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A Nuclear Resonance Vibrational Spectroscopic Study of Oxy Myoglobins Reconstituted with Chemically Modified Heme Cofactors: Insights into the Fe–O Bonding and Internal Dynamics of the Protein 2
Takehiro Ohta, Tomokazu Shibata, Yasuhiro Kobayashi, Yoshitaka Yoda, Takashi Ogura, Saburo Neya, Akihiro Suzuki, Makoto Seto, and Yasuhiko Yamamoto Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00829 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on November 14, 2018
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Biochemistry
A Nuclear Resonance Vibrational Spectroscopic Study of Oxy Myoglobins Reconstituted with Chemically Modified Heme Cofactors: Insights into the Fe–O2 Bonding and Internal Dynamics of the Protein Takehiro Ohta,†,@,* Tomokazu Shibata,‡ Yasuhiro Kobayashi,§ Yoshitaka Yoda,¶ Takashi Ogura,†,∆ Saburo Neya,# Akihiro Suzuki,& Makoto Seto§,ǁ and Yasuhiko Yamamoto‡,* †
Picobiology Institute, Graduate School of Life Science, University of Hyogo, RSC-UH LP Center, Hyogo 679-5148, Japan Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan § Institute for Integrated Radiation and Nuclear Science, Kyoto University, Osaka 590-0494, Japan ‡
ǁ
Japan Atomic Energy Agency, Hyogo 679-5148, Japan
¶
Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, Japan
#
Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8657, Japan Department of Material Engineering, National Institute of Technology, Nagaoka College, Nagaoka 940-8532, Japan
&
Supporting Information Placeholder ABSTRACT: The molecular mechanism of O2 binding to hemoglobin (Hb) and myoglobin (Mb) is a long-standing issue in the field of bioinorganic and biophysical chemistry. The nature of Fe–O2 bond in oxy Hb and Mb had been extensively investigated by resonance Raman spectroscopy, which assigned the Fe–O2 stretching bands at ~570 cm–1. However, resonance Raman assignment of the vibrational mode had been elusive due to the spectroscopic selection rule and to the limited information available about the ground-state molecular structure. Thus, nuclear resonance vibrational spectroscopy was applied to oxy Mbs reconstituted with 57Fe-labeled native heme cofactor and two chemically modified ones. This advanced spectroscopy in conjunction with DFT analyses gave new insights into the nature of the Fe–O2 bond of oxy heme by revealing the effect of heme peripheral substitutions on the vibrational dynamics of heme Fe atom, where the main Fe–O2 stretching band of the native protein was characterized at ~420 cm–1.
Myoglobin (Mb) has been serving as a paradigm for the structure-function correlation of hemoproteins, since after the first x-ray crystal structure was reported 60 years ago.1 However, understanding of the Fe–O2 bonding of oxy myoglobin (MbO2) is a long-standing issue in the field of bioinorganic chemistry.2-4 While three major electronic states of Pauling (Fe2+(S = 0)–O2(S = 0)),5 McClure-Goddard (Fe2+(S = 1)–O2(S = 1)),6,7 and Weiss (Fe3+(S = 1/2)–O2–(S = 1/2))8 mechanisms were proposed theoretically, experimental probes for the electronic structure of the Fe– O2 bond are limited.4 Moreover, mechanistic understanding of protein dynamics that controls the O2 affinity of Mb is a long-sought goal of the biophysical research.9
Figure 1. (a) Crystal structure of MbO2 (PDB ID: 2Z6S).3a Structural disorder is found in the conformation of His64, showing variation in the distance between the distal oxygen atom of O2 and Ne atom (a and b are 3.01 and 2.76 Å, respectively). (b) Molecular structures of the heme cofactors used in this study; protoheme (Proto) (R3 = R8 = CH=CH2, R7 = CH3), mesoheme (Meso) (R3 = R8 = C2H5, R7 = CH3) and 13,17-bis(2carboxylatoethyl)-3,8-diethyl-2,12,18-trimethyl-7trifluoromethylporphyrinatoiron(III)16 (7-PF) (R3 = R8 = C2 H5, R7 = CF3).
