Involvement of Propionate Side Chains of the Heme in Circular

Article pubs.acs.org/JPCB

Involvement of Propionate Side Chains of the Heme in Circular Dichroism of Myoglobin: Experimental and Theoretical Analyses Masako Nagai,*,†,§ Chika Kobayashi,‡ Yukifumi Nagai,† Kiyohiro Imai,‡ Naoki Mizusawa,†,‡ Hiroshi Sakurai,§ Saburo Neya,∥ Megumi Kayanuma,⊥ Mitsuo Shoji,# and Shigenori Nagatomo*,○ †

Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo 184-0003, Japan Department of Frontier Bioscience, Faculty of Bioscience and Applied Chemistry, Hosei University, Koganei, Tokyo 184-8584, Japan § School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa 920-0942, Japan ∥ Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Inohana, Chuo-Ku, Chiba 260-8675, Japan ⊥ Department of Computer Science, Graduate School of Systems and Information Engineering, University of Tsukuba, Tsukuba, Ibaraki 305-8573, Japan # Department of Physics, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan ○ Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan ‡

S Supporting Information *

ABSTRACT: Incorporation of the heme into globin induces a prominent circular dichroism (CD) band in the Soret region. The appearance of heme optical activity is widely believed to arise from the interaction between the heme and aromatic residues of the globin. However, hemoglobin (Hb) containing the reversed heme exhibits a CD spectrum obviously different from that of native Hb, indicating that the interactions of heme side chains with globin contribute to the appearance of heme optical activity. We examined this possibility by comparing CD spectra of native myoglobin (Mb) and those of Mb reconstituted with synthetic hemes lacking vinyl and/or propionate. Replacement of 2,4-vinyl groups with methyl induced moderate changes. In contrast, replacement of 6,7propionate groups with carboxylate resulted in complete disappearance of the positive Soret CD band. To get theoretical basis for the contributions of 6,7-side chains on the band, we investigated the CD spectra at a time-dependent density functional theory level. In the antiparallel conformation of the 6,7-side chains, the rotational strengths were calculated to be positive, on the other hand in the parallel conformation to be negative. We also found that the weak Soret CD band in 2,4-dimethyl-6,7-dicarboxyheme can be explained by canceling between different carboxyl conformers.

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observation indicates that peptide π → π* transitions are also involved to couple with heme π → π* transition, but this contribution was not predicted to be significant in holoproteins where the heme is completely surrounded by peptide residues.5 Recently we have demonstrated that the Hb with a reversed heme exhibits a prominent negative CD band in the Soret region.6 Even if the heme is reversed in Hb, the arrangement of surrounding aromatic residues relative to the heme remains unchanged. On the other hand, the reversed heme brings about an interchange of the 1,3-dimethyl groups with the 2,4-divinyl groups and possibly changes of the direction of 6,7dipropionate groups,7 suggesting that the interaction of the

INTRODUCTION

Circular dichroism (CD) is one of the powerful probes of protein structure in solution. Isolated heme is symmetrical and optically inactive. When it is bound to proteins, however, the heme-protein interaction induces Cotton effects. CD spectra of hemoproteins in the 250−650 nm region arise from its heme moiety. Most of vertebrate myoglobin (Mb) and hemoglobin (Hb) exhibit positive CD bands in the Soret (B) and visible (Q) regions, while Hbs of mollusca1 and hagfish2 show negative CD bands. Hsu and Woody 3 proposed that coupled oscillator interactions in the π → π* transition between porphyrin and aromatic residues could account for the sign and magnitude of the observed CD band of hemoproteins. However, the heme with undecapeptide without aromatic residues was found to exhibit an optical activity of the same order of magnitude as that observed for native Mb in the Soret region.4,5 This © 2014 American Chemical Society

Received: August 26, 2014 Revised: November 26, 2014 Published: December 19, 2014 1275

DOI: 10.1021/jp5086203 J. Phys. Chem. B 2015, 119, 1275−1287

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The Journal of Physical Chemistry B

Figure 1. Structure of the heme derivatives used in the present experiments: 1, protoheme; 2, mesoheme (2,4-diethyl); 3. 2,4-dimethyldeuteroheme; 4, α-ethyl-2,4-dimethyldeuteroheme; 5, 2,4-dimethyl-6,7-diacetateheme; 6, 2,4-dimetyl-6,7-dicarboxyheme; 7, etioheme; 8, meso-tetramethylheme.

heme (6),18 etioheme I (7),19 and meso-tetramethylheme (8)20 were prepared as described. We expressed each individual heme by number as defined in Figure 1. Reconstituted Mb. Horse heart Mb was purchased from Sigma. ApoMb was prepared using acid methyl ethyl ketone.21,22 ApoMb was mixed with a 1.2-fold molar excess of hemin dissolved in a small amount of 50 mM potassium hydroxide. The crude mixture was extensively dialyzed overnight against large amount of cold 10 mM Tris at pH 7.0 to remove methyl ethyl ketone and residual unbound hemin, and was loaded on a carboxymethylated cellulose (Whatman, CM 52) column equilibrated with the same buffer to remove apoMb. After apoMb was washed out by the equilibrated buffer, absorbed reconstituted Mb’s on the column were eluted with 0.1 M Tris, pH 7.0. Purities of reconstituted Mb’s were examined to compare their absorption spectra with those in references. We used the ratio of the height of Soret absorption band of aquomet-form to 280 nm optical density of protein band over 3 as one of criteria for purity. Each reconstituted Mb was expressed like Mb3, if apoMb was reconstituted with heme 3. Spectroscopic Measurements. The electronic absorption spectra were recorded on a model U3010 spectrophotometer (Hitachi, Tokyo) at 20 °C. CD spectra of the reconstituted Mb’s (40 μM in heme) in a 0.05 M phosphate buffer, pH 7.0, were obtained using a model J 820 spectropolarimeter (Jasco, Tokyo) with a cell of path length of 2 mm (UV to Soret region) or 5 mm (visible region) at 20 °C. The scan speed was 50 nm/min and 20 scans were averaged. The molar CD (Δε) is given in M−1cm−1, calculated per heme. Theoretical Calculation of the Soret CD (Computational Details). All the calculations were performed using the Gaussian 09 program package.23 The initial coordinates of hemes were taken from the X-ray structure of an oxyHb A

heme side chains with globin could also contribute to the Soret optical activity. The heme incorporated to apoMb through the Fe-His(F8) coordination bond is stabilized through hydrophobic interactions between the vinyl groups and nonpolar amino acid residues, and further fixed through the hydrogen bond networks of the heme propionate groups.8,9 X-ray crystallographic analysis of horse heart Mb has revealed that the heme6-propionate forms a salt bridge with Lys45 (Arg45 in sperm whale Mb). The heme-7-propionate interacts with His97 and Ser92, which forms a hydrogen bond with His93 (F8).10 These propionate groups are believed to be one of the major factors for the incorporation, orientation and function of the heme within hemoproteins.11−13 Recently the contribution of 2,4divinyl groups to chirality of the heme has been theoretically examined by Woody and Pescitelli.14 However, analyses of the involvement of 6,7-dipropionate groups of the heme in CD spectra remain to be pursued. In order to examine the contribution of heme side chains to optical activity of the heme and to oxygen binding function, we prepared Mb with unnatural hemes lacking the vinyl and/or propionates. The involvement of propionate side chains to the Soret CD spectra was also examined by theoretical calculations. We demonstrated for the first time experimentally and theoretically that 6,7-dipropionate groups of Mb contribute to rotational strength of the Soret CD band.

