Low-Frequency Heme, Iron-Ligand, and Ligand Modes of Imidazole

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Low-Frequency Heme, Iron-Ligand, and Ligand Modes of Imidazole and Imidazolate Complexes of Iron Protoporphyrin and Microperoxidase in Aqueous Solution. An Analysis by Far-Infrared Difference Spectroscopy Laure Marboutin,† Alain Desbois,‡ and Catherine Berthomieu*,† Laboratoire des Interactions Prote´ine Me´tal, SBVME/iBEB/DSV, CEA-Cadarache, UMR 6191 CNRS CEA UniVersite´ Aix-Marseille II, Baˆt 185, 13108 Saint-Paul-lez-Durance Cedex, France, and Laboratoire Stress Oxydant et De´toxication, SB2SM and CNRS URA 2096/iBiTec-S/DSV, CEA-Saclay, 91191 Gif-sur-YVette cedex, France ReceiVed: December 8, 2008; ReVised Manuscript ReceiVed: January 21, 2009

FTIR difference spectroscopy, notably in the far-IR domain, is appealing for the analysis of hemoproteins, since it permits us to directly probe the properties of the heme and its ligands but also those of aminoacids remote from the heme. We recently set a thin path-length electrochemical cell with diamond windows allowing the far-IR analysis of proteins in aqueous solutions using FTIR difference spectroscopy (Berthomieu, C,; Marboutin, L.; Dupeyrat, F.; Bouyer, P. Biopolymers 2006 82, 363-367). In this study, we used this cell to identify redox-sensitive low-frequency IR modes of imidazole complexes of Fe-protoporphyrin IX and microperoxidase-8 and analyzed the pH dependence of these modes. The far-IR bands of the heme and the axial imidazole ligands were assigned using 15N2-, and d3-imidazole isotopic substitution, as well as imidazole substitution by 4(5)-methylimidazole. Internal modes of the axial histidine and imidazole ligands were identified in the 670-580 cm-1 region, which are sensitive to the iron coordination (five-coordinated high-spin heme or six-coordinated low-spin heme) and the protonation states of the axial ligands. We showed that deformation modes of the heme pyrroles dominate the 420-370 cm-1 region of the difference spectra. These modes were highly sensitive to the coordination and redox states of the heme iron and the conformation of the tetrapyrrole. While no νas(Fe-axial ligand) IR mode was detected in the difference spectra of the neutral imidazole complexes of Fe-protoporphyrin and microperoxidase, a new mode at 312 and 334 cm-1 was found specific of the imidazolate complexes of Fe3+-protoporphyrin and Fe3+-microperoxidase-8, respectively. On the basis of isotope shifts observed upon ligand deuteration, this band was assigned to a mode mixing the asymmetric stretching of the axial bonds with an internal deformation of the imidazolate rings. These data set the bases for the analysis of the IR low-frequency modes of hemoproteins, and specifically the electronic properties of the heme axial histidine ligands. Introduction Hemoproteins catalyze a large range of biochemical reactions. One of the most significant structural characteristics governing the functional properties of these proteins is the coordination environment of the heme iron, including the number and type of axial ligand(s), and the geometry of the coordination sphere. The chemical nature of heme ligand(s) impacts on the iron accessibility, its reactivity. and its redox properties. A fine regulation of the heme properties is also exerted by minute differences in steric constraints on the Fe-ligand bond, the relative orientation of the ligand side-chains, and/or the hydrogenbonding interactions involving the axial ligands.1-9 Structural constraints induced by the protein scaffold on the tetrapyrrole also result in specific heme deformations. From the planar structure, the heme skeleton can be distorted to adopt domed, ruffled, and/or saddled structures (Chart 1). These distortions influence the Fe-N(pyrrole) and Fe-axial ligand(s) bond strengths as well as the electronic properties and the reactivity of hemes.10-12 * To whom correspondence should be addressed. E-mail: [email protected]. Tel: 04 42 25 43 84. Fax: 04 42 25 26 25. † SBVME/iBEB/DSV, CEA-Cadarache, and CNRS UMR 6191. ‡ SB2SM and CNRS URA 2096/iBiTec-S/DSV.

CHART 1: Schematic Representation of Planar (A), Saddled (B), Ruffled (C) and Domed Conformationsa

a The filled and open circles indicate displacements above and below the mean porphyrin plane, respectively.

Iron-protoporphyrin IX (FePP) is the cofactor found in a large number of heme proteins. The axial ligation of this b-type heme is variable, but a histidyl imidazole group is often the fifth axial ligand. In c-type cytochromes, the heme attachment is stabilized by the condensation of the vinyl groups of heme b with two cysteinyl protein residues, forming two thioether bridges. The

10.1021/jp810774g CCC: $40.75  2009 American Chemical Society Published on Web 02/27/2009

