10992
J. Phys. Chem. B 2001, 105, 10992-10999
Ultrafast Structural Relaxation of Myoglobin Following Photodissociation of Carbon Monoxide Probed by Time-Resolved Resonance Raman Spectroscopy Yasuhisa Mizutani‡ Institute for Molecular Science, Okazaki National Research Institutes, Myodaiji, Okazaki 444-8585, Japan
Teizo Kitagawa* Center for IntegratiVe Bioscience, Okazaki National Research Institutes, Myodaiji, Okazaki 444-8585, Japan ReceiVed: March 12, 2001; In Final Form: August 28, 2001
Studies on the structural relaxation of myoglobin following CO photolysis revealed that the structural change of heme itself caused by the cleavage of the Fe-CO bond is completed within the instrumental response time (∼2 ps) of the time-resolved resonance Raman apparatus used. In contrast, changes in the intensity and frequency of the iron-histidine stretching [ν(Fe-His)] mode were found to occur in the picosecond regime. The ν(Fe-His) band is absent for the CO-bound form, and its appearance upon photodissociation was not instantaneous in contrast with the changes observed in the vibrational modes of heme, suggesting appreciable time evolution of the Fe displacement from the heme plane. Same behaviors were observed for the model compound of the heme part without protein matrix. Therefore the intensity change in ν(Fe-His) is not associated with protein relaxation following the CO photodissociation. The band position of the ν(Fe-His) mode changed with a time constant of about 100 ps, whereas that of the model compound without the protein matrix showed no shift. This indicates that tertiary structural changes of the protein occurred in a 100 ps range.
I. Introduction Protein dynamics are intimately connected to the structure/ function relationship of biological systems. In numerous biological processes, the ensuing protein structural changes accompanying a reaction at a specific site must spatially extend to mesoscopic dimensions to achieve a biological function. The molecular mechanism of cooperativity in oxygen binding of hemoglobin (Hb) is one of the classical problems in this aspect.1 The binding of small molecular ligands to the hemes in Hb is a highly localized perturbation. Nonetheless, this localized perturbation initiates a sequence of propagating structural events that culminates in a change of quaternary structure, proceeding from the low affinity (T) deoxy state to the high affinity (R) ligated state. The large-amplitude motions at the quaternary level, which form a communication link at the subunit interface, are driven by changes of the tertiary structure upon ligation. In this respect, myoglobin (Mb), which is structurally very similar to a subunit of Hb, but is involved in oxygen storage in muscles, serves as a model system for the tertiary relaxation processes. Ultrafast optical absorption studies of Mb2,3 revealed that the excited-state potential energy surface of the ligated heme is dissociative regarding a ligand, so as to be able to photoinitiate a structural change from the ligated form to the deligated form within 50 fs with quantum yield of nearly unity. Protein reorganization in response to changes at the heme is expected to occur more slowly. By using CO as the ligand rather than O2, it is possible to avoid the geminate recombination complicating the reaction dynamics in the picosecond regime.4 Thus, the carbonmonoxy myoglobin (MbCO) system is unique in that * To whom correspondence should be addressed. Tel: +81-564-557340. Fax: +81-564-55-4639. E-mail:
[email protected]. ‡ Present address: Molecular Photoscience Research Center, Kobe University, Nada, Kobe 657-8501, Japan.
