Fe(TPP)(1-PhIm)(CO)] were prepared by a method similar to the preparation of [Fe(TPP)(1-MeIm)(CO)]88. Ambient temperature infrared spectra of these compounds were recorded as Nujol mulls between NaCl windows.

Myoglobin from horse heart (Sigma, St. Louis, MO) was reconstituted following the method of Teale 103 with ⁵⁷Fe-enriched protoporphyrin IX (Frontier Scientific, Logan, UT). Following concentration to 13mM in pH 8-phosphate buffer, the protein solution was agitated under 1atm of CO gas and reduced with sodium dithionite to produce the CO derivative (MbCO) 79.

NRVS measurements were performed at sector 3-ID-D of the Advanced Photon Source at Argonne National Laboratory (Lemont, IL). The sample was placed in a monochromatic x-ray beam, whose energy was scanned through the 14.4keV ⁵⁷Fe resonance using a high resolution monochromator 104 with an energy bandwidth (full width at half-maximum) of 7cm⁻¹ (0.85meV) or 8cm⁻¹ (1.0meV). X rays were distributed across a 4×1mm² beam cross section and arrived as a train of pulses 70ps wide at 154-ns intervals, with an average flux of 2×10⁹ photons/s=4μW incident on the sample. An avalanche photodiode detected photons emitted by the excited ⁵⁷Fe atoms, which arrive with a delay on the order of the 140ns ⁵⁷Fe excited-state lifetime. The counter was disabled during a time interval containing the arrival time of the x-ray pulse, to suppress the large background of electronically scattered 14.4keV photons, which arrive in coincidence with the x-ray pulse.

Polycrystalline powders or frozen solutions were loaded into polyethylene sample cups and mounted on a cryostat cooled by a flow of liquid He, with x-ray access through a beryllium dome. Single crystals were mounted on a goniometer and data was recorded in two orthogonal orientations related by rotation about an axis orthogonal to the x-ray beam. Crystal orientation was verified by x-ray diffraction before and after NRVS measurements. A stream of cold N₂ gas from a commercial cryocooler controlled crystal temperature during NRVS measurements.

Both [Fe(TPP)(1-MeIm)(CO)]·C₆H₆ and [Fe(OEP)(2-MeHIm)]·C₇H₈ crystallized in space group with a single porphyrin in the asymmetric unit. In each case, a single ⁵⁷Fe-enriched crystal [1.05×1.00×0.22mm³ for Fe(TPP)(1-MeIm)(CO) and 2.00×0.50×0.40mm³ for [Fe(OEP)(2-MeHIm)] was oriented with a selected crystallographic plane parallel both to the x-ray beam and to the goniometer rotation axis. For [Fe(TPP)(1-MeIm)(CO)]·C₆H₆, the chosen {24, −2, 1} plane lies 0.6° from the mean plane of the four pyrrole nitrogens, while the {6, 6, 17} plane selected for [Fe(OEP)(2-MeHIm)]·C₇H₈ lies 1.3° from the porphyrin plane. This procedure ensures that only Fe motion parallel to the porphyrin plane contributes to the NRVS signal recorded in the original orientation. In each case, a second data set, recorded after a 90° rotation about the goniometer axis, sampled Fe motion perpendicular to the porphyrin plane.

[Fe(TPP)1,2-Me₂Im)(CO)]·C₇H₈ crystallized in space group P2₁/n with a single porphyrin in the asymmetric unit. A single crystal with dimensions 1.50×0.50×0.54mm³ was oriented with the {12, 1, −8} plane parallel to the plane defined by the x-ray beam and the goniometer rotation axis, with the x-ray beam parallel to the planes of all porphyrins. Because the two symmetry-related porphyrin planes lie ∼77° apart, it was not possible to simultaneously orient both porphyrins orthogonal to the x-ray beam, and data was recorded only for the original in-plane orientation.

A spectrum recorded as a function of x-ray energy consists of a central resonance, due to recoilless excitation of the ⁵⁷Fe nuclear excited state at E₀=14.4keV, together with a series of sidebands corresponding to creation or annihilation of vibrational quanta of frequency which have an area proportional to the mean squared displacement of the Fe and are displaced from the recoilless absorption by an energy 85,105.