Iron Protoporphyrin and Microperoxidase in Aqueous Solution cysteinyl pair is part of a highly conserved Cys-X-X-Cys-His sequence in which the histidyl aminoacid provides the fifth axial ligand of the heme iron. The heme octapeptide microperoxidase-8 (MP8), a digestion product of horse cytochrome c (cyt c) with pepsin and trypsin, includes the Cys14AlaGlnCysHis18 heme binding motif, while the sixth coordination position is occupied by a water molecule that can be exchanged with various exogenous ligands.13 This model offers the possibility to investigate the effects of different axial ligations and heme structures on the hemoprotein properties, using a variety of spectroscopic techniques.14-20 Among these, vibrational spectroscopy is particularly well suited to analyze the properties of the Fe-N(pyrrole) and Fe-axial ligand bonds, directly linked to the porphyrin geometry. Systematic studies by resonance Raman (RR) spectroscopy on wellcharacterized metalloporphyrin analogues provided markers of the metal oxidation state, metal-ligands vibrations and structural features of the heme in proteins.1,2,21-27 Below 1000 cm-1, inplane (ip) and out-of-plane (oop) heme deformation modes were observed both in RR and in infrared (IR) spectra of metalloporphyrins.2,28,29 In particular, the oop modes of the tetrapyrrole skeleton are expected to be very sensitive to the porphyrin structure, and thus could be used to describe structural constraints generated by the polypeptide chains in heme proteins. This low-frequency region of the vibrational spectra potentially contains information about the Fe-axial ligand bonds, in particular stretching and deformation modes,1,2 whose frequencies are directly linked to the strength of the axial bonds, via various electronic and steric factors (ligand electronegativity, bonding and nonbonding interactions between heme and ligands, coordination geometry, and so forth).24,30,31 The RR-active vibrations involving the axial Fe-imidazole stretching in six-coordinated (6c) heme complexes are weakly enhanced. For bis-imidazole complexes of FePP, the symmetric stretching mode of the axial Fe-N(His or imidazole) was however located in the 200-230 cm-1 region, the frequency of this mode depending on the H-bonding and ionization states of the axial imidazole rings.18,24,32-35 More recent studies on bis-imidazole and bis-pyridine complexes of “superstructured” Fe-porphyrins also showed the influences of steric strains and heme deformations on the symmetric stretching mode of the axial bonds.11,12 As far as the IR-active modes involving the Fe-axial ligand bonds are concerned, the vibrational information is notably scarce because far-IR data remained rather limited for Feporphyrins. Although far-IR analyses of metalloporphyrins powder in films or in paraffin oil evidenced heme modes sensitive to the metal ion and vibrations involving the metal atom and its axial and equatorial ligands36-43 only few data were reported for Fe-porphyrins, in particular in the reduced form.40 Moreover, IR data are missing for heme proteins in solution. The use of IR spectroscopy as a structural probe for this protein family is therefore appealing, since it should describe vibrations difficult or even impossible to access by RR spectroscopy. In addition to technical difficulties, the major absorption of a broad water relaxation band in the low-frequency domain limited the acquisition of IR data. FTIR difference spectroscopy is however a method of choice to identify single IR modes in strong absorbing backgrounds.44 In the mid infrared region, redox-sensitive heme modes, but also modes arising from the histidine ligands, the polypeptide backbone and the amino acid side chains, have been observed for heme proteins and microperoxidase.45-48 In the far-IR domain, experiments were

J. Phys. Chem. B, Vol. 113, No. 13, 2009 4493 reported using electrochemistry coupled to FTIR difference spectroscopy until 500 cm-1 using a transmission cell with ZnSe windows.49 We recently equipped our spectrometer with an electrochemical cell containing CVD diamond windows to record FTIR difference spectra in the whole mid to far IR range.50 In this study, we used electrochemically induced FTIR difference spectroscopy in the far-IR range, until 50 cm-1, to detect IRactive modes, in particular oop modes involving the heme, its axial bonds, and/or its axial ligands. We report the results of our FTIR investigation of the far IR domain of imidazole and imidazolate complexes of FePP and MP8 in aqueous solution. We identified new redox-sensitive IR bands, highly dependent on the heme coordination and conformation, and IR modes specific of the imidazole axial ligand, sensitive to the protonation state of this ligand. Experimental Methods Preparation of the Heme Model Compounds. Microperoxidase-8, hemin chloride, l5N2-labeled imidazole (15N2-ImH, 98% enrichment), and 4(5)-methylimidazole (4MeImH) were purchased from Sigma. Deuterated imidazole (d4-ImD, 95% enrichment) was purchased from Aldrich. These compounds were used without further purification. The purity of the MP8 preparation was greater than 87%, as determined by highperformance liquid chromatography.51 The MP8 samples contained a contaminant (ca. 10%), resulting from the loss of the C-terminal glutamic acid and valine residues of the parent MP8 molecule (not shown). All other reagents were of analytical grade quality and used as received. The MP8 concentration was determined spectrophotometrically from dilute samples (ε397 ) 1.57 × 105 M-1 cm-1).50,51 The FTIR experiments were performed with 4 mM MP8 in solutions buffered with 50 mM tris(hydroxymethyl)-aminomethane (Tris)-HCl (pH 7.4 and 8.5), N-cyclohexyl-2-aminoethanesulfonic acid (Ches)-NaOH (pH 9.3), 3-cyclohexylamino-1-propanesulfonic acid (CAPS)-NaOH (pH 9.7 and 11.1) with or without cetyltrimethylammonium bromide (CTABr), and 40 µM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). CTABr was at 15-20% (w/v) for the imidazole complexes of FePP and at 2% (w/v) for the methylimidazole complexes of FePP. Concentrated solutions of ImH or 4MeImH (0.1 M) were adjusted to the desired pH in various buffers and added to the MP8 solution to reach final concentrations ranging from 10 to 20 mM in a final volume of 20 µL. In order to obtain the lowspin (LS) MP8-ligand complexes, the appropriate ImH and 4MeImH concentrations were determined using UV-visible and EPR spectroscopies as previously described.48 Alkaline samples were prepared by dissolving the ligands in deionized water (at 0.1 M concentration) and adjusting the pH value (pH 12-13) by addition of KOH. The ligand solutions were then added to alkaline MP8 solutions prepared at the desired pH. Because of sample aggregation and precipitation, we cannot evaluate the effects of pH at values higher than 13. For the experiments performed in the mid-IR region, the MP8-ligand complexes were obtained with 4 mM MP8 in the presence of 10 mM ImH or 4MeImH,48 while for the far-IR analysis, the concentrations of MP8 and ligand were twice that of the mid-IR (8 and 20 mM, respectively). Control experiments were made using electronic absorption spectroscopy in the UV-vis region48 and comparing the FTIR difference spectra of the MP8(ImH) and MP8(4MeImH) complexes formed at 4 and 8 mM in the 1800-1000 cm-1 range. The bis(ImH) complexes of Fe-protoporphyrin IX (FePP) (with unlabeled (14N2-ImH), 15N2-labeled (15N2-ImH) and deu-

