Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX
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Pressure Effect on Zero-Field Splitting Parameter of Hemin: Model Case of Hemoproteins under Pressure Tsubasa Okamoto,† Eiji Ohmichi,*,† Yu Saito,‡ Takahiro Sakurai,‡ and Hitoshi Ohta§ †
Graduate School of Science, ‡Research Facility Center for Science and Technology, and §Molecular Photoscience Research Center, Kobe University, Kobe 657-8501, Japan
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S Supporting Information *
ABSTRACT: We experimentally studied the pressure dependence of the zero-field splitting (ZFS) parameter of hemin (iron(III) protoporphyrin IX chloride), which is a model complex of hemoproteins, via high-frequency and high-field electron paramagnetic resonance (HFEPR) under pressure. Owing to the large ZFS, the pressure effect on the electronic structure of iron−porphyrin complexes has not yet been explored using EPR. Therefore, we systematically studied this effect using our newly developed sub-terahertz EPR spectroscopy system in the frequency range of 80−515 GHz, under magnetic fields up to 10 T and pressure up to 2 GPa. We observed a systematic shift of the resonance fields of hemin upon pressure application, from which the axial component of the ZFS parameter was found to increase from D = 6.9 to 7.9 cm−1 at 2 GPa. In contrast to the previous methods used to study proteins under pressure, which mainly focused on conformational changes, our HFEPR technique can obtain more microscopic insights into the electronic structures of metal ions under pressure. In this sense, our technique provides novel opportunities to study the pressure effects on biofunctional active centers of versatile metalloproteins. nitrogen atoms and one chlorine atom.8 In the case of free Fe3+ (3d5), the ground state is a sextet (S = 5/2, L = 0), but for hemin, the zero-field degeneracy of the three Kramers doublets of S = 5/2 (Sz = ±1/2, ±3/2, and ±5/2) is removed by the effect of zero-field splitting (ZFS), which yields the magnetic anisotropy of the system. In the case of S ≥ 1, ZFS is described by the following spin Hamiltonian
1. INTRODUCTION Porphyrin complexes play vital roles in biochemistry.1,2 In particular, metalloporphyrins,3 in which a metal ion is configured at the center of a porphyrin ring, is widely found in numerous proteins and enzymes, such as chlorophyll, hemoprotein, cytochrome, and cyanocobalamin. The metal ions in these complexes play essential roles in biological processes, such as redox, catalysis, and electron transfer. Thus, the electronic structures of metal ions in porphyrin complexes are crucial for understanding the functional roles of metalloproteins and metalloenzymes from a microscopic viewpoint. Hemoproteins are known to be among the most important metalloproteins in various biological systems.4,5 For example, hemoglobin plays a role in transporting oxygen molecules to individual cells. The functional center of hemoglobin is ironprotoporphyrin IX, which is also known as heme. An iron atom is located at the center of heme, and an oxygen molecule can be coordinated with the iron atom. Hemoglobin controls whether the oxygen molecule is captured or released, depending on subtle differences in the environment. During this process, the molecular structure of heme changes accordingly; thus, the microscopic study of the electronic structure of heme, especially around an iron atom, is very important.6,7 Because hemin, i.e., iron(III) protoporphyrin IX chloride,3 has the same geometric structure as functional centers of hemoproteins, this molecule is one of the model substances of hemoproteins. The Fe3+ ion located at the center of protoporphyrin IX is coordinated by five ligand atoms: four © XXXX American Chemical Society
H = DS2z + E(S2x − S2y)
(1)
where the z-axis corresponds to the direction parallel to the heme normal and the parameters D and E represent the axial and rhombic components of ZFS, respectively. Therefore, D and E primarily determine the magnetic and electronic properties of the spin system. Thus, precise determination of these parameters is fundamentally important for the elucidation of the electronic structure and functionality of these molecules. From this viewpoint, we previously performed multifrequency electron paramagnetic resonance (EPR) measurements of hemin at frequencies up to 700 GHz and unambiguously determined the D and E values as D ≈ 6.93 cm−1 and E ≈ 0.055 cm−1.9 Pressure is an important physical parameter in the study of proteins.10,11 In particular, hydrostatic pressure alters the internal and external interactions of proteins, the formation/ breaking of covalent and hydrogen bonds, and solvation. Received: April 2, 2018 Revised: June 14, 2018 Published: June 14, 2018 A