Normalization of this spectrum according to Lipkin's sum rule 106 yields the excitation probability Subtraction of the central resonance results in a vibrational excitation probability 107. Each mode contributes an area

(1)

The program PHOENIX 108 removes temperature factors, multiphonon contributions, and an overall factor proportional to inverse frequency from to yield an Fe-weighted vibrational density of states (VDOS), which defines the vibrational properties at all temperatures for a harmonic system. For a randomly oriented sample, such as a solution or polycrystalline powder, the total VDOS

(2)

(3)

Measurements on a perfectly oriented sample yield a projected VDOS

(4)

The sample temperatures are determined directly from NRVS, by requiring that the ratio equal the Boltzmann factor For randomly oriented powder and solution samples, we obtained 20K for [Fe(TPP)(1-MeIm)(CO)], 19K for [Fe(TPP)1,2-Me₂Im)(CO)], 33K for [Fe(TPP)(1-PhIm)(CO)], 25K for [Fe(OEP)(1-MeIm)(CO)], 25K for MbCO, 20K for [Fe(TPP)(2-MeHIm)], and 34K for [Fe(OEP)]. We found 230K (123K) for the in-plane (out-of-plane) orientation of the [Fe(TPP)(1-MeIm)(CO)] crystal, 89K (140K) for the in-plane (out-of-plane) orientation of the [Fe(OEP)(2-MeHIm)] crystal, and 86K for the [Fe(TPP)1,2-Me₂Im)(CO)] crystal (in-plane orientation only).

DFT calculations provided the optimized structures and detailed vibrational predictions for the six-coordinate porphyrins [Fe(TPP)(1-MeIm)(CO)] (Fig. 1) and [Fe(TPP)(2-MeHIm)(CO)]. Calculations were performed with Gaussian 98 109, using the 6-31G* basis set for N, O, C, and H atoms, Ahlrichs’ VTZ basis set for the Fe atom 110, and the Becke-Lee-Yang-Parr composite exchange correlation functional (B3LYP) 111,112. The kinetic energy distribution (KED) is determined for each vibrational mode from the relative Cartesian displacements of individual atoms, as described previously 90. The calculation describes the atomic vibrations using a set of relative atomic displacements from which the mode composition factors

(5)

For comparison with measurements on oriented samples, the kinetic energy fraction due to motion of atom j along a direction is calculated from

(6)

The mode composition factors for iron (j=Fe in Eq. (6)) are then used to determine the contributions to from the iron motion in the x, y, and z directions (Eq. (4)). These contributions are labeled with x and y being orthogonal directions in the plane of the porphyrin, and z perpendicular to the mean porphyrin plane. The total VDOS, is given by Eq. (2) with j=Fe.

Fig. 2 presents NRVS excitation probabilities recorded on three porphyrins that mimic the active site of CO-ligated heme proteins. These data and the VDOS derived from them (Figure 3bd) share some similarities with each other and with MbCO (Figure 3a), a typical heme protein with the same ligation, and contrast with the vibrational spectra of four- and five-coordinate Fe porphyrins (Figure 3cef). In particular, four- and five-coordinate molecules lack features above 450cm⁻¹ associated with the FeCO group (see following section).

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Figure 2

NRVS data for six-coordinate iron-carbonyl porphyrins. Peaks above 500cm⁻¹ involve the FeCO fragment. Error bars reflect counting statistics.

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Figure 3

Comparison of VDOS for MbCO (a), and for four-, five-, and six-coordinate porphyrins that mimic heme protein active sites (b–f). Features that appear above 500cm⁻¹ in the CO complexes (crosshatched) are due to FeCO vibrations, which are absent in the VDOS of four- and five-coordinate Fe porphyrins. Values for are equal to crosshatched areas. Panel c shows in-plane and out-of-plane contributions to the total VDOS of [Fe(OEP)(2-MeHIm)], determined from single crystal data recorded with the incident x-ray beam parallel (dashed line) and perpendicular (solid line) to the mean porphyrin plane, respectively. All other data were recorded on randomly oriented samples (frozen solution or polycrystalline powder). For the porphyrins, the frequencies of the in-plane modes decrease as the Fe-N_pyr bond lengths increase. The line in panel a corresponds to a five-point smoothing of the data.