Studies10 on Mbs reconstituted with chemically modified heme cofactors with variation of the electron density of the heme Fe atom (rFe) revealed that the O2 affinity of the protein is regulated in such a manner that the O2 affinity of the protein decreases, due to an increase in the O2 dissociation rate constant (koff), with a decrease in the rFe. The relationship between the rFe and O2 affinity could be interpreted in terms of the effect of a change in the rFe on the equilibrium between ferrous-oxy (Fe2+–O2) and ferricsuperoxide (Fe3+–O2–) states, and a decrease in the rFe stabilizes the Fe2+–O2 over Fe3+–O2– state, resulting in an increase of koff.10 However, resonance Raman analysis of the
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Scheme 2. The equilibrium between Fe3+–O2– and Fe2+–O2 states controlled by the strength of distal hydrogen bond in the MbO2(7-PF).
Figure 2. 57Fe PVDOS of MbO2 (Proto) at 43 K (bottom), MbO2(Meso) at 27 K (middle) and MbO2(7-PF) at 26 K (top).
MbO2s demonstrated that the conventionally assigned Fe–O2 stretching band at ~570 cm–1 of the protein was not largely affected by a change in the rFe.11 Thus, in order to examine the band assignment, we applied nuclear resonance vibrational spectroscopy (NRVS)12-14 at SPring-815 to MbO2s reconstituted with 57Fe-labeled Meso, Proto and 7-PF (MbO2(Meso), MbO2(Proto) and MbO2(7-PF), respectively) (Figure 1b). In general, NRVS spectra of MbO217 and related O2 adducts of Fe-porphyrin complexes18 show two Fe–O2 vibrational bands, one at ~420 cm–1 and the other at ~570 cm–1, and the present study reveals that the former vibrational band is highly sensitive to the chemical modifications of heme cofactor because of the strong band intensity. The internal dynamics of the protein is also discussed on the basis of the concept of stiffness and resilience14 of the environment of heme Fe atom. Figure 2 shows the 57Fe partial vibrational densities of states (57Fe PVDOS) of the reconstituted proteins, obtained using PHOENIX software19. NRVS study of MbO2(Proto) was first reported by Sage and coworkers,17 and the present analysis provides basically the same results as the previous one. However, our density functional theory (DFT) analyses demonstrated that the vibrational spectrum of MbO2(Proto) can be better reproduced by the spin polarized Fe3+–O2– state calculated with broken-symmetry singlet state,
Scheme 1. Schematic drawing of Fe–O2 vibrational modes obtained by DFT based normal mode analysis for the Fe3+–O2– state of MbO2. The vibrational energies are experimental ones.
