Characterization of Heme Orientational Disorder in ... - ACS Publications


Jul 31, 2017 - disorder is affected by the salt bridge associated with the heme 13-propionate ... properties of human adult Hb,48 and O2 affinity of r...
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Characterization of Heme Orientational Disorder in a Myoglobin Reconstituted with a Trifluoromethyl-Group-Substituted Heme Cofactor Yuki Kanai,† Ayaka Harada,‡ Tomokazu Shibata,† Ryu Nishimura,† Kosuke Namiki,† Miho Watanabe,† Shunpei Nakamura,† Fumiaki Yumoto,‡ Toshiya Senda,‡ Akihiro Suzuki,§ Saburo Neya,∥ and Yasuhiko Yamamoto*,†,⊥ †

Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan Structural Biology Research Center, Institute of Materials Structure Science, KEK/High Energy Accelerator Research Organization, 1-1 Oho, Tsukuba, Ibaraki 305-0801, Japan § Department of Materials Engineering, National Institute of Technology, Nagaoka College, Nagaoka 940-8532, Japan ∥ Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Chuoh-Inohana, Chiba 260-8675, Japan ⊥ Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba 305-8577, Japan ‡

S Supporting Information *

ABSTRACT: The orientation of a CF3-substituted heme in sperm whale myoglobin and L29F, H64L, L29F/H64Q, and H64Q variant proteins has been investigated using 19F NMR spectroscopy to elucidate structural factors responsible for the thermodynamic stability of the heme orientational disorder, i.e., the presence of two heme orientations differing by a 180° rotation about the 5−15 meso axis, with respect to the protein moiety. Crystal structure of the metaquo form of the wild-type myoglobin reconstituted with 13,17bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7trifluoromethylporphyrinatoiron(III), determined at resolution of 1.25 Å, revealed the presence of the heme orientational disorder. Alterations of the salt bridge between the heme 13-propionate and Arg45(CD3) side chains due to the mutations resulted in equilibrium constants of the heme orientational disorder ranging between 0.42 and 1.4. Thus, the heme orientational disorder is affected by the salt bridge associated with the heme 13-propionate side chain, confirming the importance of the salt bridge in the heme binding to the protein. “Heme orientational disorder” has been recognized as a general structural feature of b-type hemoproteins.1−18 Reaction between heme and apoprotein of sperm whale myoglobin (Mb) initially yields a holoprotein possessing structural heterogeneity such that the heme is inserted into the apoprotein in two distinct orientations that differ by 180° rotation about the 5−15 meso axis, with respect to the protein moiety (Figure 1),1 and the newly formed holoprotein initially contains an ∼1:1 mixture of the two hemes in Forms A and B (Figure 1). Form B is slowly converted to Form A through the heme reorientational reaction.19 Hence, there are two slowly interconverting protein forms in solution, and the dominant component has the same heme orientation as found in a crystal,20,21 i.e., Form A (Figure 1).22 Therefore, Forms A and B are often called normal and reversed heme orientations, respectively. The heme orientational disorder of a b-type hemoprotein has been investigated through studies of the reconstitution of wildtype and genetic variant proteins with not only native heme, but also a variety of chemically modified ones. 23−42 © XXXX American Chemical Society

NMR1−15,17−19,22−24,27−31,35,36,38−40,42 is generally used to characterize the heme orientational disorder, and circular dichroism43 and resonance Raman44,45 spectroscopies have been reported to be useful for identifying the presence of the reversed heme orientation. As functional consequences of the heme orientational disorder, pH-dependent oxygen (O2) affinity, i.e., Bohr effect, in monomeric insect Chironomus thummi thummi hemoglobins (CTT Hbs),46 reduction potential of bovine cytochrome b5 (cyt b5),47 cooperative O2 binding properties of human adult Hb,48 and O2 affinity of rainbow trout Mb45 have been reported to be affected by the heme orientation. The equilibrium constant of the heme reorientational reaction (KA/B = [Form A]/[Form B]) has been reported to depend strongly on the individual proteins such as wild-type Mb (KA/B = ∼9),3 CTT Hb III (∼0.5)2, Glycera dibranchiata hemoglobin component IV (0.10−0.15),14 and Rhodnius Received: May 13, 2017 Revised: July 28, 2017 Published: July 31, 2017 A

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Biochemistry

Figure 1. Molecular structures of protohemin (Proto), 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7trifluoromethylporphyrinatoiron(III) (7-PF), and 13,17-bis(2-carboxylatoethyl)-3,8-ethyl-2,7,12,18-tetramethylporphyrinatoiron(III) (Meso), and two heme orientations differing by a 180° rotation about the 5−15 meso axis with respect to His93, known as heme orientational disorder,1 i.e., Forms A and B. The rectangle indicates the orientation of the His93 imidazole plane, and ϕ is defined as the angle between the projection of the His93 imidazole onto the heme plane and the x-axis of the heme, which passes through NII−Fe−NIV and NI−Fe-NIII in Forms A and B, respectively.

prolixus nitrophorins 1 (∼2), 2 (∼10), and 7 (∞).18 The studies demonstrated that KA/B is determined by steric interactions of the peripheral side chains attached to pyrrole rings I and II of the heme, i.e., the side chains at positions 2, 3, 7, and 8 (Figure 1), with the surrounding amino acid residues in the heme pocket.11,14,18,29,30,36,41 In fact, KA/B has been shown to be greatly affected by replacement of the amino acid residues in close proximity to these heme side chains.14,18,41 The heme orientational disorder demonstrated that the molecular recognition upon insertion of the heme into the Mb heme pocket is not as strict as that for the formation of usual enzyme−substrate complexes.49 Furthermore, broad molecular recognition of the Mb heme pocket is clearly reflected in its capacity to accommodate a variety of guest molecules ranging from chemically modified heme complexes25−31,35,36,50−58 and analogues50,59−69 to metal complexes with salophen ligands.70 Characterization of the molecular recognition of the Mb heme pocket is a problem of particular importance for elucidating the heme-protein interaction and thus crucial for functional control of the protein. The protein moiety of Mb consists of 153 amino acid residues, arranged in eight helices, A−H, that fold into two hydrophobic cores made of the ABGH and CDEF helices.20,21,71,72 Studies on refolding of an apoprotein revealed that the ABGH core is first to form within ∼10 μs, with the CDEF one following later in a separate step.73,74 The ABGH core has been shown to be well-packed in the absence of heme, whereas formation of a stable tertiary structure of the CDEF core demands the heme binding.73−75 The binding of heme to the CDEF core is stabilized by the coordination bond between the heme Fe atom and the nitrogen atom of the proximal His (His93(F8), where F8 is the alphanumeric code that refers to the number of residue within the helices and loops of the protein), the hydrophobic interaction of heme with the surrounding amino acid residues in the heme pocket, and the formation of salt bridges between the heme propionate groups and nearby polar amino acid side chains.20,21,72 The pKa values of the heme propionic acid side chains of Mb have been

reported to be smaller than 5,76 and the heme 13-propionate side chain forms a salt bridge with Arg45(CD3) one (heme 13propionate salt bridge (13-PSB)) (Figure 2). Similarly, the

Figure 2. Hydrogen bond network in the proximal and distal sites of met-aquo form of sperm whale myoglobin (PDB ID: 1A6K). Amino acid residue participating in hydrogen bonds, heme, and Fe-bound H2O are shown and broken lines represent hydrogen bonds.