Since the NRVS signal is weighted by the Fe mean-squared displacement, vibrations involving all Fe ligands contribute prominently to the experimental VDOS. These include not only vibrations of the FeCO group in the 500–600cm⁻¹ region identified in previous investigations of heme-CO complexes 55,113, but also vibrations involving the imidazole and porphyrin ligands and low frequency molecular distortions. Quantitative information on vibrational amplitudes and directions, as well as frequencies, facilitates identification of these modes. Table 1 lists the frequencies and the corresponding areas of features observed in the VDOS of [Fe(TPP)(1-MeIm)(CO)] (Fig. 4 and two related model compounds, [Fe(TPP)1,2-Me₂Im)(CO)] and [Fe(TPP)(1-PhIm)(CO)] (Fig. 5).

Table 1 Correspondence between the observed vibrational frequencies and amplitudes for Fe in polycrystalline powders of Fe(TPP)(L)(CO) with L=1-MeIm, 1,2-Me2Im, and 1-PhIm

	1-MeIm		1,2-Me2Im		1-PhIm
	39	0.07	39	0.09
	64	0.13	87	0.09
	127	0.06	121	0.03	117	0.07
			139	0.04
	172	0.08	168	0.07	172	0.08
	204	0.04	194	0.08	197	0.06
	225	0.15	221	0.17	223	0.29
	241	0.15	233	0.08	233	0.05
	251	0.21	251	0.26	247	0.22
			280	0.19	293	0.17
	320	0.45	314	0.16	313	0.24
	326	0.14	327	0.29
	338	0.40	341	0.40	330	0.62
	388	0.02			385	0.08
	413	0.12	417	0.11	416	0.10
	466	0.11	463	0.13	468	0.14
			495	0.07	489	0.16
	507	0.37	509	0.24	502	0.16
	561	0.09	561	0.07	565	0.11
	587	0.21	585	0.19	589	0.17

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Figure 4

Single crystal, in-plane (upper panel), and out-of-plane (middle panel) orientations, and powder (lower panel) NRVS spectra for [Fe(TPP)(1-MeIm)(CO)]. Lines in the crystal data result from a five-point smoothing of the experimental data. Crosshatching indicates spectral area attributed to acoustic modes.

Comparison of powder and single crystal NRVS spectra provides information about the directions of the iron vibrations. Fig. 4 compares experimentally determined VDOS for the [Fe(TPP)(1-MeIm)(CO)] crystal with that determined for the polycrystalline powder. Inspection of these data identify out-of-plane modes in the powder spectrum at 39, 64, 127, 172, 225, 331, and 507cm⁻¹ and in-plane modes at 241, 320, 413, 466, and 587cm⁻¹. The prominent appearance of the 331cm⁻¹ out-of-plane mode (Fig. 4, middle panel) indicates unresolved contributions from both in-plane and out-of-plane modes to the 321cm⁻¹ band in the powder spectrum. Inspection of this feature reveals a high-frequency shoulder, and two peaks are required to fit this asymmetric feature (Table 1).

For [Fe(TPP)1,2-Me₂Im)(CO)], Fig. 5 (top panel) compares the in-plane component of the density of states, as determined from measurements on the oriented crystal, with the total density of states determined for the powder sample. We attribute features appearing at 121, 139, 168, 194, 221, and 509cm⁻¹ in the powder VDOS to out-of-plane vibrations, based on the absence of significant in-plane contributions at these frequencies. These features are unaffected by subtraction of the estimated in plane contribution from the powder data (Fig. 5, center panel). In contrast, we attribute features at 233 and 251cm⁻¹ to in-plane vibrations, because they are eliminated by this subtraction.

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Figure 5

(Upper panel) Comparison between the experimental VDOS for [Fe(TPP)1,2-Me₂Im)(CO)], powder (black line) and crystal, in-plane orientation (gray line). (Center panel) Positive features corresponding to out-of-plane vibrations of the Fe atom remain after subtraction of in-plane contributions, estimated by doubling the measured in-plane VDOS. Paired positive and negative features reflect in-plane asymmetries neglected in this estimate. (Lower panel) Experimental VDOS for [Fe(TPP)(1-PhIm)(CO)] powder. Cross-hatching and numerical values indicate areas attributed to FeCO vibrations.