DOI: 10.1021/acs.jpcb.8b03128 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B Moreover, proteins have extra volume, i.e., “voids”, in their structure, which often plays important roles in selective biochemical reactions with specific targets. This imperfect packing endows proteins with significant volume compressibility. As a result, conformational changes of proteins are induced, leading to denaturation at a certain pressure. Typically, protein denaturation occurs around the pressure of 0.1 GPa. Numerous techniques, including EPR,12−15 nuclear magnetic resonance (NMR),10,16−18 circular dichroism spectroscopy,15 X-ray diffraction,19 Raman spectroscopy,20,21 optical spectroscopy,22,23 and other methods,24,25 have been employed to investigate proteins and model complexes under pressure. For example, McCoy et al.12 performed X-band EPR studies of site-directed spin-labeled proteins under pressure and discussed the flexibility and conformational changes of the proteins. However, although the results of most high-pressure protein studies12−25 have been discussed in terms of pressureinduced conformational changes, all pressure effects on proteins cannot be understood from a conformational perspective alone. In metalloproteins and metalloenzymes, the pressure strongly affects not only the conformation but also the active sites and metal ions26−28 through local structural changes that might modify the protein activities. Therefore, the effects of a high pressure on the local electronic structure of metal-ion complexes are of particular interest. However, such pressure effects on biofunctional centers have been studied only indirectly. For instance, the electronic structure of iron in hemoproteins was inferred from pressure-induced changes of ligands and side chains of heme molecules via optical spectroscopy20,29 and NMR.30,31 On the other hand, EPR has a unique advantage in that it permits the electronic structures of metal ions to be studied selectively and directly with a high spectral resolution, at the ambient pressure.32−34 However, no high-pressure EPR studies directly probing the active site of hemoproteins have been reported. This is attributed to the fact that iron−porphyrin complexes generally have large ZFS parameters, necessitating high-frequency broadband EPR techniques under pressure. Currently, such broadband EPR techniques under pressure are very limited;35−40 thus, the pressure dependence of the ZFS parameter has hardly been discussed. We performed a high-frequency and high-field EPR (HFEPR) study of hemin, a model substance of hemoproteins, at the ambient pressure and under applied pressure using specially developed pressure cells41−44 and herein report for the first time the systematic pressure dependence of the ZFS parameter at pressures up to 2.0 GPa. A systematic shift of the ZFS parameter upon application of pressure was observed and analyzed via numerical simulations. Our techniques are applicable to hemoproteins and other metalloproteins/metalloenzymes and are useful for elucidating the pressure effects on their active sites from a microscopic viewpoint.
cylinder and an outer jacket made of NiCrAl and CuBe, respectively. The inner diameter and length of the pressure cell were 5 and 10 mm, respectively. A piston and a bottom backup made of alumina ceramic were employed, along with a top backup made of zirconia-based ceramic. The alumina ceramic had inferior toughness than the zirconia-based one but had higher transparency over the entire frequency region covered in this study. This combination allowed us to perform EPR measurements in a wide frequency range of 50−700 GHz at a maximum hydrostatic pressure of 2 GPa. Daphne 7373 oil (Idemitsu Kosan Co. Ltd.) was used as a pressure medium and was encapsulated with a sample in a Teflon capsule. The lowtemperature pressure value was calibrated in advance using the pressure dependence of the superconducting transition temperature of tin. As shown in Figure 1, an electromagnetic
Figure 1. Schematic of the high-pressure transmission-type HFEPR system used in this study.