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MATERIALS AND METHODS Structures of the Hemes Used for Reconstitution. Figure 1 shows the structures of the eight heme groups used in the present experiments. Protoheme (1) and mesoheme (2) were purchased from Aldrich. 2,4-Dimethyldeuteroheme (3) was prepared as reported previously.15 α-ethyl-2,4-dimethyldeuteroheme (4),16 6,7-diacetateheme (5),17 6,7-dicarboxy1276


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Figure 2. Absorption spectra of Mb’s in aquomet (Met, red), CO (blue), cyanomet (CNmet, brown) forms used for CD measurements, in 0.05 M phosphate buffer, pH 7. Concentrations of each Mb are between 28 and 55 μM.

(PDBID: 2DN1).24 We constructed four theoretical heme models, HEM3 (2,4-dimethyl-6,7-dipropionate), HEM4 (αethyl-2,4-dimethyl-6,7-dipropionate), HEM5 (2,4-dimethyl-6,7diacetate), and HEM6 (2,4-dimethyl-6,7-dicarboxylate) by substituting the peripheral side chains. Furthermore, we constructed two theoretical models for HEM4, HEM4′ and HEM4″, to characterize the nonplanarity of heme and α-ethyl groups. Each heme model contains a low-spin Fe, modified porphyrin and two axial ligands of one water molecule and 4methylimidazole as an analogue for the histidine side chain. Full geometrical optimizations were performed in vacuum under

constraints of dihedral angles of propionates and acetates for, HEM3, HEM4, and HEM5, respectively. In addition, the carboxyl groups of HEM3, HEM4, and HEM5 were protonated. These constraints on the acid side chains were found to be necessary to maintain the conformation without globin. Applied theoretical level for the geometrical optimizations was the UB3LYP functional and LANL2DZ and 6-31G* basis sets for iron atom and for other atoms, respectively. UV and CD spectra were calculated at the TDDFT level with the CAM-B3LYP and 6-311G* basis sets. In the geometrical optimizations, the iron atoms were treated as Fe(III), while for 1277


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The Journal of Physical Chemistry B Table 1. Absorption Maxima (λmax, nm) and Millimolar Extinction Coefficient (εmM, mM−1 cm−1, in Parentheses) of Reconstituted Mb’s visible (Q bands) Mb

ligand

Mb128

Aquomet CNmet CO Aquomet CO Aquomet CO Aquomet CO Aquomet CO Aquomet CO Aquomet CO CNmet CO

Mb228 Mb316 Mb416 Mb5a Mb618 Mb719 Mb829 a

ASoret(met)/A280

Soret (B band) 408 423 423 395 409 394 409 403 420 395 410 397 415 393 408 421 424

(188) (126) (201) (172) (206) (148) (183) (138) (151) (120) (188) (163) (173) (172) (221) (82) (162)

500 541 541 495 529 495 530 507 550 496 530 498 535 492 529 571 556

(11.6) (13.0) (17.8) (8.8) (14.6) (7.0) (10.7) (6.8) (9.1) (5.4) (9.4) (6.4) (11.7) (7.9) (13.2) (4.4) (5.9)

630 (4.7) 578 622 557 625 557 628

(15.6) (4.1) (11.6) (2.9) (8.0) (2.3)

625 560 629 564 624 557 609 599

(2.4) (7.8) (3.1) (8.5) (3.5) (11.0) (4.4) (4.5)

5.1 3.4 7.0 6.3 5.0 5.3 5.0 3.6 2.8

Millimolar extinction coefficients of Mb5 were determined by the present study.

280 nm globin band indicating a high purity of the reconstituted proteins. All Soret bands from Mb2 to Mb8, as compared with Mb1 (protoheme), exhibit a blue shift of 5−10 nm. Although milimolar extinction coefficients of these Mb’s were taken from references, wavelengths of the absorption maxima were almost the same with the values of references within 3 nm. After measurements of absorption and CD spectra of aquomet Mb, ferrous-CO derivatives were prepared by the addition of a small quantity of sodium dithionite powder in CO atmosphere. Modification of 2,4-Divinyl Groups. The protoheme is the prosthetic group of Mb, Hb, catalase, and cytochrome P450. The hydrophobic interaction of 2,4-divinyl side chains of the protoheme with globin and hydrogen bond networks of 6,7dipropionate groups are thought to be important for physiological function of oxygen binding.27 First, to examine the influence of the modification of these vinyl groups on the CD spectra, we prepared two Mb’s reconstituted with heme 2 (2,4-divinyl → diethyl) and heme 3 (2,4-divinyl → dimethyl). As shown in Figure 2 and Table 1, the absorption peak of the Soret band of native Mb (Mb1) in the aquomet form (408 nm) were blue-shifted in both Mb2 (395 nm) and Mb3 (394 nm) due to the decreased π conjugation pathway after removal of the heme vinyl groups. Figure 3 shows the structures of hemes 1, 2 and 3 and CD spectra in the Soret region of aquomet Mb2 and Mb3 are compared with that of native Mb (Mb1). Their peaks of the Soret CD bands were also blue-shifted reflecting their blue-shifted absorption maxima. Although both ellipticities of the Soret CD bands of Mb2 and Mb3 were decreased, they exhibited positive bands similar to that of native Mb (Mb1). These results indicate that the modification of 2,4-divinyl groups of the protoheme exerts only moderate influences on the CD spectra of native Mb. Figure 4 shows the whole CD spectra of Mb2 and Mb3 in the near-UV, Soret and visible regions in the CO and aquomet forms compared with those of Mb1. The spectra of Mb2 are very similar in shape to those of native Mb (Mb1) in the aquomet form, although the ellipticities of the Soret (B) and 260 nm (L) bands in the CO form are reduced to 60−70% of

the TDDFT calculations, the iron atoms were treated as Fe(II) to evaluate accurate CD spectra. Details of the computational conditions are summarized in the Supporting Information. TDDFT results for Fe(III) atoms were also presented in the Supporting Information.

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RESULTS AND DISCUSSION Heme Inside and Outside of Globin. Because of its symmetry, the porphyrin alone is optically inactive. When it is bound to the proteins, Cotton effects are induced by the hemeprotein interactions. In principle, the sign and magnitude of these effects should yield information about the nature of the heme binding sites. Hsu and Woody3 proposed the following three possible mechanisms leading to rotational strength in the Soret band. First, nonplanarity of the porphyrin in Mb could make the heme an inherently dissymmetric chromophore. Second, mixing of the Soret transition and d−d transition of the iron could lead to magnetic dipole character in the Soret transition. Third, the Soret Cotton effect may be due to coupling of the Soret transition and excited states of the globin backbone and side chains. The allowed π → π* transition of aromatic side chains, π → π* and n → π* transitions in the peptide backbone, and σ → σ* transitions in alkyl side chains are considered as main candidates. From the calculation of rotational strength caused by interactions with environmental electric and magnetic dipole transition moments, they concluded that the only mechanism which can account for observed Cotton effects is a coupled oscillator between the heme transition and allowed π → π* transition in near-by aromatic side chains.3 Molecular dynamics studies give a picture of nonplanar distortions of the heme and its interactions with the protein matrix, which are believed to be the source of Soret CD.25,26 Absorption Spectra. Figure 2 shows the absorption spectra of ferric (aquomet) and ferrous-CO forms of eight Mb’s in the UV−visible region. Table 1 summarizes their absorption maxima and the millimolar extinction coefficients in parentheses. As shown in the table, the magnitude of the Soret bands of aquomet Mb’s are 3−7-fold higher than that of the 1278