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terated imidazole (d3-ImH)) were obtained by dissolving 5 mM hemin chloride in a 100 mM Tris buffer, pH 8, in the presence of 100 mM KCl, 15% CTABr, and 2 M ImH. It was previously shown that this procedure produced the FePP(ImH)2 complexes.45 Electrochemistry. The thin path length electrochemical cell developed by Moss and Ma¨ntele53 was modified to adapt 1° wedged chemical vapor deposition (CVD) diamond windows (Φ ) 10 mm, midthickness ≈ 0.25 mm) purchased from Crystran Ltd. (U.K.). Wedged windows were used to avoid interferences fringes.54 The 4 µm thick gold grid working electrode (Buckbee-Mears, U.S.A.) was surface-modified by dipping it into a 5 mM pyridine-3-carboxaldehyde thiosemicarbazone (PATS-3, Lancaster Synthesis) solution heated to 80-90 °C for 5 min. Twenty microliters of the sample was deposited on the gold grid. The cell included an Ag/AgCl/3 M KCl reference electrode (Em ) 208 mV vs the normal hydrogen electrode, NHE) and a platinum counter electrode. The redox potential was varied using an EG&G potentiostat (Princeton Applied Research, model 362), triggered by the FTIR spectrometer. Reduction and oxidation of the FePP and MP8 complexes were performed as previously described. 45,48,50 Spectroscopy. The FTIR spectra were recorded using a Bruker 66 SX spectrometer equipped with KBr or 6 µm Simylar beam splitters (Bruker Optics), and DTGS-PE (absorption spectra of imidazole derivatives) or Si-Bolometer (electrochemically induced FTIR difference spectra, Infrared Laboratories USA) detectors. The spectra were recorded at a 4 cm-1 resolution. Typically, 300 scans were accumulated for each spectrum. The absorption spectrum of a protoporphyrin sample is shown in Supporting Information. Single-beam spectra recorded before and after the change in applied potential were subtracted to calculate the reduced minus oxidized (and reverse) difference spectra. For each sample, the results of 20-30 successive redox cycles were averaged. Very similar spectra were obtained for the firsts and last cycles. The IR spectra recorded with at least two different samples were averaged to improve the signal-to-noise ratio. Results FePP Imidazole Complexes in Aqueous Detergent Solution. Previous electronic absorption and vibrational studies on bis(ImH) complexes of FePP dissolved in aqueous CTABr showed that the ionization properties of the axial imidazole ligands are sequential and dependent on the oxidation state of the metal atom.55,56 At pH 7.8, the imidazole ligands of Fe2+PP and Fe3+PP are protonated (Fe2+PP(ImH)2 and Fe3+PP(ImH)2, respectively). The bis(imidazolate) complex of Fe3+PP (Fe3+PP(Im-)2) is fully formed at pH 13, while the axial ligands of Fe2+PP are only partially deprotonated (ca. 25%) under the same alkaline condition. Figure 1 shows the low-frequency regions (650-50 cm-1) of the reduced-minus-oxidized FTIR difference spectra of various bis(ImH) and bis(Im-) complexes of FePP at pH 7.8 and 13. In these spectra, positive bands correspond to the reduced form and negative bands correspond to the oxidized form of the complexes. The spectra recorded for the FePP(ImH)2 and FePP(4MeImH)2 complexes exhibit intense and well-defined difference bands at 668-666(+)/657-655(-) cm-1 (Figure 1A, spectra a and c). For the bis(ImH) complex, another difference band is clearly detected at 624(-)/614(+) cm-1. These bands are sensitive to d3-ImH labeling with a negative band at 556 cm-1 and a difference band at 528(-)/520(+) cm-1 for FePP(d3ImH)2 (Figure 1A, spectrum b). When the complexes are titrated