The imidazole removes the nominal fourfold symmetry of the Fe environment, and vibrational data show clear evidence for x–y inequivalence in the 320–350cm⁻¹ region for both compounds. Fe motion along the two directions chosen for the single crystal measurements cannot account for the 338cm⁻¹ feature in the total density of states of [Fe(TPP)(1-MeIm)(CO)] (Fig. 4), clearly indicating measurable x–y frequency splitting for the in-plane modes in this region. For [Fe(TPP)(1,2-Me₂Im)(CO)], the calculated difference (Fig. 5, center panel) reveals paired positive and negative features that suggest frequency differences between the two in-plane directions. Such difference features correspond not only to a broad feature that a fit to the powder data resolves into components at 314, 327, and 341cm⁻¹, but also to features at 585cm⁻¹ and possibly at 463cm⁻¹, for which the powder data do not resolve significant structure.

Addition of a second methyl group to the imidazole increases the Fe-Im bond length by 9pm (Table 3) and perturbs the vibrational dynamics of the Fe. Nevertheless, the combination of powder and oriented crystal data allows the identification of vibrational features in the two molecules that correspond in frequency, amplitude, and direction. We also propose corresponding vibrational features in the powder data on [Fe(TPP)(1-PhIm)(CO)] (Fig. 5, lower panel, Table 1).

In addition to intramolecular vibrations, molecular translation contributes to the VDOS 85. For the polycrystalline powders studied here, we expect low frequency acoustic lattice vibrations to be the principal translational contribution. To estimate acoustic mode contributions for [Fe(TPP)(1-MeIm)(CO)], we crosshatch the 0–173cm⁻¹ region of the in-plane VDOS (Fig. 4, upper panel), which has the area expected 85 for translation of the entire molecule along an in-plane direction. Although it is unlikely that the actual acoustic mode spectrum has a sharp cutoff frequency, the frequency separation of the crosshatched area from other spectral features nevertheless suggests that acoustic motions dominate the in-plane VDOS below 200cm⁻¹. By comparison, excess area due to additional vibrational contributions is evident in the out-of-plane VDOS, and overlay of the spectrum of acoustic modes estimated from the in-plane VDOS onto the out-of-plane and powder VDOS (Fig. 4, middle and lower panel) highlights vibrational features at 39, 64, 127, and 172cm⁻¹ (Table 1), which we attribute to out-of-plane intramolecular vibrations.

All of the CO complexes presented in Figure 2 and Figure 3 display vibrational features above 500cm⁻¹. Previous Raman measurements on proteins and model compounds 63,114,115,116,117 have associated frequencies in this range with vibrations of the FeCO fragment. These modes (crosshatched in Fig. 3) are absent in the four- and five-coordinate complexes (Figure 3cef).

The lower frequency FeCO mode ranges between 499 and 507cm⁻¹ for the compounds studied here (Table 2). Crystal data (Figure 4 and Figure 5) reveal the out-of-plane character of these modes, and confirm assignment to Fe-CO stretching. Their mode composition factors range between 0.30 and 0.37, values close to the value expected for a two body Fe-CO oscillator (see Appendix ).

Table 2 FeCO vibrations for iron porphyrins. Values for are determined directly from NRVS experiments

	Compound	ν(Fe−CO)		δ(FeCO)		ν(C−O)
	Fe(TPP) (1-MeIm)(CO)	507	0.37	561/586	0.09/0.21	1969
	Fe(TPP) (1,2-Me2Im)(CO)	506	0.31	561/583	0.07/0.19	1948/1953
	Fe(TPP) (1-PhIm)(CO)	502	0.32	565/589	0.13/0.17	1975
	Fe(OEP) (1-MeIm)(CO)	499/513	0.30/0.16	582/575	0.25/0.04	1951

We attribute higher frequency modes, ranging from 561 to 589cm⁻¹, to FeCO bending. Single crystal measurements on [Fe(TPP)(1-MeIm)(CO)] and [Fe(TPP)1,2-Me₂Im)(CO)] confirm that Fe motion is primarily parallel to the porphyrin plane for modes in this region.

Three bands appear in the FeCO region of the VDOS for iron carbonyl tetraphenylporphyrins (Figure 3d and Figure 5d). We attribute the out-of-plane feature (507cm⁻¹ for [Fe(TPP)(1-MeIm)(CO)]) to Fe-CO stretching vibration, and the two in-plane features (561cm⁻¹ and 587cm⁻¹ for [Fe(TPP)(1-MeIm)(CO)]) to FeCO bending. For MbCO and for [Fe(OEP)(1-MeIm)(CO)] (Figure 3ab), the experimentally determined VDOS resolves only a single peak in the FeCO bending region. However, the [Fe(OEP)(1-MeIm)(CO)] data (Figure 2b and Figure 3b) includes an additional band at 513cm⁻¹, on the high frequency edge of the Fe-CO stretching band at 499cm⁻¹.