heme 17-propionate side chain forms a salt bridge with His97(FG3) side chain imidazole of which pKa value was reported to be 5.6377 (heme 17-propionate salt bridge (17PSB)), together with a hydrogen bond with Ser92 side chain hydroxyl group (Figure 2). Compared with the 17-PSB, the 13PSB has been considered to be particularly important for the process of heme binding.27,34 Crystallographic studies demonstrated that the Arg45 side chain is also hydrogen-bonded to not only the Asp60(E3) side chain, but also to the distal His (His64(E7)) one through an intervening water molecule (Figure 2), stabilizing the 13-PSB through strengthening of the hydrogen-bonding network associated with the Arg45 side chain.72 Consequently, the 13-PSB important for the heme B

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containing 2.9 M ammonium sulfate and 0.1 M Bis-Trispropane HCl buffer, pH 6.3. Crystal Structure Determination and Refinement. Xray diffraction data were collected at BL-1A (Photon Factory at the High Energy Accelerator Research Organization, Tsukuba, Japan). The protein crystal was soaked in a reservoir solution containing 30% xylitol and then flash-frozen in liquid nitrogen. The protein crystal was maintained at 100 K, by a cold nitrogen stream, throughout the data collection. All diffraction data were processed with XDS.84 The structures were determined by the molecular replacement method using the program PHASER.85 A crystal structure of Mb (PDB ID: 1A6K)72 was used as a search model. Crystallographic refinement was carried out using the program Phenix.refine.86 The structures were visualized and modified using the program COOT. 87 Diffraction and refinement data statistics are summarized in Table 1.

recognition could be perturbed through replacement of His64.78,79 Replacement of His64 with Gln, i.e., the H64Q mutation, was found to result in complete breakdown of the Arg45 hydrogen bond network, and hence the 13-PSB was no longer retained in this variant protein.78 Similarly replacement of His64 with Leu, i.e., the H64L mutation, is expected to perturb the 13-PSB. In addition, replacement of Leu29(B10), adjacent to the heme and His64, with Phe, i.e., the L29F mutation, alters the His64 side chain conformation, leading to a sizable change in the 13-PSB.80 Finally, the 13-PSB is further perturbed through simultaneous replacement of Leu29 and His64 with Phe and Gln, respectively, i.e., the L29F/H64Q double mutation.79 In this study, we carried out the H64Q, H64L, L29F, and L29F/H64Q mutations of the protein in order to alter the 13PSB, and characterized the heme orientational disorder in the variant proteins reconstituted with 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7-tri fluoromethylporphyrinatoiron(III)81 (7-PF (Figure 1)). The thermodynamic stabilities of Forms A and B in the wild-type Mb reconstituted with 7-PF (Mb(7-PF)) are almost comparable to each other, as reflected in the ΔG value of ∼2.0 kJ mol−1, yielded from the value of 0.44 determined as the KA/B of Mb(7PF). Hence, the effect of changes in the heme-protein contacts, induced by the amino acid replacements, on the ΔG value between Forms A and B is sensitively reflected in the KA/B values of the proteins reconstituted with 7-PF. Furthermore, the use of 7-PF allowed ready and accurate determination of KA/B using 19F NMR. Thus, 7-PF is quite useful as a heme cofactor for characterizing the relationship between the heme orientational disorder and the heme-protein contacts. The KA/B values determined for the H64Q, H64L, L29F, and L29F/ H64Q variant proteins reconstituted with 7-PF (H64Q(7-PF), H64L(7-PF), L29F(7-PF), and L29F/H64Q(7-PF), respectively) ranged from 0.42 in L29F(7-PF) to 1.4 in H64Q(7-PF), confirming the importance of the 13-PSB in the heme binding to the protein.

Table 1. Crystallographic Data Summary of Met-aquo form of Mb(7-PF) data collection light source T (K) space group cell dimensions a, b, c (Å) α, β, γ resolution (Å) Rmerge I/σI completeness (%) redundancy resolution (Å) no. reflections Rwork Rfree no. atoms protein ligand/ion water B-factors (Å2) protein ligand/ion water rms deviations bond lengths (Å) bond angles (deg) PDB ID



MATERIALS AND METHODS Materials and Protein Samples. All reagents and chemicals were obtained from commercial sources and used as received. Sperm whale Mb was purchased as a lyophilized powder from Biozyme and used without further purification. The expression and purification of the H64Q, H64L, L29F, and L29F/H64Q variant proteins were carried out according to the methods described by Springer et al.82 7-PF was synthesized as previously described.81 The apoproteins of the wild-type and variant proteins were prepared at 4 °C according to the procedure of Teale,83 and the reconstitution of the prepared apoproteins with 7-PF was carried out as described previously.57 The reconstituted proteins were kept at 4 °C for at least 3 days for equilibration of the heme reorientation reaction. Met-cyano forms of the proteins were prepared by adding KCN (Nacalai Chemicals Ltd.) to the reconstituted proteins. The pH of each sample was measured with a Horiba F-22 pH meter equipped with a Horiba type 6069-10c electrode. The pH of a sample was adjusted with 0.1 M NaOH or HCl. Protein Crystallization. Crystallization of met-aquo Mb(7PF) was performed at 4 °C using the hanging-drop vapordiffusion method.56 The protein drops were prepared by mixing 1.5 μL of the protein solution (18 mg/mL in 10 mM potassium phosphate buffer, pH 7.0) with 1.5 μL of the reservoir solution

a

PF BL-1A 100 P21 34.26, 30.85, 64.13 90.0, 105.4, 90.0 33.04−1.25 (1.27−1.25)a 0.073 (0.366)a 15.9 (5.4)a 99.8 (99.2) 6.3 (5.9)a refinement 30.92−1.25 36 020 0.163 0.173 1189 92/15 136 10.20 7.65/16.36 18.92 0.006 0.958 5XL0

Numbers in parentheses indicate the highest resolution shell.

19 F NMR Spectroscopy. 19F NMR spectra of the reconstituted proteins were recorded on a Bruker AVANCE500 spectrometer operating at a 19F frequency of 471 MHz. Typical 19F NMR spectra consisted of about 20k transients with a 100 kHz spectral width and 16k data points. The signal-tonoise ratio of the spectra was improved by apodization, which introduced ∼30 Hz line broadening. The chemical shifts of 19F NMR spectra are given in ppm downfield from trifluoroacetic acid as an external reference.

C

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RESULTS Crystal Structure of Met-aquo Mb(7-PF). The crystal structure of met-aquo Mb(7-PF) indicated not only that the protein structure of Mb(7-PF) is essentially identical to that of the wild-type protein, but also that 7-PF is accommodated properly as for the heme of the wild-type protein (Figure 3).

Figure 3. 2mF0 − DFc map for Forms A and B of met-aquo Mb(7PF): side-view (a) and view from His93 (b). The contour levels of the maps are 1.2σ.

Figure 4. Simulated annealing omit maps for 7-PF side chain conformations of Forms A (left) and B (right) in met-aquo Mb(7-PF). Side-views from the 5-C carbon atom (top) and views from His93 (bottom). The contour levels of the maps are 3.0σ.