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native Mb (Mb1). The CD spectra of Mb3 are also similar in shape to those of normal Mb, but the ellipticities are reduced to 70% in the aquomet form and to 40% (Soret) and 60% (visible) in the CO form comparing with those of native Mb (Mb1). The ethyl side chains of heme 2 are more effective than the methyl side chains of heme 3 in induction of the optical activity. From the decrease in intensity, it seems that the length of alkyl side chain is important for induction of optical activity of the heme while the double bond in vinyl side chains at 2,4positions plays a minor role. The CD spectra of visible region correspond well with the absorption maxima. OxyMb is of the type in which two CD bands are at positions corresponding to the α and β bands, whereas the COMb spectrum exhibits splitting CD of both the α and β bands as shown in Figure 4 (upper right spectrum of Mb1 (blue)), suggesting a reflection of lowered symmetry of the heme due to a nonlinear complex.30 However, these band splittings are not obvious in COMb2 (red) and almost no splitting in COMb3 (green) (Figure 4, upper right spectra) and the CD spectra became similar to that of oxyMb of linear coordination.31 Distorted Heme. Recently, Neya et al.16 synthesized heme 4 and prepared its reconstituted Mb, Mb4. A minimum energy calculation has suggested that the heme is distorted to nonplanar.16 In present study, we confirmed nonplanarity of heme 4 by theoretical calculation, and discussed below in the Theoretical Calculations. The reconstituted Mb with distorted heme 4 showed a very low oxygen affinity.16 Although the absorption peaks of Mb4 were red-shifted in both the Soret and visible regions compared with Mb3 (Figure 2 and Table 1), the shape of absorption spectrum was similar to those of Mb3. As shown in the inset of Figure 5, the structure of heme 4 is different from heme 3 only in the ethyl attachment to the αmeso position from heme 3. Figure 5 shows CD spectra of Mb4 in the (245−480) nm and (480−680) nm regions. The distorted heme alone was also not optically active as shown in a green noisy trace in the figure. However, CD spectra of Mb4 were strikingly different from Mb3. Both the aquomet and CO forms of Mb4 exhibited negative Soret CD bands and a complex CD with the positive and negative bands in the visible region. These results indicate that the distorted heme can also induce prominent CD spectra in globin, although the heme itself has no optical activity. It should be noted that CD spectra of Mb4 with the distorted-heme were greatly different from those of undistorted one, suggesting that distortion of heme structure could cause substantial changes in the interaction between the heme side chains and globin. Theoretical calculation also supported this suggestions as shown later. Modification of 6,7-Dipropionates. We subsequently examined the contribution of 6,7-dipropionate side chains of the protoheme to the CD spectra of Mb. Figure 6 shows the structures of three modified hemes used, heme 3 (6,7dipropionates), heme 5 (6,7-diacetates), and heme 6 (6,7dicarboxylates), and Soret CD spectra of Mb’s with these modified hemes in the aquomet form at pH 7. A slight red-shift of the Soret absorption bands was observed in Mb5 and Mb6 (Figure 2 and Table 1). As can be seen, the Soret CD bands of Mb’s with both the 6,7-diacetate heme (Mb5) and the 6,7dicarboxylate heme (Mb6) were also slightly red-shifted. Onecarbon shortening from the propionate to acetate heme (Mb5) could induce the optical activity of the Soret band. In contrast, two-carbon shortening of the 6,7-dipropionates to 6,7dicarboxylates heme (Mb6) resulted in complete disappearance

Figure 3. Structures of the heme groups used for reconstitutions and CD spectra of Mb1 (blue), Mb2 (red) and Mb3 (green). (Top) Structures of hemes kept by Mb1 (2,4-divinyl), Mb2 (2,4-diethyl), and Mb3 (2,4-dimethyl). (Bottom) CD spectra; Mb1 (blue), Mb2 (red), and Mb3 (green) in the aquomet form at pH 7.0, respectively.

Figure 4. Comparison of CD spectra of Mb2 (red) and Mb3 (green) with Mb1 (blue) in the spectral region between 245 and 680 nm in the CO (upper) and aquomet (lower) forms in 0.05 M phosphate buffer, pH 7.0.

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Figure 5. CD spectra of Mb4 in the aquomet (red) and CO forms (blue) at pH 7. Inset: the structure of heme 4. CD spectrum of heme 4 alone was shown in the figure as a noisy green trace around the zero line.

Figure 7. Comparison of CD spectra of Mb5 (red) and Mb6 (green) with Mb3 (blue) in the spectral region between 245 and 650 nm in the CO (upper) and aquomet (lower) forms in 0.05 M phosphate buffer, pH 7.0.

Figure 6. CD spectra of Mb’s with modified heme at the 6,7-potitions in the Soret region. (Top) Structures of the hemes used for reconstitution: heme 3 (6,7-dipropionates); heme 5 (6,7-diacetates); heme 6 (6,7-dicarboxylates). (Bottom) CD spectra of Mb3 (blue), Mb5 (red), and Mb6 (green) in the aquomet form at pH 7.0.

although the spectral shapes were similar to Mb3 (6,7dipropionates). In contrast, Mb6 (6,7-dicarboxylates) exhibited a small negative CD bands in the aquomet form as shown in Figure 7 (lower panel). However, Mb6 shows CD bands at the 260 nm (L) and visible (Q) regions in the CO form but does not exhibit distinct Soret CD band. In the visible region, two positive CD bands at the positions corresponding to the α and β absorption bands were seen in the spectra of COMb5 and COMb6 with no band splitting. Relation between Intensity of the Soret CD and Oxygen Affinity of Mb’s. Mb is an oxygen storage protein in muscle. Oxygen, transferred from Hb to Mb, is released for the

of the positive Soret CD band and was replaced by a small negative band at 396 nm. This observation indicates that the interaction of heme propionate groups with globin seems to be responsible for the appearance of the prominent positive CD bands in the Soret region, and that the acetate side chains at 6,7-positions could interact with globin to induce appreciable optical activity of the heme in Mb. The whole CD spectra of Mb5 and Mb6 are shown in Figure 7 in comparison with those of Mb3 in both the aquomet and CO forms. As shown in Figure 7, the replacement from propionate to acetate caused a slight decrease in CD intensity, 1280