it gives access to the full range of protein relaxation processes by a single perturbation (ligand dissociation) without complicated side reactions and associated population dynamics. These features of MbCO enabled us to trigger optically the structural changes of a protein and to follow the accompanied structural relaxation using optical probes that are sensitive to different aspects of the protein’s motion. For this reason, an enormous body of work has been accumulated about relaxation processes of Mb, helping to establish general principles related to protein dynamics. In Mb, the heme is an iron-protoporphyrin IX (FeIIPPIX), in which the FeII ion is bound to the proximal histidine (His) as the only covalent link to the protein. The heme iron binds diatomic molecules, such as NO, CO, and O2, at the opposite side of the proximal His. X-ray crystallography provided extensive information about the end-point equilibrium structures.5 The magnitude of atomic displacements at the heme site that must occur on ligand binding and release, is estimated by examining the structures containing the heme with and without bound ligands. The ligated heme has a planar structure in which the low-spin iron atom is in the porphyrin plane. For MbCO, the core size, that is, the distance between the pyrrole nitrogen atoms and the heme center, is 2.005 Å.5 Upon ligand dissociation, the iron atom is converted from low to high spin (S ) 0 f S ) 2) and moves out of the porphyrin plane by about 0.3 Å. Concomitantly, the porphyrin core size expands to 2.057 Å. Resonance Raman (RR) spectroscopy has provided a considerable amount of details regarding the influence of tertiary and quaternary structures on a few selected vibrational modes of heme. In particular, the iron-proximal histidine stretching [ν(Fe-His)] frequency6,7 has been shown to couple with the tertiary and quaternary structures and also to be a direct reflection of proximal strain, a major determinant of ligand-
10.1021/jp010923w CCC: $20.00 © 2001 American Chemical Society Published on Web 10/16/2001
Ultrafast Dynamics of Myoglobin binding reactivity. The Fe-His bond is the only linkage between the heme and globin, and it thus functions as the focal point for the forces driving the structural changes. In this regard, timeresolved resonance Raman (TR3) studies of the ν(Fe-His) mode are key to elucidating the protein dynamics. Resonance Raman studies by Scott and Friedman showed that the temporal evolution of the ν(Fe-His) frequency of Hb after CO photodissociation was seen in nanosecond to microsecond regime.8 In contrast, for Mb, it was only a few picoseconds that the ν(Fe-His) band returns close to the equilibrium deoxy frequency.9 On the other hand, protein dynamics of Mb has been suggested to occur in picosecond regime.10-12 Therefore, more precise measurements on the ν(Fe-His) mode with higher time resolution are necessary to discuss the photolysis-induced structural changes of the protein, which would be functionally important. In this study, we closely examined the temporal behavior of the ν(Fe-His) band in comparison with those of other heme Raman bands to explore protein relaxation and found changes of the ν(Fe-His) band both in the intensity and in the frequency. II. Experimental Section Laser System. The details of the TR3 apparatus is described elsewhere.13 A picosecond mode-locked Ti:sapphire oscillator (Spectra-Physics, Tsunami 3950), pumped by an Ar+ ion laser (Spectra-Physics, BeamLok 2060), produced approximately 1.5 ps pulses with a repetition of 82 MHz and an average power of about 0.7 W. The seed pulse was amplified by a regenerative amplifier (Positive Light, Spitfire) operated at 1 kHz by pumping with the 527 nm output of an intracavity frequency-doubled Nd:YLF laser. This amplification unit provides 784 nm pulses, each with an energy of about 0.8 mJ, duration of 2.5 ps, and spectral width of 6 cm-1. In the pump arm, a pump pulse of 540 nm was generated with a home-built optical parametric generator (OPG) and amplifier (OPA), which were pumped with the second harmonic of the 784 nm output. In the probe arm, a probe pulse of 442 nm was generated as the first Stokes stimulated Raman scattering from compressed methane gas (50 kg/cm2) excited by the second harmonic of the 784 nm output. Components other than the first Stokes scattering were removed spectrally with a glass filter and dichroic mirrors and spatially with a Pellin-Broca prism. The energy and bandwidth of the OPG-OPA output was 30 µJ and about 3 nm, respectively. The pulses were used for the pump beam for the ps-TR3 measurements in this work after they were attenuated to 10 µJ using a Cr-coated quartz ND filter. The energy and bandwidth of the stimulated Raman scattering were between 1.0 and 1.5 µJ and 14 cm-1, respectively. The first Stokes scattering was attenuated to 0.1 