Furthermore, the heme Fe coordination structure of Mb(7-PF) was almost identical to that of the wild-type protein (see Figure S1 in the Supporting Information). These findings supported the validity of the studies on the structure−function relationship of Mb through analysis of the proteins reconstituted with chemically modified hemes possessing CF3 groups as side chains.57,88−93 The presence of both Forms A and B has been revealed by the crystal structure of Mb(7-PF). The orientation of the porphyrin moiety of 7-PF, with respect to the protein, was not greatly affected by the heme orientational disorder (Figure 3). To elucidate the structural origin of the dominance of Form B over Form A in Mb(7-PF), as reported previously,36 steric interactions of the heme side chains at positions 2, 3, 7, and 8 with the surrounding amino acid residues in the protein were examined on the basis of the crystal structure. In Form A, the CF3 group at position 7 (7-CF3) is in close contact with Leu104(G5) CδH3, and the ethyl ones at positions 3 and 8 (3and 8-C2H5, respectively) are in contact with Leu104 CδH3:Phe138(H15) CζH and Thr39(C4) CαH, respectively (see Figure S2 in the Supporting Information). Particularly, the distance between 7-CF3 fluorine and Leu104 CδH3 carbon atoms of Form A in the protein was determined to be 0.29 nm. Since the van der Waals radii of carbon and fluorine atoms are 0.147 and 0.170 nm, respectively,94 the steric contact between them is thought to contribute to destabilization of Form A in the protein. On the other hand, in Form B, no obvious steric contact was detected between the heme side chains at positions 2, 3, 7, and 8 with the surrounding amino acid residues (see Figure S2 in the Supporting Information). The crystal structure of met-aquo Mb(7-PF) provided the conformations of the 3- and 8-C2H5 groups in Form A are also fixed with χ angles of ∼ −130° and ∼ +109° for the 3- and 8C2H5 groups, respectively, and similarly those of Form B are fixed with χ angles of ∼ +111° and ∼ −98°, respectively (χ values are the dihedral angles between the planes of the (n + 1)−C−n-C−CH2 carbon atoms and the n-C−CH2−CH3 ones, where n = 3 and 8 for the 3- and 8-C2H5 groups, respectively, and positive and negative χ values indicate that the terminal CH3 groups are pointing toward and away from His93, respectively (Figure 4)).

Thus, the conformations of 3-C2H5 in Form A and 8-C2H5 in Form B (or 8-C2H5 in Form A and 3-C2H5 in Form B) could be interconverted with each other, indicating that the conformations of the C2H5 groups are primarily determined by steric contact with nearby amino acid residues. Furthermore, the conformations of the side chain propionate groups at positions 13 and 17 could be completely interconverted between Forms A and B (Figure 4). 19 F NMR Spectra of Wild-Type and Variant Proteins Reconstituted with 7-PF. We next determined KA/B values of the proteins using 19F NMR. Since the KA/B value of Mb has been shown to be essentially independent of the heme Fe oxidation, spin, and ligation states, the heme orientational disorder of the protein can be readily characterized through analysis of an NMR spectrum of its paramagnetic met-cyano form,1,3,4 as shown in Figure 5. The heme orientational disorder was clearly manifested in the observation of two signals in each of the 19F NMR spectra of the met-cyano forms of Mb(7-PF), L29F(7-PF), H64L(7-PF), L29F/H64Q(7-PF), and H64Q(7PF) at 25 °C (Figure 5). Since Form B has been shown to dominate over Form A in the carbon monoxide (CO) form of Mb(7-PF)39 (see Figure S3 in the Supporting Information), comparison of the signal intensities allowed assignment of the signals at 21.37 and 36.86 ppm to the CF3 groups of Forms A and B, respectively. Therefore, the signals at ∼21 and ∼36 ppm in the spectra of the variant proteins were similarly assigned to Forms A and B, respectively (Table 2). The observation of the two separate signals allowed the determination of KA/B through analysis of the signal intensities. The analysis yielded KA/B values of 0.42, 0.94, 1.0, and 1.4 for L29F(7-PF), H64L(7-PF), L29F/H64Q(7-PF), and H64Q(7PF), respectively. The KA/B values yielded through analysis of 1 H NMR signal intensities were essentially identical to those determined by 19F NMR (see Table S1 in the Supporting Information). These results indicated that KA/B is quite sensitive to the mutations, as expected, and that the preference of the 7-PF orientation in the protein can be controlled through structural changes of the 13-PSB induced by replacement of the nearby amino acid residues. D

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broken by the H64Q one, while the 17-PSB was essentially unaffected by the mutations. As a result, the CD-loop of the protein is slightly displaced away from the heme, as reflected in changes in the distance between the heme carbon atom at position 13 and the Arg45 Cα atom from 0.84 nm in the wildtype protein72 to ∼0.9 nm in the L29F,80 H64L,78 and L29F/ H64Q79 variant ones, and then up to 0.94 nm in the H64Q79 variant one. Due to the displacement of the CD-loop away from the heme, the thermodynamic energy levels of Forms A and B are almost identical to each other in L29F/H64Q(7-PF), as manifested in the KA/B of 1.0, and, on the other hand, the former is slightly more stabilized relative to the latter in H64Q(7-PF), as reflected by KA/B = 1.4. These results supported that the tertiary structure of the CDEF core of the protein is formed and stabilized through interaction with the heme, as reported previously.73−75 19 F NMR Shifts. The δobs value of the met-cyano Mb in Figure 5 is given by the sum of the diamagnetic (δdia) and the paramagnetic shifts (δpara), and then δpara is expressed as the sum of paramagnetic contact (δc) and pseudocontact (δpc) shifts due to Fermi contact interaction with a delocalized unpaired electron and a through-space dipolar interaction with the unpaired electron spin, respectively. Considering the shifts of signals A and B of the CO form of Mb(7-PF) at 25 °C, i.e., 30.81 and 29.03 ppm, respectively (see Figure S3 in the Supporting Information), as the δdia values for the corresponding signals of the met-cyano form of Mb(7-PF), i.e., 21.37 and 36.86 ppm, respectively (Figure 5), the δparas of signals A and B were estimated to be ∼ −9.4 and ∼ +7.8 ppm, respectively. In the case of 7-PF, delocalization of the unpaired electron from the heme Fe atom to 7-CF3 occurs through the porphyrin πsystem, and the delocalization of the unpaired electron from the π-system to the fluorine atoms occurs through hyperconjugation and spin polarization mechanisms, both of which result in positive δc values. In addition, metal-centered δpc (δpc(M)) values due to an unpaired electron spin localized at the heme Fe atom are thought to be in the range of ∼ −7 to ∼ −3 ppm for signals A and B.95 Consequently, ligand-centered δpc (δpc(L)) values due to an unpaired electron spin delocalized into p orbitals of both the pyrrole carbon atom to which 7-CF3 is attached, and 7-CF3 fluorine ones appeared to contribute to the relatively large extent to the δparas of signals A and B, i.e., ∼ − 9.4 and ∼ +7.8 ppm for the former and the latter, respectively. The KA/B value was found to increase with increasing δobs of signal B (Figure 5). This finding could be interpreted in terms of the effect of the mutations on δc. In the met-cyano form of Mb, the energy levels for the dxz and dyz orbitals are affected by the interaction with axial ligands, and a single unpaired electron resides in either the dxz or dyz orbital, whichever possesses the highest energy.95−99 Consequently, depending upon the relative energies of the dxz and dyz orbitals, π spin delocalization occurs for either pyrrole I, III or II, IV (Figure 1). The conformation of the axial His93 side chain is restricted due to the coordination bond, together with the formation of a hydrogen bond of the His93 NδH hydrogen atom with the carbonyl oxygen atom of Leu89(F4) (Figure 2), and hence, the orientation of the His93 imidazole, with respect to the heme, contributes significantly to determination of the relative energies of the dxz and dyz orbitals. In contrast, since a cyanide ion (CN−) is coordinated to heme Fe atom with its orientation nearly normal to the heme plane,100 the effect of the CN− coordination on the relative energy levels of the two orbitals is essentially negligible.101 The coordination structure of His93