Figure 1. Reduced-minus-oxidized Far-IR FTIR difference spectra of bis(imidazole) and bis(imidazolate) complexes of Fe-protoporphyrin. (A) (a) FePP(ImH)2 at pH 7.8; (b) FePP(d3-ImH)2 at pH 7.8; (c) FePP(4MeImH)2 at pH 7.8; (d) FePP(Im-)2 at pH 13; (e) FePP(d3Im-)2 at pH 13; (f) FePP(4MeIm-)2 at pH 13. (B) Enlargement of the 420-360 cm-1 regions of FePP(ImH)2 (a), FePP(d3-ImH)2 (b), and FePP(4MeImH)2 (c) at pH 7.8. (C) Enlargement of the 420-360 cm-1 regions of FePP(Im-)2 (a), FePP(d3-Im-)2 (b), and FePP(4MeIm-)2 (c) at pH 13.

at pH 13, two weak positive bands remain at 668 and 618 cm-1 for the ImH complex (Figure 1A, spectrum d) and a weak positive band at 522 cm-1 for the d3-ImH complex (Figure 1A, spectrum e). A single band is seen at 664 cm-1 for the 4MeImH complex (Figure 1A, spectrum f). All these bands originate from the reduced forms with neutral axial ligands (Fe2+PP(ImH)2, Fe2+PP(d3-ImH)2, and Fe2+PP(4MeImH)2). On the contrary, a negative band in the 312-304 cm-1 region is specific of the

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Figure 2. Reduced-minus-oxidized Far-IR FTIR difference spectra recorded with microperoxidase-8 at pH 7.4. (a) 5cHS MP8; (b) 6cLS MP8(ImH); and (c) 6cLS MP8(15N2-ImH). Samples were in 50 mM Tris/HCl pH 7.4.

imidazolate forms of Fe3+PP since its intensity largely increases at alkaline pH. Moreover, this band is sensitive to the axial ligation with an observed frequency at 312 cm-1 for the bis(Im-) complex at 307 cm-1 when the Im- ligands are deuterated (d3Im-) and at 304 cm-1 for the bis(4MeIm-) complex (Figure 1A, spectra d-f). One may add that a negative band is observed at 545 cm-1 for the Fe3+PP(d3-Im-)2 complex only (Figure 1, spectrum e). In the 420-370 cm-1 region, we observe a positive band at 412 cm-1 for Fe2+PP complexes at pH 7.8 and a series of overlapping negative bands (Figure 1B). The shape of the negative cluster appears to be sensitive to both the axial ligation and pH, but the behavior of each individual band is difficult to analyze (Figure 1B,C). A 385 cm-1 band is however observed for the Fe3+PP(ImH)2 complex and is shifted to 383 cm-1 when the ligand is partially deuterated (d3-ImH) and to 381 cm-1 for the bis(4MeImH) complexes (spectra a-c in Figure 1A,B). For the bis(imidazolate) complexes (Figure 1A, spectra d-f, and panel B), the most intense component is observed at 398-396 cm-1 and thus appears to be upshifted when compared to the 397-394 cm-1 band of the protonated forms. Complexes of the Heme c Peptide Model MP8. Using UV-vis and EPR spectroscoscopies, we showed that a monodisperse five-coordinated high-spin MP8 form (5c HS MP8) was obtained at millimolar concentrations in Tris buffer in the presence of CTABr at a micelle-forming concentration.18,48 Electrochemistry at the coated gold electrode surface, using TMPD as a redox mediator, allowed the reproducible reduction/ oxidation of this 5c HS MP8 species and, thus, the observation of reduced-minus-oxidized FTIR difference spectra in the mid-IR.48,57 Figure 2 (spectrum a) shows the corresponding spectrum recorded in the far-IR range (680-50 cm-1). Distinct bands were observed at 663(+)/652(-), 506(+), and 414(+)/ 397, 387, 373(-) cm-1.

Figure 3. Reduced-minus-oxidized far-IR FTIR difference spectra of 6cLS imidazole complexes of microperoxidase-8. (a) MP8(ImH) at pH 7.4; (b) MP8(Im-) at pH 13; (c) MP8(15N2-Im-) at pH 13; (d) MP8(d3Im-) at pH 13; and (e) MP8(4MeIm-) at pH 13.

