Article pubs.acs.org/IC
Characterization of Ground State Electron Configurations of HighSpin Quintet Ferrous Heme Iron in Deoxy Myoglobin Reconstituted with Trifluoromethyl Group-Substituted Heme Cofactors Tomokazu Shibata,† Yuki Kanai,† Ryu Nishimura,†,‡ Liyang Xu,† Yuki Moritaka,† Akihiro Suzuki,§ Saburo Neya,⊥ Mikio Nakamura,∥ and Yasuhiko Yamamoto*,†,# †
Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan Department of Materials Engineering, Nagaoka National College of Technology, Nagaoka 940-8532, Japan ⊥ Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Chuoh-Inohana, Chiba 260-8675, Japan ∥ Department of Chemistry, Faculty of Science, Toho University, Funabashi, Chiba 274-8510, Japan # Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba 305-8577, Japan §
S Supporting Information *
ABSTRACT: We introduced trifluoromethyl (CF3) group(s) as heme side chain(s) of sperm whale myoglobin (Mb) in order to characterize the electronic nature of heme Fe(II) in deoxy Mb using 19F NMR spectroscopy. On the basis of the anti-Curie behavior of CF3 signals, we found that the deoxy Mb is in thermal equilibrium between the 5B2, (dxy)2(dxz)(dyz)(dz2)(dx2−y2), and 5E, (dxy)(dxz)2(dyz)(dz2)(dx2−y2), states of the heme Fe(II), i.e., 5B2 ⇆ 5E. Analysis of the curvature in Curie plots has yielded for the first time ΔH and ΔS values of ∼−20 kJ mol−1 and ∼−60 J K−1 mol−1, respectively, for the thermal equilibrium. Thus, the 5E state is slightly dominant over the 5B2 one at 25 °C. These findings provide not only valuable information about the ground state electronic structure of the high-spin heme Fe(II) in deoxy native Mb but also an important clue for elucidating the mechanism responsible for acceleration of the spin-forbidden oxygenation of the protein.
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a mixture of Weiss,15 Pauling,14,16 and McClure forms.17−20 Triplet O2 and a high-spin quintet heme Fe(II) possess two and four unpaired electrons, respectively, and hence, depending upon the relative directions of two sets of unpaired electrons of the reactants, the product would have two or six unpaired electrons. In contrast to this consideration, a low-spin singlet heme Fe(II) of oxy Mb has no unpaired electron.16 Consequently, the oxygenation of the protein involves inversion of at least one electron spin.9 Since chemical reactions do not normally change the spin states of electrons, the reaction of O2 with heme Fe(II) in the protein is formally spin forbidden, which means that it is slow. In the heme Fe(II) system, the dx2−y2 orbital is occupied in the quintet state while it is unoccupied in triplet and singlet states. The occupation of the dx2−y2 orbital is thought to be relevant to the displacement of the Fe atom from the porphyrin plane, because of an antibonding interaction between the dx2−y2 orbital and the lone pair of pyrrole N atoms in the porphyrin
INTRODUCTION Myoglobin (Mb), an oxygen (O2) storage hemoprotein, is probably at present the best, albeit as yet incompletely, understood metalloprotein in terms of its structure−function relationship.1−7 The heme cofactor is buried inside the protein matrix composed of 153 amino acid residues, and its binding to the protein is stabilized by the coordination bond between the heme iron atom (Fe) and the nitrogen one (Nε) of proximal His (His93) (Figure 1A), together with hydrophobic interaction of the heme cofactor 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.3,4,8 O2 is reversibly bound to a ferrous heme iron (Fe(II)) on the side of the heme cofactor opposite His93 in the protein (Figure 1A). Although the reaction between O2 and heme Fe(II) of deoxy Mb is often considered as one of the simplest biological reactions, in fact, the reaction is much more complicated than it appears.9−13 Upon oxygenation of the protein, ground state triplet O2 is bound to a high-spin quintet heme Fe(II) in deoxy Mb14 to yield a low-spin singlet heme Fe(II) in oxy Mb, which is considered as © XXXX American Chemical Society
Received: June 5, 2016
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DOI: 10.1021/acs.inorgchem.6b01360 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
Figure 1. (A) Structures and numbering system for protoheme (R2 = CH3, R3 = R8 = CHCH2, R7 = CH3), 2,8-DPF (R2 = R8 = CF3, R3 = R7 = CH3), and 7-PF (R2 = CH3, R3 = R8 = C2H5, R7 = CF3), and orientation of the axial His93 imidazole, with respect to the heme cofactor, in native Mb, with hydrogen bonding between the His93 NδH hydrogen and Leu89 carbonyl oxygen atoms.3,4,8 (B) d-Electron configurations of the 5E, (dxy)(dxz)2(dyz)(dz2)(dx2−y2), and 5B2, (dxy)2(dxz)(dyz)(dz2)(dx2−y2) states of the high-spin heme Fe(II) in deoxy Mb.