Figure 5. 471 MHz 19F NMR spectra of met-cyano forms of the wildtype Mb and L29F, H64L, L29F/H64Q, and H64Q variant proteins reconstituted with 7-PF at pH 7.0 and 25 °C (Mb(7-PF), L29F(7-PF), H64L(7-PF), L29F/H64Q(7-PF), and H64Q(7-PF), respectively). Signal assignments are shown with the spectra. Circle graphs show the ratio between Forms A and B in the proteins.

Table 2. Equilibrium Constants of the Heme Reorientational Reactions (KA/B) in Mb and Variant Proteins, and 19F NMR Shifts of Signals A and B of Met-cyano Forms of the Proteins 19

F NMR shift (ppm)b

protein

KA/Ba

signal A

signal B

Mb(7-PF) L29F(7-PF) H64L(7-PF) L29F/H64Q(7-PF) H64Q(7-PF) Wild-type Mb

0.44 0.42 0.94 1.0 1.4 9.0c

21.37 21.75 21.83 21.61 21.43 N/Ad

36.86 36.37 35.74 35.60 35.41 N/Ad

The experimental errors for the KA/B were ±10%. bAt pH 7.0 and 25 °C. cObtained irom ref 3. dNot applicable.

a

The shift difference of Forms A and B (signals A and B, respectively) of Mb(7-PF) in Figure 5 is 15.60 ppm, which is due to a difference in paramagnetic shift (δpara) between them, because the difference in the shift between the signals of the diamagnetic CO adduct of Mb(7-PF) is rather small, i.e., 1.78 ppm at 25 °C (see Figure S3 in the Supporting Information). In addition, the observed shifts (δobs) of signals A and B varied over ranges of 0.46 and 1.45 ppm among the proteins, respectively (Table 2), and it is notable that δobs of signal B correlated with KA/B in such a manner that δobs decreases with increasing KA/B.



DISCUSSION Effects of the Mutations on KA/B. The KA/B value ranging from 0.42 to 1.4 in Table 2 demonstrated that the preference of the 7-PF orientation in the protein is affected by structural changes in the 13-PSB induced by the mutations. Among the side chains attached to pyrrole rings I and II of 7-PF, CF3 has the largest van der Waals volume,94 and hence exerts the largest steric hindrance with the nearby amino acid residues. According to the crystal structures reported for the wild-type and variant proteins,72,78−80 the 13-PSB is somewhat weakened by the L29F, H64L, and L29F/H64Q mutations, and is completely E

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Biochemistry imidazole, with respect to the heme, is described by the angle, ϕ, between the projection of the imidazole onto the heme plane and the x-axis of the heme, which passes through NII− Fe−NIV and NI−Fe−NIII in Forms A and B, respectively (Figure 1). With ϕ = ∼0°, as indicated by the crystal structure of met-aquo Mb(7-PF), the energy of the dyz orbital is higher than that of the dxz one, and hence, due to direct delocalization of an unpaired electron from the dyz orbital into the π system of pyrrole II, the δc of signal B is larger than that of signal A. Consequently, a slight change in the orientation of the heme, with respect to the protein moiety, leads to a small increase in ϕ, which in turn slightly increases and decreases the δc values of signals A and B, respectively. Therefore, the relationship between KA/B and δobs of signal B suggested that the steric contact between the heme and the CDEF core of the protein is a determinant of KA/B. In contrast to that of signal B, the δobs of signal A did not correlate with KA/B, possibly because the δc of signal A is not sufficiently large enough to sharply reflect its ϕdependence. Effects of the Heme Chemical Modifications on KA/B. The predominance of Form B in met-cyano form of Mb(7-PF) (KA/B = 0.44 at pH 7.0) is in contrast to the predominance of Form A in met-cyano form of Mb reconstituted with 13,17bis(2-carboxylatoethyl)-3,8-diethyl-2,7,12,18tetramethylporphyrinatoiron(III) (Meso (Figure 1)) (Mb(Meso), KA/B = 10 at pH 8.3).102 Because these two porphyrin derivatives differ only by the CF3/CH3 replacement at position 7, the substitution is responsible for the change in KA/B values, through interactions with nearby amino acid residues. In Form A, the 2-, 3-, 7-, and 8-side chains are in close proximity to Val68(E11);Tyr103(G6);Phe138(H15), Ile99(FG5);Ile107(G8); Tyr103(G4);Leu104(G5), and Phe43(CD1);Thr39(C4); Ile99(FG5), respectively (see Figure S1 in the Supporting Information).72 Since the van der Waals volumes of CH3, C2H5, and CF3 are 0.022, 0.039, and 0.043 nm3, respectively,94 the predominance of Form B over Form A in Mb(7-PF) is possibly caused by destabilization of Form A through steric hindrance between 7-CF3 and the nearby amino acid residues.



AUTHOR INFORMATION

Corresponding Author

*Phone/Fax: +81-29-853-6521. E-mail: [email protected] tsukuba.ac.jp. ORCID

Yasuhiko Yamamoto: 0000-0003-4951-3184 Funding

This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This work was in part performed as a project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) of the Japan Agency for Medical Research and Development (AMED). The 1H and 19F NMR spectra were recorded on a Bruker AVANCE-600 spectrometer at the Chemical Analysis Center, University of Tsukuba.



ABBREVIATIONS Mb, myoglobin; CTT Hb, Chironomus thummi thummi hemoglobin; cyt b5, bovine cytochrome b5; 13-PSB, heme 13propionate salt bridge; 17-PSB, heme 17-propionate salt bridge; 7-PF, 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7- trifluoromethylporphyrinatoiron(III); CO, carbon monoxide; Mb(7-PF), wild-type Mb reconstituted with 7-PF; L29F(7-PF), the L29F variant protein reconstituted with 7-PF; H64L(7-PF), the H64L variant protein reconstituted with 7PF; L29F/H64Q(7-PF), the L29F/H64Q variant protein reconstituted with 7-PF; H64Q(7-PF), the H64Q variant protein reconstituted with 7-PF; Meso, 13,17-bis(2-carboxylato-ethyl)-3,8-diethyl-2,7,12,18- tetramethylporphyrinatoiron(III); Mb(Meso), the wild-type Mb reconstituted with Meso



CONCLUDING REMARKS The crystal structure of met-aquo Mb(7-PF) revealed not only that 7-PF is accommodated properly as for the heme of the wild-type protein, but also that 7-PF is incorporated into the heme pocket with the two orientations that differ by 180° rotation about the 5−15 meso axis, with respect to the protein moiety. The protein structure of Mb(7-PF) was essentially independent of the heme orientation, and the orientations of the core porphyrin moieties of 7-PFs in Forms A and B, with respect to the protein, were almost identical to each other, supporting that KA/B is determined through steric interactions of the peripheral side chains of the heme with the surrounding amino acid residues in the heme pocket. This finding provides valuable information for elucidating the long-standing issue of the energetics of the heme orientational disorder in b-type hemoproteins.