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heme side chains, 2,4-divinyls and 6,7-dipropionates, with globin moiety contributes somewhat to physiological function of myoglobin. The optimal oxygen affinity, i.e., that of native Mb, is attained by the heme with highest CD intensity. However, the present correlation between oxygen affinity of Mb’s and intensity of Soret CD band shown in Figure 8 could not be said to be the general rule for Mb. It is known that the two orientational isomers (normal and reverse) are present in the reconstituted Mb1 and Mb2 with protoporphyrin IX heme (heme1) and heme 2, respectively. However, it is Mb1 and Mb2 only that have two orientational isomers of reconstituted Mb’s shown in Figure 8. In present experimental procedure of reconstituted Mb1, ratios of normal form to reverse form for Mb1 is almost the same one for native sperm whale Mb (normal:reverse = 92:8),32 since product (reconstituted Mb1) of aquomet form is kept in solution for 3 days after apoMb is reacted with heme1(Fe3+). Therefore, Figure 8 can be valid for Mb1 and Mb2 keeping normal orientation mainly and for Mb3, Mb4, and Mb6 having no orientational isomers, assuming that reconstituted Mb2 also has ratio of normal form to reverse form as much as reconstituted Mb1. We, of course, know that oxygen affinities of two isomers were unchanged but the intensities of Soret CD band were different.33,34 Furthermore, we separated this orientational isomers (normal and reversed) in the recombinant Hb by SPSepharose column chromatography and showed that Hb with normal heme exhibited the positive Soret CD, while that with reversed heme did the negative band.6 Oxygen affinity of Hb with the reversed heme was not significantly different from that of normal one. The differences of both orientations is only exchange of vinyl side chain at position 2 and 4 with methyl group at position 3 and 1, respectively, but vinyl groups are present in both cases indicating that vinyl side chains are necessary for normal oxygen affinity of Mb and Hb. The CD changes from positive to negative in orientational isomers may be caused from the change of the direction of propionate side chains at positions 6 and 7 as shown later. CD Spectra of Mb’s with the Heme Lacking both the 2,4-Divinyls and 6,7-Dipropionates. Heme 7 (etio) is an alkyl heme having four methyl and four ethyl groups as shown

cellular respiration. Therefore, oxygen affinity should be kept around 1 mmHg (native Mb). If oxygen affinity was higher than this level, oxygen could not be released to tissues smoothly. If oxygen affinity was lower, Mb could not store oxygen efficiently. Figure 8 shows the relation of intensity of the

Figure 8. Relation between intensity of the Soret CD band and oxygen affinity of Mb’s.

Soret CD band and oxygen affinity (partial pressure of oxygen at 50% saturation, P50) of five Mb’s with the modified heme either 2,4-divinyls or 6,7-dipropionates. As can be seen, it showed bell-shaped curve. CD intensity of COMb’s was obtained in the present work and oxygen affinities of Mb’s were from references; Mb1,19 Mb2,27 Mb3,15 Mb4,16 and Mb6.18 Mb1 (native, protoheme) shows the highest intensity of the Soret CD band, whereas Mb2 (2,4-diethyl) and Mb3 (2,4dimethyl) with high oxygen affinity exhibit low CD intensity. In addition, Mb6 (6,7-dicarboxylates) and Mb4 (distorted heme) with low oxygen affinity display also a very small or negative Soret CD band. This finding suggests that the interaction of

Figure 9. CD spectra of Mb7 (etioheme) compared with those of Mb1 (protoheme). (Inset figure) Heme structure of heme 7 (etioheme). CD spectra of Mb1 (dotted lines) and Mb7 (solid lines) in the aquomet (red) and CO (blue) forms at pH 7. 1281


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Figure 10. CD spectra of Mb8 compared with those of Mb1 in the cyanomet and CO forms at pH 7.0. Inset: The structure of heme 8. CD spectra: red, cyanometMb1; blue, COMb1; magenta, cyanometMb8; green, COMb8.

spectra of the visible region in aquomet form are different from those in CO form as shown in Figure 7. Though both CD spectra of the Soret and visible region of Mb7 are subtly positive in CO form and almost none in aquomet form, their CD intensities are much smaller than those of Mb1 as shown in Figure 9. The Soret CD bands in Mb8 are subtly negative in both cyanomet and CO forms, and CD bands in the visible region are almost none in both forms. In a similarity to Mb7, CD intensities of Mb8 are much smaller than those of Mb1 as shown in Figure 10. Moreover, it is shown that the splitting of CD band in both the α and β bands of native Mb (Mb1) in CO form was not observed in the CD spectra of Mb’s with heme lacking of vinyl side chains.38 Thus, though CD spectra in the visible region have more information than that of Soret band potentially, the spectra are too complicated to carry out detailed analyses practically. Therefore, we consider that information on π → π* transition is reflected to Soret CD bands, and in next sections we investigate properties of π → π* transition of the heme by theoretical calculations in more details.

in the inset of Figure 9. Heme 7 can bind stoichiometrically with apoMb to allow coordination of the proximal histidine.19 From a similarity of the visible absorption spectrum of Mb7 to that of native Mb (Mb1) (Figure 2), it was suggested that Mb7 takes normal iron coordination character.19 Oxygen and CO can also bind reversibly to this Mb. As shown in Figure 9, greatly diminished CD spectra of the Mb7 were observed in both the aquomet and CO forms, suggesting that both the 2,4divinyl and 6,7-dipropionate side chains are involved in induction of CD spectra of Mb. Heme 8, iron complexes of meso-tetramethylporphyrin with D4h symmetry, has no peripheral side chain on the pyrrole moiety. Neya and Funasaki20 synthesized heme 8 and prepared Mb8. 1H NMR spectra of Mb8 in the cyanomet form exhibited a single pyrrole-proton resonance, indicating that the alkyl heme of Mb8 is in dynamic free rotation about the Fe−N(HisF8) bond.35 Figure 10 shows the CD spectra of Mb8 in the cyanomet and CO forms in comparison with those of native Mb (Mb1). Mb8 exhibits a very small negative CD bands in the Soret region and no discernible CD spectra in the visible region, indicating that peripheral groups of the heme are important for induction of CD spectra of Mb. Hemes 7 and 8 do not change π-system of porphyrin but exhibit no Soret CD band. These results, taken together, also suggest that the interaction between heme side chains and the globin is surely responsible for an appearance of the CD spectra of Mb. CD Spectra of Mb’s in the Visible Region. Previous studies indicate that both CD spectra in the Soret and visible regions of Hb and Mb derive from mainly π → π* transition of porphyrin.36,37 However, contributions of other transition such as d → d transitions of Fe atoms and/or charge transfer transition between Fe atom and ligand are included in CD spectra of the visible region.36 Therefore, a sign (positive or negative) of Soret CD bands is not always consistent with CD bands of visible region. In the present study, CD bands of the Soret and visible region of Mb1, Mb2, Mb3, and Mb5 are positive in both forms (aquomet and CO forms). Whereas the Soret CD bands of Mb4 are negative in both aquomet and CO forms, but CD spectrum in the visible region exhibits a complex CD with a negative and a positive bands. On the other hand, the Soret CD bands of Mb6 are subtly negative, but CD spectra in the visible region are positive in both forms, though CD

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THEORETICAL CALCULATIONS Relation between Conformation of 6,7-Dipropionate and Rotational Strength. In order to investigate origins of the CD spectra, theoretical calculations were carried out for aquomet myoglobin reconstituted with heme 3, heme 5, and heme 6. These computational heme models were named as HEM3, HEM5 and HEM6, respectively. Conformations of propionate/acetate/carboxyl groups were generated based on the conformations observed in X-ray structures. In X-ray structures of Hb A (oxy- and/or CO-forms), the heme propionates alternate in up and down side (antiparallel) conformation or extend to the same side (parallel) conformation against heme plane in the α and β subunit, respectively.24 In most of myoglobin, the propionates are in the antiparallel conformation with respect to the heme plane. Actually, the crystal structure of sperm whale Mb indicates that two propionate side chains linked at the 6 and 7 positions of the heme interact with Arg45 and Ser92/His93, respectively.39,40 The crystal structure of oxyMb revealed two unique hydrogen bond networks, one is the distal site network of the 6propionate-Arg45-H2O-His64(E7)-bound oxygen, and the 1282