wave, introduced from the top of the pressure cell into the cell body, interacted with the sample and was finally transmitted to the other side of the cell body. The cutoff frequency was approximately 50 GHz for a 5 mm diameter. Figure 1 schematically shows the setup for the high-pressure EPR experiments, in which magnetic fields were generated by a cryogen-free superconducting magnet. Porcine hemin powder was purchased from Sigma-Aldrich (product no. 51280) and used as received. A sample weighing 66 mg was used. The intensity transmitted through the pressure cell was detected by an InSb bolometer placed at the bottom of the cryostat. An optical chopper was used to modulate the output power of the BWO, whereas an internal modulation source of the power supply was used for the Gunn oscillator. The synchronous changes were monitored by a lock-in amplifier with a time constant of 1 s. The magnetic field was swept with a typical sweep rate of 0.5 T/min. To analyze the experimental data, we calculated the resonance field with respect to the electromagnetic-wave frequency via numerical diagonalization of the following spin Hamiltonian
2. EXPERIMENTAL SECTION HFEPR measurements of hemin powder samples were performed at the ambient pressure and under applied pressure. A transmission-type EPR setup was used in this study. We used Gunn oscillators and backward-traveling wave oscillators (BWOs) as light sources. They covered the frequency ranges of 80−160 and 200−700 GHz, respectively. A specially designed pressure cell41−44 was used. The pressure-cell body was composed of two parts: an inner B
DOI: 10.1021/acs.jpcb.8b03128 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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Figure 2. (a−e) Multifrequency EPR data obtained at 4.2 K for P = 0, 0.51, 1.00, 1.52, and 1.96 GPa, respectively. The data are vertically shifted for clarity. The arrows indicate a systematic shift of the resonance field.
H = μB S·g ·B + DS2z + E(S2x − S2y)
3. RESULTS Figure 2 shows HFEPR spectra of the hemin powder sample obtained at pressures of P = 0, 0.51, 1.00, 1.52, and 1.96 GPa, respectively. All data were acquired for the same sample, and we confirmed that pressure-induced changes of the EPR spectra were reversible with a pressurization cycle. For the case of P = 0 GPa, the sample was immersed in Daphne 7373 oil at room temperature and cooled to 4.2 K without pressurization. Thus, the sample was surrounded by the frozen pressure medium. According to the Mössbauer effect,26 the ratio of ferrous iron to ferric iron in hemin increases at pressures above ∼5 GPa. All high-pressure EPR results correspond to ferric iron in this study, as expected. 3.1. Ambient-Pressure HFEPR Results. As shown in Figure 2a, we obtained the EPR spectra in the broad frequency range of 80−515 GHz. For frequencies of 80−160 GHz, an asymmetric powder-pattern spectrum, which is typically observed in X-band EPR measurements,46 was clearly observed. The most intense signal is considered to be the geff eff ≈ 6 signal (usually called gperp ). A multipeak structure
(2)
where g is the g-tensor, μB is the Bohr magneton, and B is the external magnetic field. In this calculation, intermolecular interactions such as exchange and dipolar interactions were not directly taken into account, as the estimated intermolecular exchange interaction was very small (J ≈ 0.1 cm−1)45 compared with the Zeeman term. The dipolar interaction was even smaller and can be considered negligible. The effect of the exchange interaction was partly represented by the intrinsic line width in the EPR simulation. Under hydrostatic pressure, the intermolecular exchange interaction may increase owing to the reduced intermolecular distance. However, it was difficult to quantitatively estimate this effect, because the frozen pressure medium caused the broadening of the EPR spectra, as shown later. Powder-pattern EPR spectra were numerically simulated by integrating individual EPR absorptions over the entire magnetic-field orientation. C
DOI: 10.1021/acs.jpcb.8b03128 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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Figure 3. (a) Electromagnetic-wave frequency vs resonance field derived from the raw data at the ambient pressure, together with the previously reported data in ref 9. The simulation curves are also shown. (b, c) Calculated energy levels for B//x, y and B//z, respectively, as a function of the applied magnetic field. The vertical arrows indicate the EPR transition between sublevels. The energy gaps at the zero field correspond to 2D and 4D from the ground state.