Views of met-aquo native Mb; superposition of heme, His93 and Fe-bound H2O in native Mb and those of Forms A and B of Mb(7-PF); Orientations of some amino acid residues near heme in Forms A and B of metaquo Mb(7-PF); 471 MHz 19F NMR spectrum of the carbonmonoxy form of Mb(7-PF); equilibrium constants of the heme reorientational reactions in Mb and variant proteins (PDF)



REFERENCES

(1) La Mar, G. N., Budd, D. L., Viscio, D. B., Smith, K. M., and Langry, K. C. (1978) Proton nuclear magnetic resonance characterization of heme disorder in hemoproteins. Proc. Natl. Acad. Sci. U. S. A. 75, 5755−5759. (2) La Mar, G. N., Smith, K. M., Gersonde, K., Sick, H., and Overkamp, M. (1980) Proton nuclear magnetic resonance characterization of heme disorder in monomeric insect hemoglobins. J. Biol. Chem. 255, 66−70. (3) La Mar, G. N., Davis, N. L., Parish, D. W., and Smith, K. M. (1983) 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. 168, 887−896.

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Biochemistry (4) Jue, T., Krishnamoorthi, R., and La Mar, G. N. (1983) Proton NMR study of the mechanism of the heme-apoprotein reaction for myoglobin. J. Am. Chem. Soc. 105, 5701−5703. (5) La Mar, G. N., Yamamoto, Y., Jue, T., Smith, K. M., and Pandey, R. K. (1985) 1H NMR characterization of metastable and equilibrium heme orientational heterogeneity in reconstituted and native human hemoglobin. Biochemistry 24, 3826−3831. (6) Levy, M. J., La Mar, G. N., Jue, T., Smith, K. M., Pandey, R. K., Smith, W. S., Livingston, D. J., and Brown, W. D. (1985) Proton NMR study of yellowfin tuna myoglobin in whole muscle and solution. Evidence for functional metastable protein forms involving heme orientational disorder. J. Biol. Chem. 260, 13694−13698. (7) Pande, U., La Mar, G. N., Lecomte, J. T., Ascoli, F., Brunori, M., Smith, K. M., Pandey, R. K., Parish, D. W., and Thanabal, V. (1986) NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements. Biochemistry 25, 5638−5646. (8) McLachlan, S. J., La Mar, G. N., Burns, P. D., Smith, K. M., and Langry, K. C. (1986) 1H-NMR assignments and the dynamics of interconversion of the isomeric forms of cytochrome b5 in solution. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 874, 274−284. (9) Peyton, D. H., La Mar, G. N., and Gersonde, K. (1988) A nuclear overhauser study of heme orientational isomerism in monomeric Chironomus hemoglobins. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 954, 82−94. (10) McGourty, J. L., La Mar, G. N., Smith, K. M., Ascoli, F., Chiancone, E., Pandey, R. K., and Singh, J. P. (1989) Nuclearmagnetic-resonance investigation of the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis. Molecular and electronic structure of the cyano-met derivative. Eur. J. Biochem. 184, 53−61. (11) Lee, K.-B., La Mar, G. N., Pandey, R. K., Rezzano, I. N., Mansfield, K. E., Smith, K. M., Pochapsky, T. C., and Sligar, S. G. (1991) 1H NMR study of the role of heme carboxylate side chains in modulating heme pocket structure and the mechanism of reconstitution of cytochrome b5. Biochemistry 30, 1878−1887. (12) Wu, J.-Z., La Mar, G. N., Yu, L. P., Lee, K.-B., Walker, F. A., Chiu, M. L., and Sligar, S. G. (1991) 1H NMR study of the solution molecular and electronic structure of Escherichia coli ferricytochrome b562: Evidence for S = 1/2 ⇆ S = 5/2 spin equilibrium for intact His/ Met ligation. Biochemistry 30, 2156−2165. (13) Rivera, M., Barillas-Mury, C., Christensen, K. A., Little, J. W., Wells, M. A., and Walker, F. A. (1992) Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5. Biochemistry 31, 12233− 12240. (14) Alam, S. L., Dutton, D. P., and Satterlee, J. D. (1994) Expression of recombinant monomer hemoglobins (component IV) from the marine annelid Glycera dibranchiata: evidence for primary sequence positional regulation of heme rotational disorder. Biochemistry 33, 10337−10344. (15) Nguyen, B. D., Xia, Z., Cutruzzolá, F., Allocatelli, C. T., Brunori, M., and La Mar, G. N. (2000) Solution 1H NMR study of the influence of distal hydrogen bonding and N terminus acetylation on the active site electronic and molecular structure of Aplysia limacina cyanomet myoglobin. J. Biol. Chem. 275, 742−751. (16) Shimizu, H., Park, S.-Y., Shiro, Y., and Adachi, S. (2002) X-ray structure of nitric oxide reductase (cytochrome P450nor) at atomic resolution. Acta Crystallogr., Sect. D: Biol. Crystallogr. 58, 81−89. (17) Du, W., Syvitski, R., Dewilde, S., Moens, L., and La Mar, G. N. (2003) Solution 1H NMR characterization of equilibrium heme orientational disorder with functional consequences in mouse neuroglobin. J. Am. Chem. Soc. 125, 8080−8081. (18) Berry, R. E., Muthu, D., Shokhireva, T. K., Garrett, S. A., Goren, A. M., Zhang, H., and Walker, F. A. (2014) NMR investigations of nitrophorin 2 belt side chain effects on heme orientation and seating of native N-terminus NP2 and NP2(D1A). JBIC, J. Biol. Inorg. Chem. 19, 577−593.