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used, because considerable excitations from higher spin states were observed in Fe(III) models. Resulting of Fe(III) models were shown in the Supporting Information. It is noted that qualitative features of CD spectra in the 300−450 nm region were unchanged by the iron valence state. The antiparallel and parallel conformations of the acid chains are indicated by subscripts “a” and “b”, respectively. Rotational strengths for HEM3 and HEM5 models at the two optimized conformations were calculated and shown in Figure 14. Positive peaks at 358 and 352 nm were calculated for HEM3a and HEM5a, and negative peaks at 357 nm for both HEM3b and HEM5b. Rotational strengths of a-conformers are larger than those of b-conformers. In native Mb, alternate aconformer is mainly observed in X-ray crystallographic structures.39,40 Therefore, it is expected that conformations of propionates/acetates of Mb3 and Mb5 are alternating. The CD spectra in Figure 3 also show large rotational strengths of Mb1 (native Mb), Mb2 and Mb3. Though Woody and Pescitelli reports that rotational strength of heme CD depends on dihedral angle between heme plane and vinyl group,14 the CD spectrum of Mb3 (Figures 3 and 6) and rotational strength of HEM3a based on our theoretical calculation show that alternate a-conformations of two propionates are significant for large positive rotational strengths. Rotational strength of Mb3 without vinyl group is derived from alternate a-conformations of two propionates. These results demonstrate that the conformation of propionate is also important as well as that of the heme vinyls. Furthermore, constraints of HEM3 and HEM5 were found to be necessary to maintain the conformation without the protein. These results suggest that propionates and acetates of Mb3 and Mb5 also interact with globin (protein) to keep alternate a-conformations for two acid side-chains against heme plane. Relative total energies (ΔE/kcal mol−1) calculated for aconformations and b-conformations in HEM3 and HEM5 are also shown in Table SI5. Calculated CD spectra of HEM3 and HEM5 are slightly blue-shifted by 3−10 nm compared to the calculated UV/vis absorption maxima at 361−363 nm (Figure 14 and Figure SI9). Excitations of the Soret absorption maxima are from the HOMO (HOMO−1) to the LUMO (LUMO+1) transitions, which are composed of porphyrin π and π* orbitals. Excitations for the CD peak tops contain higher energy excitations. We found that these near frontier MOs significantly depend on the propionates/acetates conformations. In HEM3 in Figure 13, LUMO and LUMO+1 of HEM3b are altered compared to LUMOs of HEM3a. In HEM5, distributions of MO167 (HOMO−2) are changed. Therefore, conformations of the propionates and acetates substantially contribute to asymmetric effects for the porphyrin π and π* MOs. All the MOs are shown in the Supporting Information (Figures SI1−SI8). Rotational strength of HEM6 models showed clear effect of the carboxyl conformations (Figure 15). Large positive rotational strength was evaluated for HEM6a, in contrast to the large negative rotational strength calculated for HEM6b. HEM6c and HEM6d have negligibly small rotational strengths. The main excitations of CD spectra are the same for absorption spectra and they are transitions from MO155 to MO162 (LUMO) and MO159 to MO163 (LUMO+1). MO155 mainly distributes on the carboxyl π group, MO159 is mainly porphyrin π orbital, and LUMO is mainly porphyrin π* orbitals. Interestingly, MOs of HEM6a are mirror images to MOs of HEM6b, where the mirror is vertical to the heme plane.

other is the proximal site network of 7-propionate-Ser92His93(F8). Accordingly, the additional characters “a” and “b” were added to designate the conformation a (antiparallel) and b (parallel) after HEM3 and HEM5 (Figure 11). In the

Figure 11. Schematic illustrations of HEM3 and HEM5 models at different conformations.

geometrical optimizations, full optimizations were performed under minimum constraints for propionate/acetate dihedral angles. For HEM6 models, four different conformations were obtained for the carboxyl groups by a series of full geometrical optimizations. Schematic views of the conformations in the theoretical models are shown in Figures 11 and 12 for HEM3, HEM5 and HEM6, respectively. All the optimized structures were summarized in Figure 13. All the geometrical optimizations were performed under the Fe(III) condition. For the rotational strength (Δε) calculations, Fe(II) state was

Figure 12. Schematic illustrations of dicarboxylheme models (HEM6s). Four different conformations of the carboxyl groups are shown. 1283


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Figure 13. Optimized structure of heme models. Imi indicates the axial ligand of 4-methylimidazole.

HEM3b (HEM5b) is symmetric. The excitations of HEM3 and HEM5 are transitions of porphyrin π orbitals, and that is not same for HEM6. However, Soret CD bands of HEM3a (HEM5a) are larger than those of HEM3b (HEM5b). Thus, the symmetric nature seems to be important. HEM6b-d conformers are less stable by 0.00, 0.88, and 0.64 kcal mol−1, respectively, than HEM6a conformer. Thermal population ratios calculated at the 293.15 K Boltzmann distributions are 1, 1.00, 0.22, and 0.33, for HEM6a−d, respectively. Rotational strength of HEM6a is positively so large, while rotational strength of HEM6b has completely opposite value. Therefore, total CD spectra of HEM6 at the thermal conditions (HEM6t in Figure 16) are canceled by

Figure 14. Calculated CD spectra for HEM3 and HEM5 models at the CAM-B3LYP/6-311G* level.

Figure 16. Calculated CD spectra for the major conformational states (HEM3a, HEM5a and HEM6t). HEM6t represent the Boltzmannaveraged spectrum at 293.15 K.

almost the same populations of HEM6a and HEM6b conformations. This explains the reason why rotational strength in CD spectra of Mb6 is very small. We show calculated rotational strength for the major conformational states (HEM3a, HEM5a) and HEM6t in Figure 16. In HEM6 (Figure 12), which has carboxylates instead of propionates, full geometrical optimizations were performed in vacuum under no constraints of dihedral angles. This assumes that interactions between two carboxyl groups of Mb6 and amino acid residue of globin are very weak. Though X-ray

Figure 15. Calculated CD spectra for HEM6 models at the CAMB3LYP/6-311G* level.