stemming from impurities contained in the zirconia parts was observed near g = 2, and the geff ≈ 2 signal (usually called geff para) eff was not clearly resolved. Here, the geff perp and gpara signals correspond to cases where an external magnetic field was applied perpendicular and parallel to the heme normal direction, respectively. As expected, the resonance fields of these signals increased with the frequency. geff perp signals were observed over the entire frequency range, whereas geff para signals were outside of the field range for frequencies beyond 293 GHz. In this study, the splitting of the geff perp signal was not observed, in contrast to the previous report.9 This is attributed to spectral broadening, which could have been due to an inhomogeneously distributed strain induced by the frozen pressure medium. In the frequency range above 293 GHz, two additional signals (hereinafter denoted as α and β) were observed in the lower-field side of the geff perp signal. The α branch shifted to the lower-field side as the frequency increased, finally approaching a zero field. On the other hand, the β signal observed above 400 GHz shifted to a higher-field region as the frequency increased. Figure 3a shows the frequency-field plot for all EPR signals at the ambient pressure, together with the results reported in ref 9. Simulation curves derived from eq 2 are also presented. Here, the solid and dotted lines correspond to the field orientation for B//x, y (θ = 90°) and B//z (θ = 0°), respectively. The experimental data agree well with the simulation curves, and the overall behavior was wellreproduced. As shown in Figure 3b,c, the observed α and β branches corresponded to the inter-Kramers transitions and directly gave the axial ZFS parameter. Using these data points, we estimated the spin Hamiltonian parameters by assuming that the system was totally axial (E ≈ 0), as follows: gx = gy = 1.93, gz = 2.05, and D = 6.90 ± 0.01 cm−1. Here, gi (i = x, y, z) represents the intrinsic g values and eff differs from geff perp and gpara, which were determined solely from eff the resonance field. The resonance fields of the geff perp and gpara signals were linear only in the low-field region and showed curvature in the high-field region owing to the existence of the D term. The D value was directly determined from the interKramers transitions (α and β) at zero field, which corresponds to the intersection in the vertical axis in Figure 3a. Because of this large D value, HFEPR is needed to directly determine the D terms of iron−porphyrin complexes. 3.2. High-Pressure HFEPR Results. Figure 4 shows a frequency-field diagram of three EPR branches for all pressure
Figure 4. Frequency-field diagram for all pressure values.