(19) La Mar, G. N., Toi, H., and Krishnamoorthi, R. (1984) Proton NMR investigation of the rate and mechanism of heme rotation in sperm whale myoglobin: Evidence for intramolecular reorientation about a heme twofold axis. J. Am. Chem. Soc. 106, 6395−6401. (20) Takano, T. (1977) Structure of myoglobin refined at 2.0 Ǻ resolution. I. Crystallographic refinement of metmyoglobin from sperm whale. J. Mol. Biol. 110, 537−568. (21) Takano, T. (1977) Structure of myoglobin refined at 2.0 Ǻ resolution. II. Structure of deoxymyoglobin from sperm whale. J. Mol. Biol. 110, 569−584. (22) Lecomte, J. T. J., Johnson, R. D., and La Mar, G. N. (1985) Characterization of heme orientational disorder in myoglobin by proton nuclear Overhauser effects. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 829, 268−274. (23) Jue, T., and La Mar, G. N. (1984) Heme orientational heterogeneity in deuterohemin-reconstituted horse and human hemoglobin characterized by proton nuclear magnetic resonance spectroscopy. Biochem. Biophys. Res. Commun. 119, 640−645. (24) Yamamoto, Y., and La Mar, G. N. (1986) 1H NMR study of dynamics and thermodynamics of heme rotational disorder in native and reconstituted hemoglobin A. Biochemistry 25, 5288−5297. (25) Miki, K., Yasuaki, Yukawa, M., Owatari, A., Hato, Y., Harada, S., Kai, Y., Kasai, N., Hata, Y., Tanaka, N., Kakudo, M., Katsube, Y., Kawabe, K., Yoshtoa, Z., and Ogoshi, H. (1986) Crystal structures of modified myoglobins. I. Heme orientation and structural changes around heme in myoglobins reconstituted with isopemptoheme, pemptoheme, 2-ethyldeuteroheme, and 4-ethyldeuteroheme. J. Biochem. 100, 269−276. (26) Miki, K., Harada, S., Hato, Y., Iba, S., Kai, Y., Kasai, N., Katsube, Y., Kawabe, K., Yoshida, Z., and Ogoshi, H. (1986) Crystal structures of modified myoglobins. II. Relation between oxygen affinity properties and structural changes around heme in myoglobins reconstituted with 2,4-diisopropyldeuteroheme, 2-isopropyl-4-vinyldeuteroheme, and 2-vinyl-4-isopropyldeuteroheme. J. Biochem. 100, 277−284. (27) La Mar, G. N., Pande, U., Hauksson, J. B., Pandey, R. K., and Smith, K. M. (1989) Proton nuclear magnetic resonance investigation of the mechanism of the reconstitution of myoglobin that leads to metastable heme orientational disorder. J. Am. Chem. Soc. 111, 485− 491. (28) La Mar, G. N., Smith, W. S., Davis, N. L., Budd, D. L., and Levy, M. J. (1989) Proton NMR study of the influence on iron oxidation/ ligation/spin state on the heme orientational preference in myoglobin. Biochem. Biophys. Res. Commun. 158, 462−468. (29) Hauksson, J. B., La Mar, G. N., Pande, U., Pandey, R. K., Parish, D. W., Singh, J. P., and Smith, K. M. (1990) 1H-NMR study of the mechanism of assembly and equilibrium heme orientation of sperm whale myoglobin reconstituted with protohemin type-isomers. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1041, 186−194. (30) Lee, K.-B., La Mar, G. N., Kehres, L. A., Fujinari, E. M., Smith, K. M., Pochapsky, T. C., and Sligar, S. G. (1990) 1H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5. Biochemistry 29, 9623−9631. (31) Santucci, R., Ascoli, F., La Mar, G. N., Pandey, R. K., and Smith, K. M. (1993) Reconstitution of horse heart myoglobin with hemins methylated at 6- or 7-positions: a circular dichroism study. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1164, 133−137. (32) Shiro, Y., Iizuka, T., Marubayashi, K., Ogura, T., Kitagawa, T., Balasubramanian, S., and Boxer, S. G. (1994) Spectroscopic study of Ser92 mutants of human myoglobin: hydrogen bonding effect of Ser92 to proximal His93 on structure and property of myoglobin. Biochemistry 33, 14986−14992. (33) Lloyd, E., Burk, D. L., Ferrer, J. C., Maurus, R., Doran, J., Carey, P. R., Brayer, G. D., and Mauk, A. G. (1996) Electrostatic modification of the active site of myoglobin: characterization of the proximal Ser92Asp variant. Biochemistry 35, 11901−11912. (34) Hunter, C. L., Lloyd, E., Eltis, L. D., Rafferty, S. P., Lee, H., Smith, M., and Mauk, A. G. (1997) Role of the heme propionates in G

DOI: 10.1021/acs.biochem.7b00457 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry the interaction of heme with apomyoglobin and apocytochrome b5. Biochemistry 36, 1010−1017. (35) Yamamoto, Y., Hirai, Y., and Suzuki, A. (2000) 19F NMR study of protein-induced rhombic perturbations on the electronic structure of the active site of myoglobin. JBIC, J. Biol. Inorg. Chem. 5, 455−462. (36) Hirai, Y., Yamamoto, Y., and Suzuki, A. (2000) 19F NMR study of the heme orientation and electronic structure in a myoglobin reconstituted with a ring-fluorinated heme. Bull. Chem. Soc. Jpn. 73, 2309−2316. (37) Arnoux, P., Haser, R., Izadi-Pruneyre, N., Lecroisey, A., and Czjzek, M. (2000) Functional aspects of the heme bound hemophore HasA by structural analysis of various crystal forms. Proteins: Struct., Funct., Genet. 41, 202−210. (38) Hu, B., Hauksson, J. B., Tran, A.-T. T., Kolczak, U., Pandey, R. K., Rezzano, I. N., Smith, K. M., and La Mar, G. N. (2001) 1H and 13C NMR investigation of the influence of nonligated residue contacts on the heme electronic structure in cyanometmyoglobin complexes reconstituted with centro- and pseudocentrosymmetric hemins. J. Am. Chem. Soc. 123, 10063−10070. (39) Yamamoto, Y., Nagao, S., Hirai, Y., Inose, T., Terui, N., Mita, H., and Suzuki, A. (2004) NMR investigation of the heme electronic structure in deoxymyoglobin possessing a fluorinated heme. JBIC, J. Biol. Inorg. Chem. 9, 152−160. (40) Nagao, S., Hirai, Y., Suzuki, A., and Yamamoto, Y. (2005) 19F NMR characterization of the thermodynamics and dynamics of the acid-alkaline transistion in a reconstituted sperm whale metmyoglobin. J. Am. Chem. Soc. 127, 4146−4147. (41) Yang, F., Zhang, H., and Knipp, M. (2009) A one-residue switch reverses the orientation of a heme b cofactor. Investigations of the ferriheme NO transporters nitrophorin 2 and 7 from the blood-feeding insect Rhodnius prolixus. Biochemistry 48, 235−241. (42) Nagao, S., Osuka, H., Yamada, T., Uni, T., Shomura, Y., Imai, K., Higuchi, Y., and Hirota, S. (2012) Structural and oxygen binding properties of dimeric horse myoglobin. Dalton Trans. 41, 11378− 11385. (43) Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) Functional effects of heme orientational disorder in sperm whale myoglobin. J. Biol. Chem. 262, 46−54. (44) Rwere, F., Mak, P. J., and Kincaid, J. R. (2008) The impact of altered protein-heme interactions on the resonance Raman spectra of heme proteins. Studies of heme rotational disorder. Biopolymers 89, 179−186. (45) Howes, B. D., Helbo, S., Fago, A., and Smulevich, G. (2012) Insights into the anomalous heme pocket of rainbow trout myoglobin. J. Inorg. Biochem. 109, 1−8. (46) Gersonde, K., Sick, H., Overkamp, M., Smith, K. M., and Parish, D. W. (1986) Bohr effect in monomeric insect haemoglobins controlled by O2 off-rate and modulated by haem-rotational disorder. Eur. J. Biochem. 157, 393−404. (47) Walker, F. A., Emrick, D., Rivera, J. E., Hanquet, B. J., and Buttlaire, D. H. (1988) Effect of heme orientation on the reduction potential of cytochrome b5. J. Am. Chem. Soc. 110, 6234−6240. (48) Nagai, M., Nagai, Y., Aki, Y., Imai, K., Wada, Y., Nagatomo, S., and Yamamoto, Y. (2008) Effect of reversed heme orientation on circular dichroism and cooperative oxygen binding of human adult hemoglobin. Biochemistry 47, 517−525. (49) Pincus, M. R., and Scheraga, H. A. (1981) Theoretical calculations on enzyme-substrate complexes: the basis of molecular recognition and catalysis. Acc. Chem. Res. 14, 299−306. (50) Neya, S. (2013) Dynamic motion and rearranged molecular shape of heme in myoglobin: structural and functional consequences. Molecules 18, 3168−3182. (51) Hayashi, T., and Hisaeda, Y. (2002) New functionalization of myoglobin by chemical modification of heme-propionates. Acc. Chem. Res. 35, 35−43. (52) Neya, S., and Funasaki, N. (1987) Proton NMR study of the cyanide metmyoglobin reconstituted with meso-tetraalkylhemins. J. Biol. Chem. 262, 6725−6728.