On the other hand, HEM6c and HEM6d are related to be mirror symmetry for the heme plane, if the axial ligands are neglected (Figure 12). The mirror symmetry in HEM6c and HEM6d does not contribute to large rotational strength. From the viewpoint of mirror symmetry vertical to the porphyrin plane, HEM3a (HEM5a) is asymmetric, while 1284


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QM-MM interactions. Even without the QM−MM interactions, i.e. QM model in vacuum condition, CD spectra are still positive maximized at 409 nm and the strength of the CD peak top decreased to about half. Therefore, our QM/MM model of heme 3-substituted Mb, HEM3@Mb, well reproduced the experimental CD spectra qualitatively. Figure SI15 shows a whole of QM/MM system of HEM6substituted Mb (HEM6a@Mb) including protein and water molecule (A) and enlarged QM regions (HEM6a) (B). Calculated CD spectra of HEM6@Mb showed a strong positive for HEM6a@Mb as shown in Figure SI16. Electrostatic contributions from the MM backgrounds were analyzed by neglecting interactions from the water solvent environment (NoMMWater) or by neglecting all the interactions from the MM region (NoMM). In HEM6a@Mb, water solvent contributed to a red-shift and the Mb protein environment increased the CD strength. Therefore, theoretical models supported the point that CD values are highly affected by the conformations of the propionate and carboxylate groups. In summary, our QM/MM models of heme-substituted Mb’s well characterized the CD spectra features, and it is supported that the conformations of the propionate and carboxylate groups strongly affected the CD spectra as shown by their QM models. Relation between Conformation of Nonplanarity of Heme (Mb4) and Soret CD. It is considered that nonplanarity of heme is also significant for a rotational strength of Soret CD. However, though heme 4 alone also indicates nonplanarity, its rotational strength of Soret CD is inactive, different from that of Mb4, reconstituted with heme 4. To investigate the reason, we carried out theoretical calculations. We used HEM4, HEM4′, and HEM4″ models as computational heme models. HEM4 is a full model of heme 4′. HEM4′ is a truncated model of heme 4′, where 6,7-dipropionate groups were substituted with 6,7-dimethyl groups to remove the effect of 6,7-dipropionate groups above-mentioned. It is because influences from the carboxyl group are minimized. HEM4″ is an all truncated model of heme 4′, where all the side chains in heme 4 are replaced to hydrogen atoms maintaining the other atomic coordinates. Therefore, only the heme nonplanarity can be examined using the HEM4″ model. Structures of HEM4, HEM4′, and HEM4″ are shown in parts A−C of Figure SI17, respectively. CD spectra calculated for the heme 4 models (HEM4, HEM4′, and HEM4″) are shown in Figure SI18. We demonstrated that the CD spectra of HEM4′ and HEM4″ are strongly affected by two factors: (1) heme nonplanarity and (2) conformation of side chains, α-ethyl and propionate groups. We showed that HEM4″ model has a nonzero (small positive) CD peak, indicating that the heme-nonplanarity contributes to the CD spectra. We also showed that the HEM4′ model has a negative CD peak, indicating that the α-ethyl group also substantially contributes to the CD spectra. In the case of heme 4 alone, it is supposed that net rotational strength may become achiral, because the conformation of the α-ethyl group is averaged for arbitrary direction. HEM4 model has positive CD peak at 352 nm. It is suggested that the effect of antiparallel conformation of 6,7-dipropionates causes the positive CD peak, because 6,7-dipropionates of HEM4 are constrained at antiparallel conformation (a-conformation of HEM3). Therefore, negative Soret bands of Mb4 would be attributed to parallel conformation (b-conformation of HEM3) of 6,7-dipropionates.

crystallographic structure of Mb6 shows that a carboxylate of two carboxyl groups interacts with Arg45, His64, Ser92, and His97, many of interactions are indirect through water molecules.18 Only two main interactions are found; the two distances between Nη (Arg45) and O (heme carboxylate) and between Nδ (His64) and O (heme carboxylate) are 2.83 and 4.09 Å, respectively.18 It is guessed that these interactions are weak in solution, since crystalline water molecules are expected to be much mobile, though Mb6 binds CN− as a sixth ligand to exhibit CD different from that of aquomet Mb6.18 It is supposed that release of crystalline water promotes free rotation around the Cβ (6 or C7 positions)−C (carboxyl) axis. Thus, the present theoretical calculations revealed the role of heme acid groups to control the rotational strength of the Mb Soret CD and accordingly explained the observed characteristic CD spectra of Mb3 (propionate), Mb5 (acetate), and Mb6 (carboxylate). Effect of Protein (Globin) for the Soret CD. Thus, far, we discussed a relation between Soret CD band and conformation of the 6,7-side chains and found that conformation of the 6,7-side chains is important for Soret CD band. Here, we investigate a contribution of protein for Soret CD band and show that its contribution for Soret CD band does not change a sign of Soret CD band. Before showing theoretical calculated results, we compared X-ray crystallographic structure of native Mb (Mb1)40 with that of Mb6.18 Note that though X-ray crystallographic structure of Mb1 is in aquomet form, that of Mb6 is in cyanomet form. Xray structures of native Mb (Mb1) and heme 6-substituted Mb (Mb6) were reported at atomic resolution. Superimposition of the Mbs showed that overall Mb structures are almost identical, and the root-mean-square deviation (RMSD) on the backbone Cα positions is 0.3 Å (PDB IDs are 1A6M40 and 1IOP18) as shown in Figure SI12. Side chain conformations of Mb (Mb1) and heme 6-substituted Mb (Mb6) are also very similar to each other, though only the side chain of Arg 45 is shifted from a close position to carboxyl group of heme to a distant position by shorting of the carboxyl group in heme 6. Therefore, except for the small Arg shift, electrostatic interactions from Mb1 are unchanged by the heme substitution. We performed QM/MM calculations to theoretically evaluate the influences (of shortening of the chain length of 6,7-propionate groups) affected by the heme substitutions. We constructed two QM/MM models of heme-substituted Mbs. First model is heme 3-substituted Mb (abbreviation: HEM3@ Mb). Original atomic coordinates were taken from the X-ray structure of normal Mb (PDBID: 1A6M) and the vinyl groups in protophyrin IX are substituted to methyl groups as with heme 6. The conformation of heme propionate groups is in the antiparallel arrangement. Therefore, this QM/MM model corresponds to a more realistic HEM3a (abbreviation: HEM3a@Mb = HEM3@Mb). Figure SI13 shows a whole of QM/MM system of HEM3a@Mb including protein and water molecule (A) and enlarged QM regions (B). Second model is heme 6-substituted Mb (abbreviation: HEM6a@Mb). Original atomic coordinates were taken from the X-ray structure of Mb6 (PDBID: 1IOP 18 ). The conformation of heme carboxyl groups corresponding to the HEM6a form shown by Figure 12 corresponds to HEM6a@ Mb in this model. Figure SI14 shows a result of QM/MM calculation of HEM3@Mb. Calculated CD spectra of HEM3@Mb showed positive peak maximized at 386 nm by full consideration of the 1285



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In our current study, it is suggested that the characteristic negative CD peak in Mb4 is due to both the heme nonplanarity and the conformation of the α-ethyl group. As another possibility, parallel conformation of 6,7-dipropionates may be also responsible for the negative CD peak.