µJ using the Cr-coated quartz ND filter. The pump and probe beams were made collinear and coaxial using a dichroic mirror. The polarization of the pump beam was rotated by 55° relative to that of the probe beam to minimize the effects of molecular rotations on the observed kinetics. Both beams were always monitored with photodiodes (Hamamatsu Photonics, S2387-1010R) and found to be stable within (10%. A crosscorrelation trace of the pump and probe pulses was measured with a 1 mm BBO crystal, which indicated a width of 2.3 ps. The 0.0 ps of delay time (uncertainty < 0.2 ps) was calibrated using sum frequency mixing in the same crystal. Data Acquisition. The sample solution was contained in a 10 mmφ NMR tube and spun with a spinning cell device that was designed to minimize the off-center deviation during rotation.14 The sample was spun at 3400 rpm in the spinning cell configured for 135° backscattering illumination and col-
J. Phys. Chem. B, Vol. 105, No. 44, 2001 10993 lection. This configuration is important to minimize the effects of molecular rotations on the observed kinetics.15 Spherical and cylindrical lenses were used to focus the pump and probe beams on the sample in line-focusing condition. Laser flux of the pump and probe pulses are ∼500 and ∼7 MW/cm2, respectively. The spectral features of photoproducts in the pump-probe spectra were also shown to be invariant to a 3-fold change of the probe power. The pump power was selected so that no saturation effect or spectral changes occurred by a tripling of the pump power. The TR3 data acquisitions were carried out as follows. In the forward scan of delay time, delay times were changed by increasing them. At each delay time, Raman signals were collected for three 20 s exposures with both the pump and probe beams present in the sample. This was followed by equivalent exposures for pump-only, probe-only, and dark measurements. Measurements in the backward scan of delay time were done in the same procedure except for the direction of change in delay time. The transient Raman spectra were obtained by averaging the data for 10 entire cycles for Stokes spectra and 90 entire cycles for anti-Stokes spectra. This method enabled us to avoid the errors caused by a slow drift of laser power and to obtain quantitatively reproducible spectra from 1 day to the next, which is possible because of the excellent long-term stability of this laser system. The pump-only spectrum was directly subtracted from the pump and probe spectrum, yielding the “probe-withphotolysis” spectrum. The dark spectrum was directly subtracted from the probe-only spectrum, yielding the “probe-withoutphotolysis” spectrum (MbCO). The probe-without-photolysis spectrum was subtracted from the probe-with-photolysis spectrum to yield the photoproduct spectrum. The subtraction parameter was determined by subtracting the probe-withoutphotolysis spectrum from the probe-with-photolysis spectrum until negative features were seen at the location of prominent bands of MbCO. The subtraction parameter was then reduced until these negative peaks were just eliminated, thereby accounting for the depletion of MbCO caused by the pump pulse. The scattering intensity for the change in the optical absorption of the sample at each time point was corrected by normalizing the data to the intensity changes of the 982 cm-1 line of sulfate ions dissolved into the sample. Intensity of the sulfate band decreased by 8% upon the photoexcitation due to the effect of self-absorption and was nearly constant in the range where the time delay was positive. The moment analysis needs no assumption about the band shape and, therefore, provides general information even if the band shape is asymmetric. The band intensity and position were calculated in terms of the zeroth and first moments of the band, respectively. Raman scattering was collected by a doublet achromat (80 mm focal length (FL), f/2) and were imaged onto the 200 µm entrance slit of a single spectrometer (Spex, 500M) by a doublet achromat (200 mm FL, f/5). A dichroic short-pass filter was placed between the lenses to remove the scattered pump beam. A holographic notch filter (Kaiser Optical Systems, HSNF441.6-1.0) was used to reject the unshifted scattering. A polarization scrambler was placed at the entrance slit to remove the effects of polarization on the spectrograph throughput. The spectrograph is equipped with a blazed-holographic grating (2400 grooves/mm) that enables measurements of a spectrum as wide as about 1000 cm-1 in the Soret region and with a spectral slit width of approximately 8 cm-1. The dispersed light was detected by a liquid-nitrogen-cooled CCD detector (Princeton Instruments, CCD-1100PB). Raman shifts were calibrated with cyclohexane, benzene, or carbon tetrachloride. The peak positions of Raman bands are accurate within (2 cm-1.