Addition of ImH to MP8 leads to the formation of a sixcoordinated low-spin (6c LS) complex.18,48 The FTIR spectrum of this LS complex (Figure 2, spectrum b) exhibits a difference band at 664(+)/655(-) cm-1 and a cluster at 414(+)/388, 375(-) cm-1, features very close to those observed for the 5c HS MP8 form (Figure 2, spectrum a). However, additional lines are detected at 640 (+), 619(-) and 583(+) cm-1. These frequencies are downshifted by 2-8 cm-1 upon 15N2-labeling of the imidazole ligand (Figure 2, spectrum c). The negative band at 655 cm-1 is also downshifted by 7 cm-1 (Figure 2, spectra b and c). The pH titration of the MP8(ImH) complex induces important spectral changes (Figure 3, spectra a and b, and spectra not shown). The 664(+), 655(-), and 414(+) bands are bleached at pH 13. On the contrary, a 334(-) cm-1 band becomes active when the pH value is increased. The frequency of this line is downshifted to 333 cm-1 when the imidazole is 15N2-labeled (Figure 3, spectrum c) and to 331 cm-1 when the imidazole is deuterated (d3-Im-, Figure 3, spectrum d). A band appears at 329 cm-1 when ImH is replaced by 4MeImH. In this case, additional negative bands are clearly observed at 621 and 273 cm-1 (Figure 3, spectrum e). Discussion Using the CVD diamond electrochemical cell, we could analyze heme model compounds in aqueous solution in the farIR region, until 50 cm-1. We observed a number of new IR bands sensitive to the iron redox state and to the electronic

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Figure 4. Far-IR absorption spectra of imidazole and 4(5)-methylimidazole in Nujol. (a) ImH; (b) 15N2-ImH; (c) d3-ImH; and (d) 4MeImH.

properties and/or the mass of the axial ligands. An assignment of these modes will be very useful for the interpretation of the far-IR spectra of heme proteins. Bands of the 670-500 cm-1 Region. For ImH in solution, a couple of strong IR-active modes is detected at 659 and 619 cm-1. These bands are downshifted upon 15N2-labeling (649 and 612 cm-1) or deuteration (548 and 522 cm-1) of the imidazole ring (Figure 4). For 4MeImH, they are observed at higher frequency, 668 and 625 cm-1, respectively, indicating a small sensitivity to ring methylation (Figure 4). The bands at ca. 660 and 620 cm-1 were previously assigned to ring puckering coupled with CH wagging.58-61 We propose very similar origins for the modes observed in the same region of the FTIR difference spectra of the FePP and MP8 complexes (Table 1). A couple of bands at 668-663(+)/657-648(-) cm-1 is visible in all the spectra of imidazole complexes of FePP and MP8 at pH 7-8. Only a weak positive band is detected at 664-669 cm-1 when the imidazole complex of FePP is formed at pH 13. Considering the pKa values of the bound imidazoles for each oxidation state of the heme iron,32 the 668-663 and 657-648 cm-1 bands seen at pH 7-8 correspond to IR contributions of the ferrous and ferric forms, respectively, of the FePP(ImH)2, MP8 and MP8-ImH species. In addition to redox sensitivity, these bands are thus also sensitive to the axial ligation and observed in the reduced-minus-oxidized spectra for neutral ImH ligands only. A difference signal is also observed at 664-663(+)/ 655-648(-) cm-1 in the spectra of both the 5c HS MP8 and 6c LS MP8(ImH) MP8 complexes (Figure 2, spectra a and b). These bands can therefore be assigned to the puckering modes of the intramolecular histidylimidazole ligand of MP8. Additional strong positive bands at 640-638 cm-1 and negative ones at 619-616 cm-1 (in Figure 2 spectrum b) are specific to the imidazole coordination to MP8 and are sensitive to 15N2ImH labeling. These bands can thus be assigned to puckering modes of the sixth imidazole ligand of the 6c LS MP8(ImH) complex. Similarly, the positive band at 583 cm-1 for the imidazole complex of Fe2+-MP8 at neutral and alkaline pH, downshifted to 575 cm-1 upon isotopic substitution of the