Article

ASSOCIATED CONTENT

S Supporting Information *

Computational details and optimized structures of HEM3a, HEM3b, HEM5a, HEM5b, HEM6a, HEM6b, HEM6c, and HEM6d, Table SI1, UV/vis absorption lines calculated at the CAM-B3LYP/6-311G* level at Fe(II), Table SI2, CD lines calculated at the CAM-B3LYP/6-311G* level at Fe(II), Table SI3. UV/vis absorption lines calculated at the CAM-B3LYP/6311G* level at Fe(III), Table SI4, CD lines calculated at the CAM-B3LYP/6-311G* level at Fe(III), Table SI5, relative total energies (ΔE / kcal mol−1) calculated for the side chain conformations, Figure SI1, MO of HEM3a (Fe(II)), Figure SI2, MO of HEM3b (Fe(II)), Figure SI3, MO of HEM5a (Fe(II)), Figure SI4, MO of HEM5b (Fe(II)), Figure SI5, MO of HEM6a (Fe(II)), Figure SI6, MO of HEM6b (Fe(II)), Figure SI7, MO of HEM6c (Fe(II)), Figure SI8, MO of HEM6d (Fe(II)), Figure SI9, calculated UV/vis and CD spectra with the electronic excitation lines for HEM3a at Fe(II), Figure SI10, CD of HEM3 and HEM5 at Fe(III), Figure SI11, CD of HEM6 at Fe(III), Figure SI12, superimposed views of Mb and HEM6-substituted Mb, Figure SI13, HEM3-substituted myoglobin, Figure SI14, calculated CD spectra for the HEM3substituted myoglobin, Figure SI15, HEM6-substituted myoglobin, Figure SI16, calculated CD spectra for the HEM6substituted myoglobin, Figure SI17, Heme 4 models (HEM4, HEM4′, and HEM4″), Figure SI18, calculated CD spectra for the heme 4 models (HEM4, HEM4′, and HEM4″), and complete reference23. This material is available free of charge via the Internet at http://pubs.acs.org.

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CONCLUSION Our present findings have shown that heme side chains play important roles for both the induction of optical activity and function of Mb. In a role of heme side chain (propionate) for the induction of optical activity, we have demonstrated from the theoretical calculations that 6,7-diacid (dipropionate and diacetate) side chain conformations in heme substantially affect the Soret CD band. In addition, two different conformers of 6,7-diacid (dipropionate and diacetate) side chains, antiparallel and parallel, showed positive and negative rotational strength of Soret bands, respectively, in the heme models with propionates and acetates substitutions (HEM3 and HEM5). From the molecular orbitals analyses, these CD excitations are mainly contributed to the electronic transitions between near frontier molecular orbitals. In the antiparallel conformation (a-conformer) of the side chain, the MOs are perturbed asymmetrically vertical to the heme plane. Therefore, asymmetric perturbations seem to be important for large rotational strength. On the other hand, HEM6 (2,4-dimetyl-6,7dicarboxylate heme model) can take four carboxyl conformers. In the antiparallel conformations, HEM6a and HEM6b, positively and negatively large CD spectra were calculated, respectively, and in other conformations, HEM6c and HEM6d, negligible small CD spectra were calculated. The thermal average of these spectra (HEM6t) showed that CD band almost disappears by canceling of opposite contributions from carboxyl conformers. Therefore, calculated CD spectra for HEM3a, HEM5a and HEM6t can correspond to the CD spectra observed in Mb3, Mb5, and Mb6. Regarding a role of heme side chains for the function, it was shown experimentally that Mb with the highest chirality in the Soret region gave an appropriate oxygen affinity. This indicates that both the vinyl and propionate side chains of the protoheme are critical for the induced optical activity and function of Mb. In this study, contributions from the 6,7-diacid side chains to rotational strength of Soret bands were intensively investigated through both experimental and theoretical approaches. It is noted that CD spectra in the Soret region are not completely determined only by the 6,7-diacid side chain conformations. However, we consider that the 6,7-diacid side chain conformations strongly affect the rotational strength of Soret bands. To investigate a contribution of protein for Soret CD band, we performed QM/MM calculation and showed that its contribution for Soret CD band does not change a sign of the Soret CD band. Finally, we emphasize that CD spectra of hemes contain valuable information for the 6,7-diacid side chain conformations and the motilities. The Soret CD band will be greatly valuable to obtain fine structural changes of hemoproteins. In particular, we will stress that CD spectra of Mb3 and Mb5 enable us to determine a conformation of propionate and acetate against heme plane. Moreover, we demonstrated that nonplanarity of heme and conformations of α-ethyl group may cause a strong negative Soret CD spectrum. Nonplanarity of heme and conformations of α-ethyl group are also significant for a rotational strength of the Soret CD.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +81-42-387-5120. Fax: +81-42-387-5121. E-mail: [email protected] (M.N.). *Telephone: +81-29-853-5768. Fax: +81-29-853-6503. E-mail [email protected] (S.N.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Science and Sport, and Technology of Japan to K.I. (22570217) and S.N. (Saburo Neya) (23590040) and by a research grant from the Research Center for Micro-Nano Technology, Hosei University (to M.N., K.I., and N.M.).

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ABBREVIATIONS CD, circular dichroism; CO, carbon monoxide; Mb, myoglobin; Hb, hemoglobin; oxyMb, oxygenated myoglobin; COMb, carbon monoxide myoglobin; oxyHb A, oxygenated human adult hemoglobin; TDDFT, time-dependent density functional theory; MO, molecular orbital; UB3LYP, unrestricted density functional theory with the B3LYP (Becke 3-parameter Lee− Yang−Parr) hybrid functional; LANL2DZ, Los Alamos National Laboratory 2 double-ζ effective core potential; CAMB3LYP, coulomb attenuating method based on B3LYP

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REFERENCES

(1) Boffi, A.; Wittenberg, J. B.; Chiancone, E. Circular Dichroism Spectroscopy of Lucina I Hemoglobin. FEBS Lett. 1997, 411, 335− 338. 1286