values. At frequencies below 350 GHz, the geff perp signals showed little pressure dependence, but above 350 Hz, they systematically shifted to the lower-field side as the pressure increased. For this reason, it has been difficult to observe a pressureinduced shift of the resonance field in hemin using conventional EPR techniques. In addition to geff perp, the α branch was shifted to the higher-field side. The relationship between the frequency and the resonance field was well-fitted by a straight line at all pressures, but the slope varied slightly under pressure, as discussed later. On the other hand, the β branch was shifted to the lower-field side as the pressure increased. Thus, the intersection of the α and β branches extrapolated to the zero field was shifted upward. This result clearly indicates an increase in the axial ZFS parameter D of hemin upon pressure application. For the β branch, a large pressure effect was found near the zero field, but it became small as the frequency increased, because the effect of the ZFS parameter D decreased within the high-magnetic-field limit (D ≪ gμBB). This trend is indicated by the simulation curves of the β branch in Figure 4. Figure S1 in the Supporting Information shows the frequency-field diagram for a wider frequency range up to 1000 GHz. Figure 5 shows the pressure dependence of the D term and the intrinsic gz value. The D values were estimated from the numerical simulations shown in Figure 4. The contribution of the E term was neglected in this study because of its small value compared to D (E/D < 0.01). Once the D values were determined, gx and gy were estimated to reproduce the experimentally observed geff perp signals. In addition, the gz values were estimated from the slope of the α branch. These analyses revealed that the D value increased substantially, from 6.9 to 7.9 cm−1, upon pressure application to 2 GPa. This D
DOI: 10.1021/acs.jpcb.8b03128 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The electron configuration of ferric hemin is 3d5, and the ground state is in the high-spin state (S = 5/2). Thus, there is no degree of freedom in the orbital momentum and magnetic anisotropy is not expected in the usual sense. Magnetic anisotropy in metalloporphyrins has been studied theoretically,49,53−55 and the spin−orbit interaction is widely recognized as the dominant source of the ZFS parameters compared with the spin−spin interaction. The spin−orbit interaction couples the ground state with excited states having different spin configurations; thus, the multiplet energy diagram must be considered (see Figure 6). The ground state (6A1 state) in the presence of a ligand field corresponds to the 6S state for the free 3d5 ion, and its electron configuration is (dxy)1(dyz)1(dxz)1(dx2 − y2)1(dz2)1. The lowest excited 4T1 (S = 3/2) state is composed of three degenerated quartet states in the cubic symmetry. When the symmetry is decreased, the 4T1 state splits into the 4E′ and 4A2′ sublevels in the tetragonal symmetry, and 4E′ is further split into Ex and Ey in the orthorhombic symmetry. The electron configurations of the Ex, Ey, and 4A2′ states are represented as (dxy)1(dyz)2(dxz)1(dz2)1, (dxy)1(dyz)1(dxz)2(dz2)1, and (dxy)2(dyz)1(dxz)1(dz2)1, respectively, for hemin. In these three excited states, one electron in the dx2 − y2 orbital moves to one of the t2g orbitals (dxy, dyz, or dxz). Here, we define the energy difference between the 6A1 state and the Ex, Ey, or 4A2′ state as Δ(Ex), Δ(Ey), or Δ(4A2′), respectively. The ZFS parameters D and E are written as ÄÅ É Å ij 1 yzÑÑÑÑ ζ 2 ÅÅÅ 2 1 j z zzÑÑÑ ÅÅ − jj + D= Δ(Ey) zzÑÑÑÑ 10 ÅÅÅÅ Δ(4 A ′ ) jj Δ(Ex) (3) 2 k {Ö Ç
Figure 5. Pressure dependence of D and the intrinsic gz value.
anomalously large shift was observed for the first time using our broadband HFEPR technique under pressure. We also found that the intrinsic gz values decreased slightly, from 2.06 to 2.00, as the pressure increased, although it was difficult to precisely estimate small changes in the intrinsic gx and gy values, owing to the broad EPR spectra. Because the β branches obtained at different pressures tended to merge together in the high-field region, we inferred that the intrinsic gx and gy values did not shift substantially upon pressure application. Therefore, we used previously estimated values (gx = gy = 1.93) for all simulation curves.
4. DISCUSSION 4.1. ZFS Parameters. There have been numerous studies on the axial ZFS parameter of hemin, D,34,47−52 although the rhombic ZFS parameter of hemin, E, was only very recently reported by our group.9 The axial component was large (D ≈ 6.9 cm−1) compared to those of other metalloporphyrins. We next discuss the electronic structure of hemin with regard to ligand field theory.