compared with the Fe2+–O2 closed-shell singlet state, in terms of the vibrational energies and peak intensities of the major bands of Fe-pyrrole (Fe–NPyr) and Fe–O2 ligand modes, thus giving new spectroscopic insights into the Fe–O2 bonding mechanism (vide infra) (Figure S1 and Table S1 in the Supporting Information). The normal mode analysis by DFT indicated that an intense band at 420 cm–1 and a small one at 576 cm–1 in the Fe–O2 ligand mode region are assignable to the Fe–O2 stretching and Fe–O-O bending modes, respectively (Figure S2 and Table S2 in the Supporting Information). As shown in Scheme 1, in the Fe–O2 stretching mode, the two oxygen atoms move in-phase, while the motion is out-of-phase in the bending mode (angle between the displacement vectors of two oxygen atoms is 174° as listed in Table S2). However, single crystal NRVS study of oxygenated ‘picket fence’ porphyrins18 suggested that significant mixing of the in-plane motion of Fe atom in both ~420 and 570 cm–1 bands may not allow their definitive assignments. Indeed, DFT based normal mode analysis of MbO2(Proto) in the Fe2+–O2 state suggested that a lower energy band computed at 459 cm–1 may be interpreted as Fe–O-O bending mode, while a higher energy band at 659 cm–1 as Fe–O2 stretching (Figure S7 and Table S7 in the Supporting Information). However still, the agreement between the experimental and theoretical 57Fe PVDOS in the present study supported the assignments of the bands at ~420 and ~570 cm–1 to the Fe–O2 stretching and Fe–O-O bending modes, respectively. Furthermore, the higher vibrational energy of Fe–O-O bending compared to that of the Fe–O2 stretching is reminiscent of the Fe–ligand vibration of carbon monoxy (CO) adduct of heme, where the Fe–C-O bending energy is higher than the Fe–CO stretching one.20-22 While resonance Raman spectroscopy of MbO2 did not show a band associated with Fe–O2 ligand vibration at 420 cm–1, other hemoproteins such as hemoglobin,23 heme oxygenase24 and cytochrome c oxidase25 exhibited oxygenisotope sensitive bands in a similar energy region, attributed to Fe–O-O bending mode. However, the assignments of the mode by resonance Raman studies were done by normal mode analyses using an Fe–O-O three body oscillator with assuming empirical force constants, which may bring significant uncertainty in the characterization of
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Biochemistry the vibrational modes. The weak resonance Raman enhancement of the mode at ~420 cm-1 may be due to the Raman selection rule which diminishes the Fe–O2 stretching band intensity that includes out-of-plane motions of iron porphyrin.
the Supporting Information) indicated that the force constants of stiffness are 369 ± 7, 374 ± 14, and 400 ± 36 pN/pm for MbO2(Proto), MbO2(Meso), and MbO2(7-PF), respectively, which are typical values for six-coordinate low-spin heme Fe atom.14,17
Deconvolution of the Fe–O2 stretching band region of the
The intensity of the 57Fe PVDOS below 100 cm–1 of MbO2(Proto) (Figure S3 (a), left, in the Supporting Information) indicated that translational motion of heme cofactor is larger in the native protein compared to those in MbO2(Meso) and MbO2(7-PF). Accordingly, the resilience14,17,29 of the native protein should be smaller than those of MbO2(Meso) and MbO2(7-PF). The conformational flexibility of the native protein would be associated with the appearance of multiple conformations of the Fe–O2 bond.
57Fe PVDOS of MbO2(Proto), i.e., 400-430 cm–1, gave at least