plane.21,22 Upon the O2 binding, an electron in the dx2−y2 orbital flips its spin state and moves to the dyz one through the spin− orbit interaction.21,22 Heme Fe is thought to play a crucial role by enhancing the spin-transition probability for accelerating the spin-forbidden oxygenation.9 The high-spin heme Fe(II) of deoxy Mb has been suggested to have excited states with unpaired electrons close in energy to the ground state, enhancing crossover probability through spin−orbit coupling of Fe to allow reactions with triplet O2.9 Thus, elucidation of the ground state electronic nature of heme Fe(II) in deoxy Mb is essential for understanding the oxygenation of the protein. Despite extensive efforts, however, the ground state electronic structure of the high-spin heme Fe(II) in deoxy Mb remains to be elucidated. Theoretical studies indicated that the low-lying electronic terms of the high-spin heme Fe(II) in deoxy Mb are 5 B2, 5E, 3E, and 1A with 5B2 or one of the 5E components as the ground state (Figure 1B).23−29 Investigation of the Mössbauer effect of deoxy Mb demonstrated that the ground state is one of the components of the 5E term split by rhombic distortions.26 The d-orbital splittings derived from the 5E, (dxy)(dxz)2(dyz)(d z 2 )(d x 2 −y 2 ) or (d xy )(d xz )(d yz ) 2 (d z 2 )(d x 2 −y 2 ), and 5 B 2 , (dxy)2(dxz)(dyz)(dz2)(dx2−y2) states for the high-spin heme Fe(II) are thought to be close in terms of energy.23−34 In addition, in the 5E state, the relative energies of the dxz and dyz orbitals are affected by the interaction between the heme Fe dπ orbital and His93 Nε out-of-plane p orbital, leading to the removal of the degeneracy of the energy levels for the two configurations of the 5E term.30−33 With the orientation of the His93 side chain imidazole with respect to the heme cofactor, as illustrated in Figure 1A, the energy level of (dxy)(dxz)2(dyz)(dz2)(dx2−y2) is lower than that of (dxy)(dxz)(dyz)2(dz2)(dx2−y2). The contributions of the multiple electronic states to the electronic and magnetic natures of deoxy Mb involve complicated data interpretation. The temperature dependence of quadruple splitting, ΔEQ, measured in Mössbauer experiments has been used as a probe for the characterization of the ground state electronic structure of the high-spin heme Fe(II) in the protein.26 Although the ΔEQ value sensitively reflects the differences in Coulomb repulsion, associated with the d electrons, regarding the states, elucidation of the electronic state of the high-spin heme Fe(II) in deoxy Mb at ambient temperature through interpretation of the data obtained for frozen samples remains to be performed.
Electron paramagnetic resonance (EPR) has been also utilized for such characterization.33 However, in addition to the ultralow sample temperatures used for the measurements, samples with high integer S values such as the high-spin heme Fe(II) present severe technical difficulties for such measurements. In studies of high-spin heme Fe(II) model complexes, X-ray structure determination, nuclear resonance vibrational spectroscopy, and magnetic circular dichroism have also been used to differentiate the ground state d-electron configurations.31,32,34 In this study, taking advantage of the high sensitivity and resolution of 19F NMR as to the heme electronic structure,35,36 we established a methodology for characterizing the mixing of the d-electron configurations of the 5E and 5B2 states of the heme Fe(II) of deoxy Mb at ambient temperature. 19F labeling of the protein has been performed through incorporation of trifluoromethyl group(CF3)-substituted heme cofactors, i.e., 13,17-bis(2-carboxylatoethyl)-3,7,12,18-tetramethyl-2,8-bis(trifluoromethyl)-porphyrinatoiron(III) (2,8-DPF)37 and 13,17-bis(2-carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7trifluoromethylporphyrinatoiron(III) (7-PF)38 (Figure 1A and also see the insets of Figure 2), into the proteins to yield reconstituted Mbs, i.e., Mb(2,8-DPF) and Mb(7-PF), respectively. Native heme (protoheme), 2,8-DPF, and 7-PF differ in the numbers of methyl (CH3), vinyl (CHCH2), ethyl (C2H5), and CF3 side chains at positions 2, 3, 7, and 8 (Figure 1A). 2,8-DPF possesses 2-CF3, 3-CH3, 7-CH3, and 8-CF3 side chains, and 7-PF has 2-CH3, 3-C2H5, 7-CF3, and 8-C2H5 ones, while protoheme has 2-CH3, 3-CHCH2, 7-CH3, and 8-CH CH2 ones. These CF3-substituted heme cofactors of Mb(2,8DPF) and Mb(7-PF) have been shown to be accommodated properly as for protoheme in the native protein.37 The mixing of the d-electron configuration between the 5E and 5B2 states in deoxy Mb(2,8-DPF) and Mb(7-PF) was clearly manifested in the anomalous temperature dependence of the 19F NMR signals. Analysis of the temperature dependence of the 19F NMR shifts allowed qualitative characterization of the thermodynamic properties of the d-electron configuration equilibrium between the two states of the proteins.