(53) Neya, S., Funasaki, N., and Imai, K. (1989) Etiohemin as a prosthetic group of myoglobin. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 996, 226−232. (54) Neya, S., Funasaki, N., and Imai, K. (1988) Structure and function of the myoglobin containing octaethylhemin as a prosthetic group. J. Biol. Chem. 263, 8810−8815. (55) Neya, S., Funasaki, N., Sato, T., Igarashi, N., and Tanaka, N. (1993) Structural analysis of the myoglobin reconstituted with iron porphine. J. Biol. Chem. 268, 8935−8942. (56) Harada, K., Makino, M., Sugimoto, H., Hirota, S., Matsuo, T., Shiro, Y., Hisaeda, Y., and Hayashi, T. (2007) Structure and ligand binding properties of myoglobins reconstituted with monodepropionated heme: functional role of each heme propionate side chain. Biochemistry 46, 9406−9416. (57) Shibata, T., Nagao, S., Fukaya, M., Tai, H., Nagatomo, S., Morihashi, K., Matsuo, T., Hirota, S., Suzuki, A., Imai, K., and Yamamoto, Y. (2010) Effect of heme modification on oxygen affinity of myoglobin and equilibrium of the acid-alkaline transition in metmyoglobin. J. Am. Chem. Soc. 132, 6091−6098. (58) Juillard, S., Chevance, S., Bondon, A., and Simonneaux, G. (2011) Dynamics of heme in hemoproteins: proton NMR study of myoglobin reconstituted with iron 3-ethyl-2-methylporphyrin. Biochim. Biophys. Acta, Proteins Proteomics 1814, 1188−1194. (59) Neya, S., Kaku, T., Funasaki, N., Shiro, Y., Iizuka, T., Imai, K., and Hori, H. (1995) Novel ligand binding properties of the myoglobin substituted with monoazahemin. J. Biol. Chem. 270, 13118−13123. (60) Neya, S., Suzuki, M., Ode, H., Hoshino, T., Furutani, Y., Kandori, H., Hori, H., Imai, K., and Komatsu, T. (2008) Functional evaluation of iron oxypyriporphyrin in protein heme pocket. Inorg. Chem. 47, 10771−10778. (61) Neya, S., Funasaki, N., Hori, H., Imai, K., Nagatomo, S., Iwase, T., and Yonetani, T. (1999) Functional regulation of myoglobin by iron corrphycene. Chem. Lett. 28, 989−990. (62) Neya, S., Nakamura, M., Imai, K., and Funasaki, N. (2001) Functional analysis of the iron(II) etiocorrphycene incorporated in the myoglobin heme pocket. Chem. Pharm. Bull. 49, 345−346. (63) Hayashi, T., Dejima, H., Matsuo, T., Sato, H., Murata, D., and Hisaeda, Y. (2002) Blue myoglobin reconstituted with an iron porphycene shows extremely high oxygen affinity. J. Am. Chem. Soc. 124, 11226−11227. (64) Neya, S., Imai, K., Hori, H., Ishikawa, H., Ishimori, K., Okuno, D., Nagatomo, S., Hoshino, T., Hata, M., and Funasaki, N. (2003) Iron hemiporphycene as a functional prosthetic group for myoglobin. Inorg. Chem. 42, 1456−1461. (65) Neya, S., Imai, K., Hiramatsu, Y., Kitagawa, T., Hoshino, T., Hata, M., and Funasaki, N. (2006) Significance of the molecular shape of iron corrphycene in a protein pocket. Inorg. Chem. 45, 4238−4242. (66) Hayashi, T., Murata, D., Makino, M., Sugimoto, H., Matsuo, T., Sato, H., Shiro, Y., and Hisaeda, Y. (2006) Crystal structure and peroxidase activity of myoglobin reconstituted with iron porphycene. Inorg. Chem. 45, 10530−10536. (67) Matsuo, T., Ito, K., Nakashima, Y., Hisaeda, Y., and Hayashi, T. (2008) Effect of peripheral trifluoromethyl groups in artificial iron porphycene cofactor on ligand binding properties of myoglobin. J. Inorg. Biochem. 102, 166−173. (68) Hayashi, T., Morita, Y., Mizohata, E., Oohora, K., Ohbayashi, J., Inoue, T., and Hisaeda, Y. (2014) Co(II)/Co(I) reduction-induced axial histidine-flipping in myoglobin reconstituted with a cobalt tetradehydrocorrin as a methionine synthase model. Chem. Commun. 50, 12560−12563. (69) Morita, Y., Oohora, K., Sawada, A., Doitomi, K., Ohbayashi, J., Kamachi, T., Yoshizawa, K., Hisaeda, Y., and Hayashi, T. (2016) Intraprotein transmethylation via a CH3-Co(III) species in myoglobin reconstituted with a cobalt corrinoid complex. Dalton Trans. 45, 3277−3284. (70) Abe, S., Ueno, T., Reddy, P. A. N., Okazaki, S., Hikage, T., Suzuki, A., Yamane, T., Nakajima, H., and Watanabe, Y. (2007) Design and structure analysis of artificial metalloproteins: selective coordinaH