Marboutin et al. exogenous ligand (14N2-ImH/15N2-ImH), is assigned to a ring mode of the sixth imidazole ligand of the ImH-MP8 complex. This band most probably corresponds to the puckering mode observed at 619 cm-1 for ImH in solution. The coordination to reduced MP8 thus induces a strong downshift of this mode by 30 cm-1. This mode is assigned at 624(-)/614(+) cm-1 for FePP(ImH)2. Indeed, these bands are downshifted by ca. 95 cm-1 at 528(-)/520(+) cm-1 when the axial ligands are substituted by d3-ImH (Figure 1, spectra a and b). A very similar behavior is seen for the 619 cm-1 IR band of ImH in Nujol, that is shifted at 522 cm-1 upon deuteration (-97 cm-1) (Figure 4, spectra a and c). The observation of strong 624/614 cm-1 bands in the FTIR spectra of FePP(ImH)2 is specific of protonated ligands since no homologue bands are observed in the spectra of the imidazolate compounds of FePP (Figure 1, spectrum d). It is interesting to add that the 624 cm-1 band of Fe3+PP(ImH)2 and the 528 cm-1 bands of Fe3+PP(d3-ImH)2 are slightly upshifted when compared to the frequency of the free ligand in Nujol (619 and 522 cm-1, respectively), while the frequency of this mode is decreased upon complex formation with the reduced form Fe2+PP (614 and 520 cm-1). These opposite “redox” effects, also observed for the puckering mode at 668-663 cm-1 (red) or 657-648 cm-1 (ox) clearly illustrate different electronic interactions, that is, σ-bonding and π-backbonding, of the axial ImH rings with the metal atom. The IR spectra of Im- and 4MeIm- in KOH solution present an intense band at 689 cm-1 (Figure 5, spectra a and c). This line is not observed in the FTIR spectra of the Im- and 4MeImcompounds of FePP and MP8 since the noise level is too high in this region.62 This band occurs at 562 cm-1 for d3-Im- in the KOH solution (Figure 5, spectrum b), and it is detected at 545-536 cm-1 for d3-Im- complexes of FePP and MP8 (Figure 1, spectrum e and Figure 3, spectrum d). As discussed above for imidazole, an electronic effect of the imidazolate binding is also observed on the ring mode of imidazolate, when the 562 cm-1 band of free d3-Im- is compared to the 545 cm-1 band of Fe3+PP(d3-Im-)2 and the 536 cm-1 band of MP83+(d3-Im-). In addition as a diagnostic criterion of the oxidation state of heme iron, the observation of IR bands in the 670-520 cm-1 region will be thus very useful as a diagnostic purpose of the ionization state of the axial ligands of bis(His)-coordinated hemes in proteins and to describe the electronic interactions of the imidazole ligands with the heme iron. Bands of the 420-370 cm-1 Regions. The IR spectra of imidazole and imidazolate show no band in the 400-200 cm-1 region (Figures 4a and 5a). Therefore, the bands observed in the same region of the FTIR difference spectra of the imidazole and imidazolate complexes of FePP and MP8 do not involve any major contribution of internal modes of the bound imidazole or imidazolate rings and thus originate from the heme and/or its axial bonds. In the IR spectra of metalloporphyrins, intense bands contribute in the 420-350 cm-1 region. These bands were assigned to Fe-pyrrole stretching (ν(Fe-N4(Pyr)) and oop pyrrole tilting/folding.2,36,40,42,63-66 In the reduced-minus-oxidized FTIR difference spectra of the FePP(ImH)2 and FePP(4MeImH)2 complexes, the reduced form was characterized by a positive band at 412 cm-1, while strong negative contributions due to the oxidized forms contain three main bands at 398-394, 386-380, and 378-375 cm-1 (Table 1). Bands at similar frequencies were observed on thin films of hemin-imidazole complexes.42 The effect of d3-Im labeling on these bands were different depending on the acquisition temperature of the spectra (300 and 25-30K), indicating that the modes are not dominated

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TABLE 1: Frequencies (cm-1) of the Observed Low-Frequency IRTF Modes of Heme Complexes in This Study L ) ImH red FePP(L)2

MP(L)

L ) Im-

L ) 4MeImH ox

668 614 (-94)a 412 (0)a

657 624 395 385 375

664 (-1)c

655 (-7)c

640 (-2)c 583 (-8)c 414 (0)c

619 (-3)c

a

(–101) (-96)a (0)a (-2)a

red

ox

red

666

655 545 394 388 375

668 618 (-96)b

412

L ) 4MeImox

red

ox

664 397 388 380 312

(+1)b (-2)b (-2)b (–5)b

396 383 375 304 662 662

588 (-13)d 388 (0)c

403 (0, 0)b,d 384 (+2, +2)b,d 334 (-3, -1) b,d

403 389 329

tentative assignment imidazole ring imidazole ring heme pyrrole heme pyrrole heme pyrrole νas(Fe-L2-) + δ(Imidazolate ring) imidazole ring MeIm- ring imidazole ring imidazole ring heme pyrrole heme pyrrole νas(Fe-L2-) + δ(Imidazolate ring)

a Isotope sensitivity to h3-ImH /d3-ImH substitution. b Isotope sensitivity to h3-Im- /d3-Im- substitution. c Isotope sensitivity to N2-ImH substitution. d Isotope sensitivity to 14N2-Im-/15N2-Im- substitution.

14

N2-ImH/

15

Figure 5. Far-IR absorption spectra of imidazolate and 4(5)-methylimidazolate in a KOH aqueous solution. (a) Im-; (b) d3-Im-; (c) 4MeIm-. The presented spectra are the results of a subtraction of the absorption spectrum of the KOH aqueous solution.

by a νas(Fe-(ImH)2) contribution.42 In aqueous solution, these bands were not significantly affected by 15N2-imidazole labeling or imidazole deuteration (Figures 1a,b, and 2b,c). Considering that the macrocycle of Fe2+PP(ImH)2 is planar while that of Fe3+PP with the same axial ligands is ruffled, the 412 and ca. 395 cm-1 bands can be assigned to tilting or folding modes of pyrroles of FePP under distinct heme conformations.67 The same assignment can be made for the 417-414(+) cm-1 bands of the reduced MP8(ImH) complex and the 403(-) cm-1 contribution observed for the oxidized MP8(Im-) form (Table 1). The two additional negative bands observed in the 400-370 cm-1 region can be also assigned to oop pyrrole modes of the ferriheme of FePP and MP8 and associated with the conformational flexibility of the oxidized hemes when compared to the relative rigidity of the reduced hemes. The 389-380 cm-1 bands of the ImH, Im-, 4MeImH, and 4MeIm- complexes of Fe3+PP and MP83+ are more intense in the MP8 series. In particular, the contribution of the 388 cm-1 component is increased upon ImH binding. This is shown by the comparison of FTIR difference spectra recorded with MP8 in the absence