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MATERIALS AND METHODS
Materials and Protein Samples. All reagents and chemicals were obtained from commercial sources and used as received. Sperm whale B
DOI: 10.1021/acs.inorgchem.6b01360 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry δobs = δdia + δpara
(1)
where δdia and δpara are the diamagnetic and paramagnetic shifts, respectively. According to the Curie law, δpara is proportional to 1/T, and hence, δobs in eq 1 converges to δdia as T → ∞.44 Hence, the δpara values of observed CF3 19F signals of the deoxy proteins in the 5E and 5 B2 states, δ(5E) and δ(5B2), respectively, are given by
δ(5E) = E /T
(2)
δ(5B2 ) = B /T
(3)
where E and B are temperature-independent constants and T is absolute temperature. Since the interconversion between the two states is faster than the NMR time scale, the δpara value observed for a given CF3 signal is expressed as δpara = δ(5E)α + δ(5B2 )(1 − α)
(4)
where α represents the fraction of the E state. The equilibrium constant K (= [5E]/[5B2]) is expressed in terms of α as follows 5
K = α /(1 − α)
(5)
The temperature dependence of K is described by the van’t Hoff equation, ln K = −(ΔH/R)(1/T) + ΔS/R, where ΔH and ΔS are the enthalpy and entropy changes of the process and R is the gas constant. Using eqs 1−5, the van’t Hoff equation is written as
Figure 2. 19F NMR (471 MHz) spectra of deoxy Mb(2,8-DPF) (left) and Mb(7-PF) (right) at pH 6.5 in 90% H2O/10% 2H2O and the indicated temperatures. Molecular structures of 2,8-DPF and 7-PF, together with two different orientations of 7-PF relative to His93, i.e., the m and M forms,48 are illustrated in insets. Signal assignments are shown with the spectra.
ln[{B − T (δobs − δdia)}/{T (δobs − δdia) − E}] = − (ΔH /R )(1/T ) + ΔS /R
(6)
The nonlinear Curie plots of the signals were fitted to eq 6 to yield the optimal B and E values, together with the ΔH and ΔS values, using a curve-fitting program previously reported by Neya and Funasaki.43
Mb was purchased as a lyophilized powder from Biozyme and used without further purification. 2,8-DPF37 and 7-PF38 were synthesized as previously described. The apoprotein of Mb (apoMb) was prepared at 4 °C according to the procedure of Teale,39 and reconstituted Mbs were prepared as described previosuly.37,40 Deoxy Mb was prepared from metmyoglobin, which had been evacuated and flushed with nitrogen gas several times, by adding Na2S2O4 (Nacalai Chemicals Ltd.). The 2H2O content of the samples was ∼10%. The pH of a sample was adjusted using 0.1 M NaOH or HCl. The pH of each sample was measured with a Horiba F-22 pH meter equipped with a Horiba type 6069-10c electrode. 1 H and 19F NMR Spectroscopies. 1H and 19F NMR spectra of deoxy Mb(2,8-DPF) and Mb(7-PF) were recorded on a Bruker AVANCE-400 spectrometer operating at the 1H frequency of 400 MHz and a Bruker AVANCE-500 spectrometer operating at the 19F frequency of 471 MHz, respectively. Typical 1H and 19F NMR spectra consisted of about 20k transients with a spectral width of 100 kHz and 16k data points. The signal-to-noise ratio of the spectra was improved by apodization, which introduced 100 Hz line broadening. A 1H−19F two-dimensional heteronuclear Overhauser effect (HOESY)41 spectrum of the carbon monoxide (CO) adduct of Mb(2,8-DPF) in 10% H2O/90% 2H2O at pH 7.0 and 25 °C was recorded on a Bruker AVANCE-600 spectrometer operating at the 19F frequency of 565 MHz. Quadrature detection in the phase-sensitive mode with States time-proportional phase incrementation42 was employed for the measurement using 18 