DOI: 10.1021/acs.biochem.7b00457 Biochemistry XXXX, XXX, XXX−XXX

Article

Biochemistry tion of His64 to copper complexes with square-planar structure in the apo-myoglobin scaffold. Inorg. Chem. 46, 5137−5139. (71) Kendrew, J. C., Bodo, G., Dintzis, H. M., Parrish, R. G., Wyckoff, H., and Phillips, D. C. (1958) A three-dimensional model of the myoglobin molecule obtained by x-ray analysis. Nature 181, 662−666. (72) Vojtechovský, J., Chu, K., Berendzen, J., Sweet, R. M., and Schlichting, I. (1999) Crystal structures of myoglobin-ligand complexes at near-atomic resolution. Biophys. J. 77, 2153−2174. (73) Hughson, F. M., Wright, P. E., and Baldwin, R. L. (1990) Structural characterization of a partly folded apomyoglobin intermediate. Science 249, 1544−1548. (74) Jennings, P. A., and Wright, P. E. (1993) Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science 262, 892−896. (75) Cocco, M. J., and Lecomte, J. T. J. (1990) Characterization of hydrophobic cores in apomyoglobin: a proton NMR spectroscopy study. Biochemistry 29, 11067−110672. (76) Krishnamoorthi, R., and La Mar, G. N. (1984) Identification of the titrating group in the heme cavity of myoglobin. Evidence for the heme-protein π-π interaction. Eur. J. Biochem. 138, 135−140. (77) Bashford, D., Case, D. A., Dalvit, C., Tennant, L., and Wright, P. E. (1993) Electrostatic calculations of side-chain pKa values in myoglobin and comparison with NMR data for histidines. Biochemistry 32, 8045−8056. (78) Quillin, M. L., Arduini, R. M., Olson, J. S., and Phillips, G. N., Jr. (1993) High-resolution crystal structures of distal histidine mutants of sperm whale myoglobin. J. Mol. Biol. 234, 140−155. (79) Zhao, X., Vyas, K., Nguyen, B. D., Rajarathnam, K., La Mar, G. N., Li, T., Phillips, G. N., Jr, Eich, R. F., Olson, J. S., Ling, J., and Bocian, D. F. (1995) A double mutant of sperm whale myoglobin mimics the structure and function of elephant myoglobin. J. Biol. Chem. 270, 20763−20774. (80) Carver, T. E., Brantley, R. E., Jr, Singleton, E. W., Arduini, R. M., Quillin, M. L., Phillips, G. N., Jr, and Olson, J. S. (1992) A novel sitedirected mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate. J. Biol. Chem. 267, 14443−14450. (81) Toi, H., Homma, M., Suzuki, A., and Ogoshi, H. (1985) Paramagnetic 19F n.m.r. spectra of iron(III) porphyrins substituted with CF3 groups and reconstituted myoglobin. J. Chem. Soc., Chem. Commun., 1791−1792. (82) Springer, B. A., Egeberg, K. D., Sligar, S. G., Rohlfs, R. J., Mathews, A. J., and Olson, J. S. (1989) Discrimination between oxygen and carbon monoxide and inhibition of autooxidation by myoglobin. Site-directed mutagenesis of the distal histidine. J. Biol. Chem. 264, 3057−3060. (83) Teale, F. W. J. (1959) Cleavage of the haem-protein link by acid methylethylketone. Biochim. Biophys. Acta 35, 543. (84) Kabsch, W. (2010) XDS. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 125−132. (85) McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658−674. (86) Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H., and Adams, P. D. (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr., Sect. D: Biol. Crystallogr. 68, 352−367. (87) Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 486−501. (88) Shibata, T., Matsumoto, D., Nishimura, R., Tai, H., Matsuoka, A., Nagao, S., Matsuo, T., Hirota, S., Imai, K., Neya, S., Suzuki, A., and Yamamoto, Y. (2012) Relationship between oxygen affinity and autoxidation of myoglobin. Inorg. Chem. 51, 11955−11960. (89) Nishimura, R., Shibata, T., Tai, H., Ishigami, I., Ogura, T., Nagao, S., Matsuo, T., Hirota, S., Imai, K., Neya, S., Suzuki, A., and Yamamoto, Y. (2013) Relationship between the electron density of the heme Fe atom and the vibrational frequencies of the Fe-bound carbon monoxide in myoglobin. Inorg. Chem. 52, 3349−3355.

(90) Nishimura, R., Shibata, T., Ishigami, I., Ogura, T., Tai, H., Nagao, S., Matsuo, T., Hirota, S., Shoji, O., Watanabe, Y., Imai, K., Neya, S., Suzuki, A., and Yamamoto, Y. (2014) Electronic control of discrimination between O2 and CO in myoglobin lacking the distal histidine residue. Inorg. Chem. 53, 1091−1099. (91) Nishimura, R., Matsumoto, D., Shibata, T., Yanagisawa, S., Ogura, T., Tai, H., Matsuo, T., Hirota, S., Neya, S., Suzuki, A., and Yamamoto, Y. (2014) Electronic control of ligand-binding preference of a myoglobin mutant. Inorg. Chem. 53, 9156−9165. (92) Kanai, Y., Nishimura, R., Shibata, T., Yanagisawa, S., Ogura, T., Matsuo, T., Hirota, S., Neya, S., Suauki, A., and Yamamoto, Y. (2016) Effects of Heme Electronic Structure and Distal Polar Interaction on Functional and Vibrational Properties of Myoglobin. Inorg. Chem. 55, 1613−1622. (93) Shibata, T., Kanai, Y., Nishimura, R., Xu, L., Moritaka, Y., Suzuki, A., Neya, S., Nakamura, M., and Yamamoto, Y. (2016) Characterization of ground state electron configurations of high-spin quintet ferrous heme Iron in deoxy myoglobin reconstituted with trifluoromethyl group-substituted heme cofactors. Inorg. Chem. 55, 12128−12136. (94) Bondi, A. (1964) van der Waals volumes and radii. J. Phys. Chem. 68, 441−451. (95) Emerson, S. D., and La Mar, G. N. (1990) NMR determination of the orientation of the magnetic susceptibility tensor in cyanometmyoglobin: a new probe of steric tilt of bound ligand. Biochemistry 29, 1556−1566. (96) Shokhirev, N. V., and Walker, F. A. (1998) The effect of axial ligand plane orientation on the contact and pseudocontact shifts of low-spin ferriheme proteins. JBIC, J. Biol. Inorg. Chem. 3, 581−594. (97) Shokhirev, N. V., and Walker, F. A. (1998) Analysis of the temperature dependence of the 1H and 13C isotropic shifts of horse heart ferricytochrome c: explanation of Curie and anti-Curie temperature dependence and nonlinear pseudocontact shifts in a two-level framework. J. Am. Chem. Soc. 120, 981−990. (98) Walker, F. A. (2000) The porphyrin handbook (Kadish, K. M., Smith, K. M., and Guillard, R., Eds.), Vol. 5, pp 81−183, Academic Press, San Diego. (99) Tachiiri, N., Hemmi, H., Takayama, S. J., Mita, H., Hasegawa, J., Sambongi, Y., and Yamamoto, Y. (2004) Effects of axial methionine coordination on the in-plane asymmetry of the heme electronic structure of cytochrome c. JBIC, J. Biol. Inorg. Chem. 9, 733−742. (100) Arcovito, A., Benfatto, M., Cianci, M., Hasnain, S. S., Nienhaus, K., Nienhaus, G. U., Savino, C., Strange, R. W., Vallone, B., and Longa, S. D. (2007) X-ray structure analysis of a metalloprotein with enhanced active-site resolution using in situ x-ray absorption near edge structure spectroscopy. Proc. Natl. Acad. Sci. U. S. A. 104, 6211−6216. (101) La Mar, G. N. (1973) NMR of paramagnetic molecules, principles and applications (La Mar, G. N., Horrocks, W. D., Jr., and Holm, R. H., Eds.), Chapter 3, pp 85−126, Academic Press, New York. (102) Kolczak, U., Hauksson, J. B., Davis, N. L., Pande, U., de Ropp, J. S., Langry, K. C., Smith, K. M., and La Mar, G. N. (1999) 1H NMR investigation of the role of intrinsic heme versus protein-induced rhombic perturbations on the electronic structure of low-spin ferrihemoproteins: effect of heme substituents on heme orientation in myoglobin. J. Am. Chem. Soc. 121, 835−843.

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DOI: 10.1021/acs.biochem.7b00457 Biochemistry XXXX, XXX, XXX−XXX