(5cHS) and in the presence of ImH (6cLS). This change in both coordination and spin states influences the Fe-N(pyrrole) bond lengths and the conformation of the tetrapyrrole macrocycle. As far as the MP83+(Im-) complex is concerned, the porphyrin is saddled as in its parent molecule, cyt c.68,69 The 388 cm-1 component could therefore represent an oop pyrrole mode in the context of a saddled heme, the 373 cm-1 line of the 5cHS species being representative of an oop pyrrole mode in a domed 5c heme.18,70 The profiles of the bands of the 420-370 cm-1 region appear to illustrate the conformational equilibrium of the heme that is naturally sensitive to the oxidation and ligation states of its metal atom. Bands of the 340-300 cm-1 Regions. Two modes involving antisymmetric stretching of the axial ligands (νas(Fe-(L)2) were detected at 385 and 319 cm-1 in the far-IR spectra of the bis(ImH) complex of Fe3+-octaethylporphyrin (OEP).41 Because of a strong mode mixing of this axial stretch with pyrrole tilts, these two modes are in fact weakly sensitive to the imidazole perdeuteration (1-1.5 cm-1).41 In the far-IR spectra of a film of imidazole complex of hemin in sodium docecylsulfate, four bands at 396, 386, 378, and 317 cm-1 were assigned to modes involving (νas(Fe-(L)2).42 However, in the absence of pH control, the investigated complex could be a mixture of Fe3+PP(ImH)2, Fe3+PP(ImH)(Im-)2, and Fe3+PP(Im-)2.32 In our FTIR difference spectra of the Fe2+/Fe3+PP(ImH)2 derivatives, we observe no difference band assignable to modes involving νas(Fe-(ImH)2). Like in the case of the Fe3+OEP(ImH)2 complex, these modes are obviously expected to be in the same 390-310 cm-1 region and IR-active. In this region, we detect dominating contributions from pyrrole tilting and folding modes. A lack of observation of this mode possibly originates from an insensitivity of the νas(Fe-(ImH)2) modes to the oxidation state of the iron atom combined to the use of a difference technique. The first part of this hypothesis is strongly supported by the comparison of the Raman-active symmetric counterparts. In fact, the frequencies of the νs(Fe-(ImH)2) modes are practically the same for Fe2+PP(ImH)2 and Fe3+PP(ImH)2 (203-200 cm-1).32,41,71-73 This electronic effect appears to be very similar for the reduced and oxidized forms of MP8(ImH). The situation may be different for the imidazolate complexes. Because of the negative charge on the ligands, the νs(Fe-(Im-)2) mode has a different frequency for Fe2+PP(Im-)2 and Fe3+PP(Im-)2 (212 and 226 cm-1, respectively).32 In the FTIR spectra of the bis(imidazole) and bis(4-methylimidazole) com-

4498 J. Phys. Chem. B, Vol. 113, No. 13, 2009 plexes of Fe3+PP at pH 13, we observe a specific negative band at 312 and 304 cm-1, respectively. Considering the pKa values of the imidazole rings bound to a ferriporphyrin,32,56 the axial ligands of the bis(ImH) and bis(4MeImH) complexes of Fe3+PP are essentially deprotonated at pH 13. The bands at 312 and 304 cm-1 are thus specific of the binding of the axial ligands under an anionic form. As far as the MP8 complexes are concerned, the 334 cm-1 band corresponds to the mono(Im-) complex of MP83+, since this negative band is observed when the pH value is raised (Figure 3). Taking into account both (i) the activation of the 312-304 cm-1 band of the Fe3+PP complexes and the 334-329 cm-1 band of the MP83+ complexes upon imidazolate binding, (ii) the sensitivities to ligand mass of the 312-304 cm-1 band of the Fe3+PP complexes (312, 307, and 304 cm-1 for the bis(h3Im-), bis(d3-Im-), and bis(4MeIm-) complexes, respectively) and the 334-329 cm-1 band of the MP8 complexes (334, 333, 331, and 329 cm-1 for the mono(h3-Im-), mono(15N2-Im-), mono(d3-Im-), and mono(4MeIm-) complexes, respectively), and (iii) the fact that no internal ring mode was observed or calculated in the 500-200 cm-1 region of the IR spectra of free Im- and d3-Im- (Figure 5, spectra a and b),60 these bands can be assigned to asymmetric stretching modes of the axial ligands: νas(Fe-(Im-)2) and νas(Fe-(4MeIm-)2) for the Fe3+PP complexes, and νas(His--Fe-Im-) and νas(His--Fe-4MeIm-) for the MP83+ complexes. However, the 5 cm-1 downshift of the 312 cm-1 band of Fe3+PP(Im-)2 as well the 3 cm-1 downshift of the 334 cm-1 band of MP8-(Im-) upon d3-Im labeling appear to be too large to account for a mode involving major νas(Fe(L-)2.74 Thus, we consider that a strong mixing of νas(Fe-(L-)2) with a deformation ring mode activated upon imidazolate binding to ferriheme is a most probable assignment. Whatever the precise assignment of the 334-304 cm-1 band, its presence is clearly a marker of the anionic state of the axial imidazole rings of heme. This feature will constitute an important indicator in the FTIR spectra of heme proteins. Another interesting point concerns the 22-25 cm-1 difference observed in the frequencies of the imidazolate compounds of Fe3+PP and MP83+. This important shift can be related to different heme-ligand interactions in the two heme systems. In particular, the porphyrin ruffling in Fe3+PP(Im-)2 favors short axial and equatorial bonds. Long axial bonds were detected for saddled Fe-porphyrins like in MP83+(Im-) when compared to planar or ruffled Fe-porphyrins (Chart 1).75-77 Because of different nonbonding interactions between the ligand rings and the nonplanar tetrapyrrole, the positioning of the imidazolate ligand rings with respect to the Fe-N(pyrrole) bonds is different for the two Fe-porphyrin conformers. The frequency of a mode mixing asymmetric stretching of the axial bonds and imidazole ring deformation can be thus sensitive to the relative and absolute position of the imidazolate rings. Conclusion The present study has characterized a series of new redoxsensitive low-frequency IR modes, common to the b- and c-type hemes, and detected using FTIR difference spectroscopy in aqueous solution. We expect that this investigation will facilitate the development of a detailed understanding of the FTIR spectra of heme proteins, in particular concerning the ionization state of the axial histidine ligand(s) and the tetrapyrrole structure. Acknowledgment. This work was financed in part by the French program Toxicologie Nucle´aire Environnementale. Andre´ Verme´glio is gratefully acknowledged for his support to this project.