ppm 1H and 2 ppm 19F spectral widths, 256 (1H) × 1k (19F) data points, a 1.5 s relaxation delay, and a mixing time of 150 ms, A phase-shifted sine-squared window function was applied to both dimensions before two-dimensional Fourier transformation. The chemical shifts of 1H and 19F NMR spectra are given in ppm downfield from the residual 1H2HO as an internal reference and trifluoroacetic acid as an external reference, respectively. Analysis of Temperature Dependence of 19F NMR Shifts. The temperature dependence of the observed shifts of the 19F signals of deoxy Mb(2,8-DPF) and Mb(7-PF) was analyzed using a program reported by Neya and Funasaki43 in order to determine the thermodynamic parameters of the interconversion between the 5E and 5B2 states in the proteins. The observed shift (δobs) of a signal is expressed as
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RESULTS Temperature Dependence of 19F NMR Signals of Deoxy Mb(2,8-DPF) and Mb(7-PF). 19F NMR spectra of deoxy Mb(2,8-DPF) and Mb(7-PF) recorded at various temperatures are shown in Figure 2. The appearance of two signals in the spectrum of deoxy Mb(2,8-DPF) is due to removal of the chemical equivalence of the two CF3 groups of 2,8-DPF through the asymmetric heme−protein interaction in the protein.45,46 On the other hand, the observation of the two signals, i.e., signals m and M, in the spectrum of Mb(7-PF) is due to the heme orientational disorder, i.e., the presence of isomers possessing two heme orientations differing by 180° rotation about the 5,15-meso axis (see inset of Figure 2),47 and the signal intensities indicated the presence of m and M forms in a ratio of m:M = 7:3.48 The considerable broadening of the signals with decreasing temperature is due to Curie spin relaxation.49,50 Interestingly, with increasing temperature, the 2-CF3 and 8CF3 signals of deoxy Mb(2,8-DPF) exhibited upfield and downfield shifts, respectively (Figure 2). The Curie plots of the 2-CF3 and 8-CF3 signals, i.e., plots of the δobs values against the reciprocal of absolute temperature (1/T), exhibited not only apparent positive and negative slopes, respectively, but also curvature (Figure 3 and see Figure S1 in the Supporting Information). In order to obtain the δdia values for the 2-CF3 and 8-CF3 signals of deoxy Mb(2,8-DPF), the signals observed at 29.68 and 29.21 ppm in the 19F NMR spectrum of the diamagnetic CO adduct of Mb(2,8-DPF) were assigned using 1 H−19F HOESY (Figure 4). In the 1H NMR spectrum of the CO adduct of Mb, the Cγ′H3 signal due to Val68, located in close proximity to pyrrole I (see Figures S2 and S3 in the Supporting Information),4 was resolved in the upfield-shifted C
DOI: 10.1021/acs.inorgchem.6b01360 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry
Figure 3. Curie plots, i.e., plots of the δobs values against the reciprocal of absolute temperature (1/T), of the 19F NMR signals of deoxy Mb(2,8-DPF) (left) and Mb(7-PF) (right) at pH 6.5 in 90% H2O/ 10% 2H2O, together with fitting the data to eq 6 using a curve-fitting program previously reported by Neya and Funasaki.43 Plots for the 2CF3 signal of deoxy Mb(2,8-DPF) and signal M of deoxy Mb(7-PF) were fitted with the parameters yielded on analysis of the plots for 8CF3 and signal m, respectively. ΔH and ΔS values obtained on analysis are shown with the plots. Curie plots for the signals of the proteins in the 5E and 5B2 states, i.e., 5E(i) and 5B2(i), where i = 2-CF3, 8-CF3, m, or M, determined using the E and B values yielded on curve fitting, are shown as dot-and-dash and broken lines, respectively.