three peaks at 402, 421 and 436 cm–1 (Figure S3 in the Supporting Information), indicative of the presence of multiple conformations. A hydrogen bonding interaction between Fe-bound O2 and a nearby amino acid residue, for example, distal histidine (His64) in native protein (distal hydrogen bond) is thought to contribute to stabilize the Fe3+–O2– over the Fe2+–O2 state. In addition, resonance Raman studies of O2 bound hemoproteins showed that their Fe–O2 bonds are weakened by the formation of the distal hydrogen bond.26,27 Thus, the presence of a low energy band at ~400 cm–1 would indicate a formation of a strong distal hydrogen bond in a conformational sub-state of MbO2(Proto). The Fe2+–O2 bond is highly covalent and stronger than the Fe3+–O2–bond, where a coulombic interaction plays a dominant role (Figure S6 and Table S5 in the Supporting Information). In the spectra of MbO2(Meso) and MbO2(7-PF), the pseudo Fe–O2 stretching modes were observed in 400–430 cm–1, showing that the vibrational energy and the band shape were affected by the heme chemical modifications. Notably, the spectrum of MbO2(7-PF) showed two bands at 417 and 430 cm–1 (Figure S3 (a), right, in the Supporting Information), while that of MbO2(Meso) exhibited only a single peak at 420 cm–1. Thus, the bands of MbO2(7-PF), observed at 417 and 430 cm–1, may be derived from species formally described as Fe3+–O2– and Fe2+–O2, respectively (Figure S5 and Table S4 in the Supporting Information). The high energy region (550–650 cm–1) of MbO2(7-PF) includes additional weak band intensities compared to that of MbO2(Meso), implying a population of Fe2+–O2 state in MbO2(7-PF). The spectral feature of the Fe–Npyr region (300-380 cm–1) of MbO2(7-PF) and DFT simulation also suggest presence of at least two discrete forms. Thus, the two bands would not arise from Fermi doublet, but from structural inhomogeneity. In light of recent x-ray crystal structural analysis of met form of Mb reconstituted with 7-PF,28 where the presence of heme orientational disorder was observed, the strength of the distal hydrogen bond may be altered depending on the heme orientations (Scheme 2). DFT computations suggested that 7-PF is more accessible the Fe2+–O2 state than Proto due to a smaller energy gap between the Fe3+– O2– and Fe2+–O2 states (Table S6 in the Supporting Information). Further analysis of the 57Fe PVDOS provided effective force constant of stiffness,14,29 which gives the measure of the interaction between heme Fe atom and ligands, where the dominant features in 57Fe PVDOS at 250–450 cm–1 make the most important contribution. The theoretical calculation from experimental 57Fe PVDOS (see pp. S5-S6 in
In summary, the Fe–O2 stretching band at ~420 cm–1 can be a useful probe of the nature of the Fe–O2 bonding in oxygenated hemoproteins. Investigation of this mode with NRVS may also solve a problem of missing correlations between the Fe–O2 and O–O stretching frequencies.26,30 The softness and flexibility of the native MbO2 protein were reduced when heme was chemically modified, implicating a critical role of low-frequency dynamics that controls the reactivity of Mb.9
ASSOCIATED CONTENT Supporting Information. Experimental and computational sections, Tables S1 – S4, and Figures S1 – S5. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author Takehiro Ohta:
[email protected] Yasuhiko Yamamoto:
[email protected] ORCID Takehiro Ohta: 0000-0003-4140-5293 Yasuhiko Yamamoto: 0000-0003-4951-3184 Note The authors declare no competing financial interest. Present address: Department of Applied Chemistry, Faculty of Engineering, Sanyo-Onoda City University, Sanyo-Onoda, Yamaguchi 756-0884, Japan ∆ Deceased on July 23, 2017 @
ACKNOWLEDGMENT This work is dedicated to the memory of Prof. Takashi Ogura. This study was financially supported by JSPS KAKENHI (No. 16K05850 to T.O.) and (No. 24221005 to M.S.) and in part performed as a project of Y.Y. 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). NRVS measurement was performed at BL09XU of SPring-8, which was approved by JASRI (Proposal No. 2015A1433 and 2015B1148). DFT
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calculations were carried out at the Research Center for Computational Science, Okazaki, Japan.