NRVS measurements on single crystals reveal additional out-of-plane modes. The feature near 222cm⁻¹ in the out-of-plane contribution to the [Fe(OEP)(2-MeHIm)] VDOS (Figure 3c) can be fit with two peaks, with frequencies 217cm⁻¹ and 233cm⁻¹, and areas and 0.20, respectively. The total approaches the value expected for a two-body Fe-Im oscillator. Previously, single crystal measurements on [Fe(TPP)(2-MeHIm)] identified out-of-plane modes at 216, 228, and 246cm⁻¹, with 0.06, and 0.19, respectively 87,93. The total is somewhat smaller than for [Fe(OEP)(2-MeHIm)], but an empirical normal mode calculation 87 identified Fe-Im contributions to each of these three modes.

Oriented single crystal measurements on [Fe(TPP)(1-MeIm)(CO)] (Fig. 4 and [Fe(TPP)1,2-Me₂Im)(CO)] (Figure 5a) reveal substantial out-of-plane character for several experimental peaks with frequencies ranging from 39cm⁻¹ to 331cm⁻¹. The area of each individual feature is substantially less than expected for an Fe-Im oscillator, as can be seen by comparison with the area associated with Fe-Im stretching in [Fe(OEP)(2-MeHIm)] (Fig. 3).

The highest frequency out-of-plane feature at 331cm⁻¹ in [Fe(TPP)(1-MeIm)(CO)] is resolved from neighboring in-plane vibrations only in the single crystal data. Competing in-plane vibrations impeded the identification of analogous modes in the other CO complexes. However, modes appearing at 168, 209, and 222cm⁻¹ in the [Fe(OEP)(1-MeIm)(CO)] powder data appear to correspond to the out-of-plane modes identified at 172, 204, and 225cm⁻¹ in [Fe(TPP)(1-MeIm)(CO)] (Figure 3bd), suggesting that these modes are weakly perturbed by peripheral substituents. Single crystal measurements help to identify additional out-of-plane modes at 39, 64, and 127cm⁻¹ for [Fe(TPP)(1-MeIm)(CO)].

Each of the VDOS in Fig. 3 has a maximum in the 200–400cm⁻¹ region. We previously associated prominent modes in this region with vibrations of the in-plane Fe-N_pyr bonds in deoxyMb 79, [Fe(TPP)(NO)] 85,86,90, [Fe(TPP)(2-MeHIm)] 87,93, and [Fe(TPP)(1-MeIm)(CO)] 93,88. NRVS measurements on oriented single crystals of CO-ligated porphyrins (Figure 4a and Figure 5a) confirm the in-plane character of these modes.

In particular, the absence of the 320 and 338cm⁻¹ features, which dominate the powder spectrum, in the out-of-plane VDOS of [Fe(TPP)(1-MeIm)(CO)] (Fig. 4) demonstrates the predominant contribution of in-plane Fe motion to the associated vibrational modes. Of these, only the 320cm⁻¹ peak contributes to the experimental in-plane spectrum. The latter spectrum represents only one of two orthogonal in-plane directions, and these data suggest that the 338cm⁻¹ mode primarily involves Fe motion along the other in-plane direction. For additional pairs of in-plane modes at 241, 413, 466, and 587cm⁻¹, the frequency difference appears to be too small to resolve experimentally.

Further insight into the vibrational dynamics results from comparing the experimental results on [Fe(TPP)(1-MeIm)(CO)] with DFT predictions on [Fe(TPP)(1-MeIm)(CO)] and [Fe(TPP)(2-MeHIm)(CO)] (Fig. 6). A number of DFT calculations have been reported for six-coordinate imidazole-CO complexes of porphine 30,38,39,95,98,118,119,120,121 rather than tetraphenylporphyrin. However, experimental data in Fig. 3 indicates that the porphyrin peripheral groups significantly influence the Fe vibrational dynamics, as we previously found for five-coordinate iron nitrosyl porphyrins 90. The influence of the peripheral groups is also apparent upon comparison of NRVS spectra recorded on a model compound and on MbCO, which contains protoporphyrin IX (Figure 3abd). Therefore, it is crucial to perform DFT calculations on the complete molecule rather than on a truncated model system. Comparison with NRVS data provides a detailed, rigorous, test of DFT vibrational predictions, as previously shown for [Fe(TPP)(NO)] 90.