Article

The Journal of Physical Chemistry B (2) Sugita, Y.; Dohi, Y.; Yoneyama, Y. Circular Dichroism of Lamprey and Human Hemoglobins. Biochem. Biophys. Res. Commun. 1968, 31, 447−452. (3) Hsu, M.-C.; Woody, R. W. The Origin of the Heme Cotton Effects in Myoglobin and Hemoglobin. J. Am. Chem. Soc. 1971, 93, 3515−3525. (4) Urry, D. W.; Pettegrew, J. W. Model Systems for Interacting Heme Moieties. II. The Ferriheme Octapeptide of Cytochrome c. J. Am. Chem. Soc. 1967, 89, 5276−5283. (5) Blauer, G.; Sreerama, N.; Woody, R. W. Optical Activity of Hemoproteins in the Soret Region. Circular Dichroism of the Heme Undecapeptide of Cytochrome c in Aqueous Solution. Biochemistry 1993, 32, 6674−6679. (6) Nagai, M.; Nagai, Y.; Aki, Y.; Imai, K.; Wada, Y.; Nagatomo, S.; Yamamoto, Y. Effect of Reversed Heme Orientation on Circular Dichroism and Cooperative Oxygen Binding of Human Adult Hemoglobin. Biochemistry 2008, 47, 517−525. (7) Perutz, M. F. Myoglobin and Haemoglobin: Role of Distal Residues in Reactions with Haem Ligands. Trends Biochem. Sci. 1989, 14, 42−44. (8) Hargrove, M. S.; Wilkinson, A. J.; Olson, J. S. Structural Factors Governing Hemin Dissociation from Metmyoglobin. Biochemistry 1996, 35, 11300−11309. (9) Takano, T. Structure of Myoglobin Refined at 2.0 Å Resolution. II. Structure of Deoxymyoglobin from Sperm Whale. J. Mol. Biol. 1977, 110, 569−584. (10) Evans, S. V.; Brayer, G. D. High-resolution Study of the Threedimensional Structure of Horse Heart Metmyoglobin. J. Mol. Biol. 1990, 213, 885−897. (11) Hunter, C. L.; Lloyd, E.; Eltis, L. D.; Rafferty, S. P.; Lee, H.; Smith, M.; Mauk, A. G. Role of the Heme Propionates in the Interaction of Heme with Apomyoglobin and Apocytochrome b5. Biochemistry 1997, 36, 1010−1017. (12) Hayashi, T.; Hitomi, Y.; Suzuki, A.; Takimura, T.; Ogoshi, H. Molecular Recognition of Horse Heart Apomyoglobin to Monopropionate Hemin: Thermodynamic Determination of Two Orientational Isomers by 1H NMR Spectra. Chem. Lett. 1995, 24, 911−912. (13) La Mar, G. N.; Emerson, S. D.; Lecomte, J. T. J.; Pande, U.; Smith, K. M.; Craig, G. W.; Kebres, L. A. Influence of Propionate Side Chains on the Equilibrium Heme Orientation in Sperm Whale Myoglobin. Heme Resonance Assignments and Structure Determination by Nuclear Overhauser Effect Measurements. J. Am. Chem. Soc. 1986, 108, 5568−5573. (14) Woody, R. W.; Pescitelli, G. Z. The Role of Heme Chirality in the Circular Dichroism of Heme Proteins. Z. Naturforsch., A 2014, 69, 313−325. (15) Chang, C. K.; Ward, B.; Ebina, S. Kinetic Study of CO and O2 Binding to Horse Heart Myoglobin Reconstituted with Synthetic Hemes Lacking Methyl and Vinyl Side Chains. Arch. Biochem. Biophys. 1984, 231, 366−371. (16) Neya, S.; Suzuki, M.; Hoshino, T.; Ode, H.; Imai, K.; Komatsu, T.; Ikezaki, A.; Nakamura, M.; Furutani, Y.; Kandori, H. Molecular Insight into Intrinsic Heme Distortion in Ligand Binding in Hemoprotein. Biochemistry 2010, 49, 5642−5650. (17) Clezy, P. S.; Lim, C. L.; Shannon, J. S. Chemistry of Pyrrole Compounds XXVII. Aust. J. Chem. 1974, 27, 1103−1120. (18) Neya, S.; Funasaki, N.; Igarashi, N.; Ikezaki, A.; Sato, T.; Imai, K.; Tanaka, N. Structure and Function of 6,7-Dicarboxyhemesubstituted Myoglobin. Biochemistry 1998, 37, 5487−5493. (19) Neya, S.; Funasaki, N.; Imai, K. Etiohemin as a Prosthetic Group of Myoglobin. Biochim. Biophys. Acta 1989, 996, 226−232. (20) Neya, S.; Funasaki, N. A Facile Synthesis of the Lowest Homologues of meso-Tetraalkylporphyrin. J. Heterocyclic Chem. 1997, 34, 689−690. (21) Teale, F. W. Cleavage of the Haem-protein Link by Acid Methylethylketone. Biochim. Biophys. Acta 1959, 35, 543. (22) Asakura, T. Hemoglobin Porphyrin Modification. Methods Enzymol. 1978, 52, 447−455.

(23) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V. ; Mennucci, B.; Petersson, G. A. et al.. Gaussian 09, Revision D.01, Gaussian, Inc.: Wallingford CT, 2013. (24) Park, S.-Y.; Yokoyama, T.; Shibayama, N.; Shiro, Y.; Tame, J. R. 1.25 Å Resolution Crystal Structures of Human Haemoglobin in the Oxy, Deoxy and Carbonmonoxy Forms. J. Mol. Biol. 2006, 360, 690− 701. (25) Kiefl, C.; Sreerama, N.; Haddad, R.; Sun, L.; Jentzen, W.; Lu, Y.; Qiu, Y.; Shelnutt, J. A.; Woody, R. W. Heme Distortions in Spermwhale Carbonmonoxy Myoglobin: Correlations between Rotational Strengths and Heme Distortions in MD-generated Structures. J. Am. Chem. Soc. 2002, 124, 3385−3394. (26) Sreerama, N.; Woody, R. W. Computation and Analysis of Protein Circular Dichroism Spectra. Methods Enzymol. 2004, 383, 318−351. (27) Tamura, M.; Woodrow, G. V., III; Yonetani, T. Hememodification Studies of Myoglobin. II. Ligand Binding Characteristics of Ferric and Ferrous Myoglobins Containing Unnatural Hemes. Biochim. Biophys. Acta 1973, 317, 34−49. (28) Tamura, M.; Asakura, T.; Yonetani, T. Heme Modification Studies of Myoglobin. I. Purification and Some Optical and EPR Characteristics of Synthesized Myoglobins Containing Unnatural Hemes. Biochim. Biophys. Acta 1973, 295, 467−479. (29) Neya, S.; Funasaki, N. Proton NMR Study of the Myoglobin Reconstituted with meso-Tetra(n-propyl)hemin. Biochim. Biophys. Acta 1988, 952, 150−157. (30) Garnier, A.; Bolard, J.; Danon, J. Circular Dichroism of Hemoprotein Derivatives in the Visible Region. Chem. Phys. Lett. 1972, 15, 141−143. (31) Myers, Y. P.; Pande, A. In The porphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; Vol. III, pp 312−318. (32) La Mar, G. N.; Davis, N. L.; Parish, D. W.; Smith, K. M. Heme Orientational Disorder in Reconstituted and Native Sperm Whale Myoglobin. Proton Nuclear Magnetic Resonance Characterizations by Heme Methyl Deuterium Labeling in the Met-cyano Protein. J. Mol. Biol. 1983, 168, 887−896. (33) Aojula, H. S.; Wilson, M. T.; Morrison, I. G. Functional Consequences of Haem Orientational Disorder in Sperm-Whale and Yellow-Fin-Tuna Myoglobins. Biochem. J. 1987, 243, 205−210. (34) Aojula, H. S.; Wilson, M. T.; Drake, A. Characterization of Haem Disorder by Circular Dichroism. Biochem. J. 1986, 237, 613− 616. (35) Neya, S.; Funasaki, N. Proton NMR Study of the Cyanide Metmyoglobin Reconstituted with meso-Tetraalkylhemins. Dynamic Free Rotation of the Synthetic Hemins in the Heme Pocket. J. Biol. Chem. 1987, 262, 6725−6728. (36) Eaton, W. A.; Hanson, L. K.; Stephens, P. J.; Sutherland, J. C.; Dunn, J. B. R. Optical Spectra of Oxy- and Deoxyhemoglobin. J. Am. Chem. Soc. 1978, 100, 4991−5003. (37) Geraci, G.; Parkhurst, L. J. Circular Dichroism Spectra of Hemoglobins. Method Enzymol. 1981, 76, 262−275. (38) Sugita, Y.; Nagai, M.; Yoneyama, Y. Circular Dichroism of Hemoglobin in Relation to the structure Surrounding the Heme. J. Biol. Chem. 1971, 246, 383−388. (39) Phillips, S. E. V. Structure and Refinenment of Oxymyoglobin at 1.6 Å Resolution. J. Mol. Biol. 1980, 142, 531−554. (40) Vojtĕchovský, J.; Chu, K.; Berendzen, J.; Sweet, R. M.; Schlichting, I. Crystal Structures of Myoglobin-ligand Complexes at Near-atomic Resolution. Biophys. J. 1999, 77, 2153−2174.

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Involvement of Propionate Side Chains of the Heme in Circular

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