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J. Phys. Chem. B, Vol. 113, No. 13, 2009 4499 possible to detect absorption bands in concentrated samples, the noise level is too high in the FTIR difference spectra. (63) Cornilsen, B. C.; Nakamoto, K. J. Inorg. Nucl. Chem. 1974, 36, 2467. (64) Hu, S. Z.; Mukherjee, A.; Piffat, C.; Mak, R. S. W.; Li, X-Y.; Spiro, T. G Biospectroscopy 1995, 1, 395. (65) Kozlowski, P. M.; Rush, T. S.; Jarzecki, A. A.; Zgierski, M. Z.; Chase, B.; Piffat, C.; Ye, B.-H.; Li, X.-Y.; Pulay, P.; Spiro, T. G. J. Phys. Chem. A 1999, 103, 1357. (66) Paulat, F.; Praneeth, V. K. K.; Nather, C.; Lehnert, N. Inorg. Chem. 2006, 45, 2835. (67) An assignment corresponding to a major ν(Fe-N4(Pyr)) stretching in the normal mode can be excluded. The frequencies of the 412(+)/394398(-) cm-1 bands do not match with the lengths of the Fe-N(pyrrole) bonds that are longer in planar ferrohemes than in ruffled ferrihemes. The frequency of a ν(Fe-N4(Pyr)) mode would be higher for the ferric species than for the ferrous species. We propose a folding or tilting mode of the pyrroles, but we cannot exclude a minor contribution of the Fe-N coordinates. (68) Takano, T.; Dickerson, R. E. J. Mol. Biol. 1981, 153, 95. (69) Berghuis, A. M.; Brayer, G. D. J. Mol. Biol. 1992, 223, 959. (70) Scheidt, W. R.; Lee, Y. J. Struct. Bonding (Berlin, Ger.) 1987, 64, 1. (71) Desbois, A.; Lutz, M. Biochim. Biophys. Acta 1981, 671, 168. (72) Desbois, A.; Lutz, M. In Hemoglobin; Schnek, A. G., Paul, C., Eds.; Editions de l’Universite´ de Bruxelles: Brussels, Belgium, 1983; pp 285-298. (73) Choi, S.; Spiro, T. G. J. Am. Chem. Soc. 1983, 105, 3683. (74) Considering the axial coordination of the FePP(Im-)2 complexes as a linear triatomic oscillator (L-Fe-L) and masses of 56, 67, and 70 amu for Fe, h3-Im-, and d3-Im-, respectively, a downshift of-2.7 cm-1 is calculated. For a linear triatomic oscillator simulating the axial ligation of the MP8(Im-) complex (His-Fe-L) and masses of 56, 81, 67, and 70 amu for Fe, His-, h3-Im- and d3-Im-, respectively, a 1.4 cm-1 downshift is estimated. These frequency shifts are moreover expected to be significantly decreased when a mixing of the νas mode with a pyrrole tilt is considered.41. (75) Collins, D. M.; Countryman, R.; Hoard, J. L. J. Am. Chem. Soc. 1972, 94, 2066. (76) Scheidt, W. R.; Osvath, S. R.; Lee, Y. J. J. Am. Chem. Soc. 1987, 109, 1958. (77) Quinn, R.; Strouse, C. E.; Valentine, J. S. Inorg. Chem. 1983, 22, 3934.

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