ÄÅ ÉÑ ζ 2 ÅÅÅÅ 1 1 ÑÑÑÑ ÅÅ ÑÑ E= − 10 ÅÅÅ Δ(Ex) Δ(Ey) ÑÑÑ Ç Ö
(4)
Figure 6. (a) Multiplet energy states for the 3d5 system. In the case of a free ion, the 6S and 4G states are the lowest and second-lowest multiplets. In the presence of a ligand field, as the symmetry is reduced, the multiplet energy levels are split. The energy difference between the ground and excited states are indicated by three arrows. (b) Spin configurations of 6A1 and 4A2′ and Ex or Ey (4E′ in the tetragonal symmetry) multiplet energy states. E
DOI: 10.1021/acs.jpcb.8b03128 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry B where ζ is the spin−orbit interaction constant. As mentioned previously, the E term could not be resolved in this study, owing to the broad EPR spectra, and is simplified as zero in the following discussion. 4.2. Pressure Effect on Electronic Structure. The effect of pressure on the electronic structures of iron−porphyrin complexes is not well-understood. One reason for this is that these compounds have large ZFS values exceeding the energy scale of X-band EPR spectrometers, and another reason is that eff geff perp and gpara signals, which are usually discussed in the context of X-band experiments, exhibit little frequency change below 350 GHz upon application of pressure, as shown in Figures 3 and 4. Therefore, a high-pressure broadband EPR technique is needed to investigate the pressure-induced changes in the electronic structures of these complexes. Upon the application of pressure, the crystal structure is compressed but the compressibility is generally not uniform because of the anisotropic nature of the molecular structures. For hemin, a porphyrin ring is composed of covalent bonds of carbon atoms and is rather rigid within the plane. Therefore, it is easier to compress the molecular structure normal to the plane and the bond length between iron and chlorine is thought to be more sensitive than that between iron and nitrogen to the applied pressure. This anisotropic behavior was also observed in high-pressure vibrational studies of hemoproteins,20,56,57 where the skeletal modes of heme were slightly changed, while the axial ligands of heme were more affected upon pressure application. In this situation, it is expected that the energy level of the dxy orbital changes little, whereas those of the dyz and dxz orbitals shift upward upon pressure application. This is because the bonding between iron and chlorine atoms is π-antibonding; thus, reducing the iron− chlorine distance increases the energy of the dyz and dxz orbitals. Accordingly, the energy difference Δ(4E′) increases and the negative contribution to D decreases, according to eq 3. This is a qualitative explanation of the pressure-induced increase of ZFS, which is consistent with previously reported Mössbauer results obtained under pressure.26 To study the origins of the ZFS parameter, one can systematically change either the molecular structures or the anions of the complexes. The ZFS parameters of some hemin derivatives have been studied. Recently, iron(III) tetraphenylporphyrin with different anions (F−, Cl−, Br−, and I−) was studied via inelastic neutron scattering49 and the observed ZFS parameter was discussed on the basis of the systematic change of the anions. However, the interpretation of the results is not straightforward, owing to the complicated origin of ZFS parameters. As discussed in ref 49, not only the ligand field but also numerous additional parameters must be considered to determine the ZFS parameters in quantum chemical calculations, e.g., the covalency, spin−orbit coupling constant, and relativistic effect. In this sense, situations differ among samples and it is difficult to compare different complexes. On the other hand, pressure is another way to control the ZFS parameters, which is distinct from chemical modification. Pressure is often called a clean physical parameter because it allows for reversible and continuous changes while maintaining the same atoms with the same connectivity. Therefore, our novel high-pressure HFEPR technique provides a unique and beneficial approach to exploring the origins and nature of the ZFS parameters in versatile transition-metal-containing complexes, including metalloproteins/metalloenzymes.