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Figure 6

Comparison between the measured powder VDOS for [Fe(TPP)(1-MeIm)(CO)] (upper panel), and predicted VDOS for [Fe(TPP)(1-MeIm)(CO)] (center panel) and [Fe(TPP)(2-MeIm)(CO)] (lower panel). In-plane contribution is shown in shaded representation, and out-of-plane contribution in solid representation. Predicted VDOS are convolved with an 8cm⁻¹ Lorentzian.

The optimized structure correctly reproduces several structural parameters (Table 3), including the linear FeCO geometry 29,30,33,35,38,122. All 3N−6=267 predicted vibrational normal modes have real frequencies, ranging between 10 and 3317cm⁻¹. Just 24 modes contribute 93% of the area of the total predicted Fe VDOS. for the modes above 800cm⁻¹ is smaller than 0.02, suggesting that the experimental range 0–800cm⁻¹ is large enough to offer a comprehensive picture of the Fe vibrational dynamics.

There is overall agreement between the experimental and the predicted vibrational spectra on the same absolute vertical scale without frequency scaling (Fig. 6). We previously suggested a correspondence between the predicted modes and the major experimental features 93 for [Fe(TPP)(1-MeIm)(CO)].

In general, DFT predictions for [Fe(TPP)(1-MeIm)(CO)] and [Fe(TPP)(2-MeHIm)(CO)] (Fig. 6) confirm the character of the vibrational modes determined experimentally from NRVS spectra. In addition to vibrational frequencies and amplitudes, the vibrational predictions are consistent with information on the direction of Fe motion derived from single crystal measurements. Modes above 500cm⁻¹ are associated with the FeCO group, in-plane Fe-N_pyr vibrations dominate the 300–400cm⁻¹ region, and Fe motion is predominantly out-of-plane for modes with frequencies below 220cm⁻¹. The most apparent difference is the 474cm⁻¹ frequency predicted for the Fe-CO stretching mode of [Fe(TPP)(1-MeIm)(CO)], 33cm⁻¹ lower than the observed value. Similarly, the Fe-NO vibration was the most significant error in the previously published [Fe(TPP)(NO)] calculation 90. Somewhat unexpectedly, an out-of-plane mode is predicted at 320cm⁻¹, in a region otherwise dominated by in-plane modes. Observation of an out-of-plane 331cm⁻¹ peak in the single crystal data (Fig. 4, middle panel) is consistent with this prediction.

Fig. 7 describes the predicted KED over the Fe and axial ligands of in [Fe(TPP)(1-MeIm)(CO)] in terms of the values and the sum over all 12 atoms of the 1-MeIm group. Colors highlight modes with significant involvement of the axial ligands. Green bars represent the 157, 182, and 214cm⁻¹ modes, which involve significant imidazole motion (i.e., ) as well as significant Fe motion perpendicular to the porphyrin plane (). Red bars identify the predicted FeCO stretching mode at 474cm⁻¹ and modes at 560, 566, 580, and 583cm⁻¹ with FeCO bending character (Fig. 8). Also shown in red are the in-phase bend-tilt FeCO modes 38,98 at 74, 74.5, and 75.3cm⁻¹. The 320cm⁻¹ mode involves Fe-CO translation against the rest of the molecule (Fig. 9).

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Figure 7

Predicted KED over Fe, carbonyl C and O, and imidazole atoms (which includes all the 12 atoms on the 1-MeIm group) for [Fe(TPP)(1-MeIm)(CO)]. The heights of the individual bars equal the fraction of the kinetic energy associated with each atom or group. The lower panel shows the sum of the mode composition factors for Fe, C, and O, and gives the fraction of mode energy localized on the FeCO fragment. Green bars indicate Fe-Im modes, and red bars FeCO modes.

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Figure 8

Predicted high frequency vibrational modes of the FeCO fragment of [Fe(TPP)(1-MeIm)(CO)] with frequencies (a) 474cm⁻¹, (b) 560cm⁻¹, and (c) 580cm⁻¹. The relative phase of the FeCO bending and porphyrin motions reverses between the 560cm⁻¹ and 580cm⁻¹ modes. In this and subsequent figures, each arrow is 100(m_j/m_Fe)^1/2 times longer than the zero-point vibrational amplitude of atom j, and the bonds to the Fe are omitted to enhance the visibility of the Fe motion.