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16 Toi, H., Homma, M., Suzuki, A., and Ogoshi, H. (1985) Paramagnetic 19 F n.m.r spectra of iron(III) porphyrins substituted with CF3 groups and reconstituted myoglobin, J. Chem. Soc., Chem. Commun. 1791–1792. 17 Zeng, W., Barabanschikov, A., Wang, N., Lu, Y., Zhao, J., Sthurhahn, W., Alp, E. E., and Sage, J. T. (2012) Vibrational dynamics of oxygenated heme proteins, Chem. Commun. 48, 6340-6342. 18 Li, J., Peng, Q., Barabanschikov, A., Pavlik, J. W., Alp, E. E., Sturhahn, W., Zhao, J., Schultz, C. E., Sage, J. T., and Scheidt, W. R. (2011) New perspectives on iron-ligand vibrations of oxyheme complexes, Chem. Eur. J. 17, 11178-11185. 19 Sturhahn, W. (2000) CONUSS and PHOENIX: Evaluation of nuclear resonant scattering data, Hyperfine Interact. 125, 149–172. 20 Hu, S., Vogel, K. M., and Spiro, T. G. (1994) Deformability of heme protein CO adducts: FT-IR assignment of the FeCO bending mode, J. Am. Chem. Soc. 116, 11187-11188. 21 Rai, B. K., Durbin, S. M., Prohofsky, E. W., Sage, J. T., Ellison, M. K., Roth, A., Scheidt, W. R., Sturhahn, W., and Alp, E. E. (2003) Direct determination of the complete set of iron normal modes in a porphyrin-imidazole model for carbonmonoxyheme proteins: [Fe(TPP)(CO)(1-MeIm)], J. Am. Chem. Soc. 125, 6927-6936. 22 Ohta, T., Liu, J.-G., Saito, M., Kobayashi, Y., Yoda, Y., Seto, M., and Naruta, Y. (2012) Axial ligand effects on vibrational dynamics of iron in heme carbonyl studied by nuclear resonance vibrational spectroscopy, J. Phys. Chem. B 116, 13831-13838. 23 Jeyarajah, S., Proniewicz, L. M., Brondrer, H., and Kincaid, J. R. (1994) Low frequency vibrational modes of oxygenated myoglobin, hemoglobins, and modified derivatives, J. Biol. Chem. 269, 31047-31050. 24 Takahashi, S., Ishikawa, K., Takeuchi, N., Ikeda-Saito, M., Yoshida T., and Rousseau, D. L. (1995) Oxygen-bound hemeheme oxygenase complex: Evidence for a highly bent structure of the coordinated oxygen, J. Am. Chem. Soc. 117, 6002-6006. 25 Hirota, S., Ogura, T., Appelman, E. H., Shinzawa-Itoh, K., Yoshikawa, S., and Kitagawa, T. (1994) Observation of a new oxygen-isotope-sensitive Raman band for oxyhemoproteins and its implications in heme pocket structures, J. Am. Chem. Soc. 116, 10564-10570. 26 Das, T. K., Couture, M., Ouellet, Y., Guertin, M., and Rousseau, D. L. (2001) Simultaneous observation of the O–O and Fe–O2 stretching modes in oxyhemoglobins, Proc. Natl. Acad. Sci. U.S.A. 98, 479-484. 27 Ohta, T., Yoshimura, H., Yoshioka, S., Aono, S., and Kitagawa, T. (2004) Oxygen-sensing mechanism of HemAT from Bacillus subtilis: A resonance Raman spectroscopic study, J. Am. Chem. Soc. 126, 15000-15001. 28 Kanai, Y., Harada, A., Shibata, T., Nishimura, R., Namiki, K., Watanabe, M., Nakamura, S., Yumoto, F., Senda, T., Suzuki, A., Neya, S., and Yamamoto, Y. (2017) Characterization of heme orientational disorder in a myoglobin reconstituted with a trifluoromethyl-group-substituted heme cofactor, Biochemistry 56, 4500-4508. 29 Leu, B. M., Zhang, Y., Bu, L., Straub, J. E., Zhao, J., Sturhahn, W., Alp, E. E., and Sage, J. T. (2008) Resilience of the iron environment in heme proteins, Biophys. J. 95, 5874-5889. 30 Ohta, T., and Kitagawa, T. (2005) Resonance Raman investigation of the specific sensing mechanism of a target molecule by gas sensory proteins, Inorg. Chem. 44, 758-769.
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TOC
HO
O
430
577 420
576
0.01 0.00
Proto
0.02
Proto: R 3 = R 8 = CH=CH2, R 7 = CH3 Meso: R 3 = R 8 = C2H 5, R 7 = CH3) 7-PF: R 3 = R 8 = C2H 5, R 7 = CF3)
0.01
576
O
Meso
0.02
367
HO
0.00
420
CH3
339
N
Fe PVDOS / cm
N
367
N Fe 2+
H 3C
345
0.01
335
N
7-PF
0.02
R8
417
R7
R3 H 3C
368
NRVS of oxy myoglobin with chemically modified hemes
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0.00 0
100
200
300 400 –1 Energy / cm
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600