region at ∼−2 ppm.51 In Figure 4, the Val68 Cγ′H3 proton signal at −2.11 ppm exhibited a HOESY cross-peak only with the 19F signal at 29.68 ppm, allowing the assignment of this signal to 2-CF3 and hence the signal at 29.21 ppm to 8-CF3 (see Figure S4 in the Supporting Information). Considering the δdia values of 29.68 and 29.21 ppm for the 2-CF3 and 8-CF3 signals, respectively (see Figure S4 in the Supporting Information), the negative slope of the Curie plots for the 8CF3 signal was clearly considered as evidence that this signal does not obey the Curie law, i.e., anti-Curie behavior (see Figure S1 in the Supporting Information). Similarly, the Curie plots for signals m and M of deoxy Mb(7-PF) exhibited curvature and apparent positive and negative slopes, respectively. The linear fitting of the Curie plots for signals m and M yielded y intercepts at 1/T = 0 (δ1/T=0) of 44.2 and 53.2 ppm, respectively, which are far from their δdia values, i.e., 29.03 and 30.81 ppm, respectively42 (see Figure S1 in the Supporting Information). These results confirmed the anti-Curie behavior of the CF3 signals, indicating the temperature dependence of the molecular or/and electronic structure.52,53 In the absence of conformational freedom of CF3 groups in 2,8-DPF and 7-PF, the anti-Curie behavior of the CF3 signals of deoxy Mb(2,8DPF) and Mb(7-PF) is solely due to the temperature dependence of the ground state electron configurations. Temperature Dependence of His93 NδH Proton Signals of Deoxy Mb(2,8-DPF) and Mb(7-PF). In the 1H NMR spectrum of deoxy Mb(2,8-DPF), the His93 NδH proton signal is observed at ∼75 ppm and the signal of deoxy Mb(7PF) is observed as a 7:3 doublet peak due to the heme orientational disorder (see Figures S5 and S6 in the Supporting Information).48,54 The δ1/T=0 values of the Curie plots for the His93 NδH proton signals, i.e., 12.0 and 15.8 ppm for the signals of deoxy Mb(2,8-DPF) and Mb(7-PF), respectively, yielded through linear fitting, were a little far from the shifts
Figure 4. 1H−19F HOESY spectrum of the CO adduct of Mb(2,8DPF) in 10% H2O/90% 2H2O at pH 7.0 and 25 °C. Val68 Cγ′H3 proton signal at −2.11 ppm exhibited a HOESY cross-peak only with the 19F signal at 29.68 ppm, allowing assignment of this signal to 2-CF3 and hence the signal at 29.21 ppm to 8-CF3.
observed for the CO adduct of the native protein, i.e., 9.38 ppm.51
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DISCUSSION Ground State d-Electron Configuration of the Heme Fe in Deoxy Mb. Considering the δpara values of ∼30 and ∼10 ppm for the 2-CF3 and 8-CF3 signals of deoxy Mb(2,8-DPF), respectively, these signals exhibited relatively large splitting, i.e., a shift difference of 20.61 ppm at 25 °C. As reported previously,30,54 this finding indicated the significant contribution of the π spin delocalization mechanism of the high-spin quintet heme Fe(II) in the 5E state. Similarly, the relatively large splitting, i.e., ∼20 ppm, for the relatively small δpara values, i.e., up to ∼30 ppm, for signals m and M of deoxy Mb(7-PF) were also consistent with the 5E ground state. Using δdia = 29.21 ppm, the fitting of the Curie plots for the 8-CF3 signal of deoxy Mb(2,8-DPF) with eq 6 yielded values of −20 ± 10 kJ mol−1 and −57 ± 38 J K−1 mol−1 for ΔH and ΔS, respectively (Figure 3). Although the fitting of the Curie plots for the 2-CF3 signal of the protein was less obvious, the Curie plots were well reproduced with the ΔH and ΔS values obtained for the 8-CF3 signal. Similar analyses of the Curie plots of signal m of deoxy Mb(7-PF) yielded ΔH = −17 ± 6 kJ mol−1 and ΔS = −56 ± 21 J K−1 mol−1, and the Curie plots for D
DOI: 10.1021/acs.inorgchem.6b01360 Inorg. Chem. XXXX, XXX, XXX−XXX
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Inorganic Chemistry signal M could be fitted with these thermodynamic parameters (Figure 3). The similarity in the thermodynamic parameters between the two protein systems suggested that the d-electron configuration equilibrium between the 5E and the 5B2 states of the high-spin quintet heme Fe(II) in deoxy Mb is not largely affected by the number of CF3 groups introduced into the heme cofactor. Hence, the energy levels of the two quintet states were found to be affected to almost the same degree by alteration of the electron density of the heme Fe atom due to the introduction of CF3 groups. Furthermore, the energy levels of the two quintet states would not be largely affected by the withdrawal of electron density from the porphyrin π system toward the CF3 groups, possibly due to relatively weak electronic interaction between the Fe orbitals and the porphyrin π system in pentacoordinated high-spin heme Fe(II), as manifested in small δpara (