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Figure 9

Predicted vibrational modes of [Fe(TPP)(1-MeIm)(CO)] with frequencies (a) 157cm⁻¹, (b) 182cm⁻¹, (c) 214cm⁻¹, and (d) 320cm⁻¹ involve Fe-Im motion.

Two low-frequency [Fe(TPP)(1-MeIm)(CO)] modes resemble vibrations also predicted for [Fe(TPP)(NO)] 90. A “doming” mode predicted at 96cm⁻¹ involves translation of the [Fe(TPP)(1-MeIm)(CO)] fragment coupled with swiveling of the four pyrrole groups to allow the nitrogens to follow the motion of the Fe. A predicted mode with a 26cm⁻¹ frequency involves translation of the entire porphyrin core, accompanied by motion of the four phenyl groups in the opposite direction.

The predictions for [Fe(TPP)(1-MeIm)(CO)] and [Fe(TPP)(2-MeHIm)(CO)] are similar overall (Fig. 6, Table 3), but closer examination reveals both structural and vibrational differences. In particular, the Fe-Im bond length increases by 9 pm in the optimized [Fe(TPP)(2-MeHIm)(CO)] structure, presumably to minimize nonbonded interactions between the porphyrin and the methyl group on the imidazole. Vibrational modes involving the axial ligands have altered frequencies and Fe amplitudes. The predicted 5cm⁻¹ increase of the Fe-CO frequency to 479cm⁻¹ in [Fe(TPP)(2-MeHIm)(CO)] reflects a 1 pm decrease in the Fe-CO bond length (Table 3).

On the other hand, the 9 pm increase of the Fe-Im bond length is correlated with modest decreases of the 182, 214, and 320cm⁻¹ frequencies predicted for [Fe(TPP)(1-MeIm)(CO)]. The frequencies predicted for the first two vibrations are 179 and 212cm⁻¹ for [Fe(TPP)(2-MeHIm)(CO)], while an unresolved pair of modes (317 and 319cm⁻¹) replace the 320cm⁻¹ mode predicted for [Fe(TPP)(1-MeIm)(CO)]. The splitting of the latter mode reflects vibrational interaction with a porphyrin vibration.

The DFT calculations predict frequency changes for several modes involving rotation of the Im ligand, from 157, 263, 283, and 379cm⁻¹ in [Fe(TPP)(1-MeIm)(CO)] to 163, 268, 280, and 398cm⁻¹ in [Fe(TPP)(2-MeHIm)(CO)], perhaps reflecting the increased steric interaction of the methyl group with the porphyrin. The Fe amplitudes predicted for some of these modes also change. For [Fe(TPP)(1-MeIm)(CO)], the predicted Fe amplitude is slightly smaller for the 157cm⁻¹ than for the 181cm⁻¹ mode (Table 4), while for [Fe(TPP)(2-MeHIm)(CO)] the mean-squared amplitude predicted for the 163cm⁻¹ mode () is greater than four times larger than for the 179cm⁻¹ mode (). Substitution with 2-MeHIm also increases the Fe amplitude predicted for the 268 and 280cm⁻¹ modes with respect to the corresponding modes (263cm⁻¹ and 283cm⁻¹) in [Fe(TPP)(1-MeIm)(CO)].

Table 4 Predicted ImFeCO kinetic energy distribution for Fe(TPP)(1-MeIm)(CO)

	Frequency (cm−1)						Frequency (cm−1)
		0.05	0.01	0.01	0.09	0.16	321	0.02	0.00	0.00	0.01	0.03
	50	0.01	0.02	0.08	0.04	0.15	323	0.18	0.02	0.00	0.04	0.24
	52	0.01	0.02	0.08	0.00	0.11	329	0.37	0.04	0.01	0.10	0.52
	55	0.01	0.01	0.08	0.24	0.34	379	0.02	0.02	0.00	0.90	0.94
	64	0.01	0.01	0.14	0.28	0.44	412	0.05	0.01	0.00	0.00	0.06
	96	0.08	0.02	0.03	0.27	0.39	413	0.05	0.01	0.00	0.01	0.06
	157	0.05	0.01	0.01	0.68	0.74	467	0.04	0.05	0.01	0.00	0.09
	182	0.07	0.01	0.01	0.52	0.61	469	0.05	0.04	0.01	0.00	0.10
	214<