4.3. Application to Hemoproteins and Other Metalloproteins/Metalloenzymes. Studies on the effects of a high pressure on proteins have mainly focused on conformational changes that are closely related to the protein activities. For instance, NMR,10,16−18 X-ray,19 infrared, and Raman spectroscopy20,21 have been reported. On the other hand, studies on the pressure effects at the active site of a metalloprotein have been limited, even though the electronic structure of metal ions plays crucial roles in protein activities. The Mössbauer effect under pressure26−28 was reported in relation to the local environments of iron atoms, but its application is limited to iron-containing systems. In contrast, our HFEPR technique can in principle detect any type of EPR-active spin species and is expected to have more versatile applications. HFEPR techniques are very sensitive to subtle changes in the local environment, such as changes in molecular structure, valence, and motional degrees of freedom, and have been used to investigate the active sites of metalloproteins/metalloenzymes at the ambient pressure with a high spectral resolution.32−34 Thus, our HFEPR technique provides unique opportunities to study the effects of a high pressure on such protein active sites. It is noted that the previously reported HFEPR technique under pressure39,40 used a diamond anvil cell, which cannot be practically applied to low-concentration samples due to the limited sample space. In metalloproteins, metal ions are often coordinated by amino-acid residues, which were a part of the protein structure. Upon pressure application, conformational changes are induced and accordingly the coordination of the amino-acid residues are affected. Such ligands are more susceptible to pressure than the chlorine atom of hemin, and more pressureinduced changes in the EPR spectra are expected for these proteins compared with model complexes. The EPR detection of ferrous iron states under pressure is also of particular interest, as most hemeproteins are active in the ferrous state. Thus, ferrous hemoproteins58 and hemin59 under pressure are worthwhile to study in the future. The spin sensitivity of our HFEPR system was estimated according to the signal-to-noise ratios of EPR signals of 2diphenyl-1-picrylhydrazyl (DPPH) polycrystals. DPPH was widely used as an EPR standard sample and showed a temperature-independent g value. Compared with conventional transmission-type experiments,32 several factors were unfavorable for achieving a high sensitivity in our setup. First, the attenuations of the electromagnetic wave during transmission through the pressure cell must be considered. A portion of the electromagnetic waves was reflected at the inlet of the pressure cell, and another portion was absorbed by zirconium parts inside the pressure cell. The narrow inner diameter of the cell body also causes unfavorable attenuations owing to the skin effect. Nevertheless, the spin sensitivity achieved in this study was typically 1013 spins/Gauss. With this sensitivity, a sample of 1.6 × 10−10 mol concentration will be detected in the case of an EPR line width of 1 mT. This quantity corresponds to a sample mass of 10 μg for a molecular weight of 60 kDa and is acceptable for practical sample preparation. For increasing the sensitivity, quasi-optical polarization-sensitive detection will be useful, in combination with a corrugated light pipe.60
5. SUMMARY We developed a broadband high-frequency EPR system operating under pressure and experimentally studied the F
DOI: 10.1021/acs.jpcb.8b03128 J. Phys. Chem. B XXXX, XXX, XXX−XXX
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pressure dependence of the ZFS parameter of hemin, a model complex of hemoproteins, in a frequency range up to 515 GHz and at pressures up to 2 GPa. Our data clearly indicate a 30% increase of the axial ZFS parameter, D, which is explained by the anisotropic compressibility of the hemin molecule. Our analysis was based on a ligand field theory as the crystal structure under pressure was not available. We hope that this work will stimulate further high-pressure structural investigations and subsequent quantum chemical calculations under pressure. Because pressure is recognized as a continuous and reversible physical parameter for controlling the electronic structure, the effect of pressure on the ZFS parameter provides unique opportunities to elucidate the origins and nature of the ZFS compared with other approaches based on structural modifications. To date, the effects of pressure on proteins have been studied mainly from the viewpoint of conformational changes. On the other hand, the pressure effects on the electronic structures of metal ions in metalloproteins/metalloenzymes have been unexplored owing to experimental difficulties, even though these ions play crucial roles in protein activities. Therefore, our high-pressure HFEPR technique will be extended to metalloproteins/metalloenzymes in the future. In this study, we achieved a spin sensitivity on the order of 1013 spins/Gauss, which is sufficient for 1 × 10−10 mol EPR species with a typical line width of 1 mT.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.8b03128. Expanded frequency-field diagram of hemin (