10994 J. Phys. Chem. B, Vol. 105, No. 44, 2001
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Sample Preparation. Horse skeletal Mb (Sigma, M-0630) was dissolved into a buffer (50 mM Tris‚HCl, pH 8.0) and filtrated through a 0.45 µm PTFE membrane (Millipore) to make a 300 µM solution. A total of 1 mL of the Mb solution was placed into an airtight NMR tube (10 mmφ). The remaining oxygen in the solution was removed by more than five cycles of degassing and back-filling with CO. Finally, the solution was equilibrated under 1 atm of CO. The samples were reduced with a 5-fold stoichiometric amount of sodium dithionite in a small amount of solution (approximately 50 µL). The sample solution in the cell was continuously spun during the measurements to prevent multiple probing of the same portion of sample. The sample was replaced with a fresh one every 3 h. Sample integrity was confirmed with UV-vis absorption spectra after the TR3 measurements. The samples of deoxyMb were prepared by the same procedure with MbCO preparation, except for back-filling with N2 and the equilibration of the sample under 1 atm of N2. Hemin (Sigma, H-2250) was dissolved into buffer (50 mM Tris‚ HCl, pH 8.0), containing 0.1% CTAB and 15 mM 2-methylimidazole, and filtrated through a 0.45 µm PTFE membrane to make a 300 µM solution. The procedure for the preparation of the CO-bound form of the complex was the same as that for MbCO. III. Results Raman Spectral Change of the Heme. Figure 1 shows the Stokes TR3 difference spectra of photodissociated Mb for various values of delay times of the probe pulse with respect to the pump pulse. In these spectra, the contribution of unreacted species has been subtracted. The fraction of photolyzed MbCO was estimated to be 6% based on the intensity loss of the Raman bands of MbCO. This number was the same for all the delay times shown in Figure 1. The TR3 spectrum in the 1000-1700 cm-1 region for a 1 ps delay (Figure 1) contains only the bands arising from the in-plane vibrations of heme at 1352 (ν4), 1560 (ν2), and 1617 (ν10) cm-1.16,17 These bands exhibited appreciable narrowing and frequency upshifts in the first few picoseconds, but no further changes occurred after that. The TR3 spectrum for a 2 ps delay closely resembles the spectrum of deoxyMb, indicating that the photodissociated heme has relaxed to the equilibrium structure within the instrument response time (∼2 ps). Most interestingly, the ν2 band, whose frequency is wellknown to be sensitive to the core size of the porphyrin ring,18-21 appeared at a position close to that of the ν2 band of equilibrium deoxyMb. This demonstrates that expansion of the core size is instantaneous upon CO photodissociation. This is in contrast to the results by Dasgupta et al.22 which suggested that some residual core relaxation in picoseconds is still required to achieve the heme structure in the deoxy Mb. There is no Raman band that can be assigned to the excited electronic state. The involvement of the excited electronic state in the relaxation pathway cannot be ruled out, and indeed, a few reports suggested this to be likely.23-25 However, the results in this work and other studies26,27 indicate that a majority of the population is directly channeled into the vibrational manifolds of the ground electronic state and minor channels may lead to an appearance of small amounts of long-lived (picosecond time scale) intermediate states.26 The intensity invariance in the spectra for delay times between 5 and 50 ps is consistent with the fact that recombination of CO to the heme takes place in the time regime of microseconds to milliseconds28 in contrast with the cases for NO and O2.4 Accordingly, these results show the picture that the heme in Mb is led to the ground electronic state and expanded to the equilibrium core size within the instrument response time.
Figure 1. TR3 Stokes spectra of photodissociated MbCO in the 11001700 cm-1 region. Stokes spectra of the equilibrium deoxyMb and MbCO are depicted at the bottom for comparison.
Figure 2 shows the TR3 spectrum in the 150-850 cm-1 region for delay times between -7 and 1000 ps. The bands at 220, 301, 369, and 671 cm-1 in the spectrum for a 1000 ps delay are assigned to the vibrations of the heme. The large peak at about 220 cm-1 arises from the stretching mode of the covalent bond between the heme iron and the N of proximal His, ν(FeHis).29 The peak at 301 cm-1 is an out-of-plane mode (γ7; methine wagging).30 The peak at 369 cm-1 is a substituent mode, δ(CβCcCd), involving deformation of the propionate methylene groups.30 The peak at 671 cm-1 is an in-plane mode (ν7; breathing-like mode of the porphyrin inner ring). Similar to the spectra in the high- frequency region, RR bands exhibited appreciable frequency upshifts in the first few picoseconds, but no further large changes occurred after that. Raman Spectral Change of the Fe-His Stretching Mode. The band intensity and position were calculated in terms of the zeroth and first moments of the band, respectively. To draw baselines, pixels where the band intensities were nearly zero were selected in both sides of the band as illustrated with a broken line for the 220 cm-1 band of deoxyMb spectrum in Figure 2 as an example. The averaged intensities over five pixels around the channel were calculated. The baselines were drawn by connecting the two points. The moments were calculated for signal intensities above the baseline for each pixel. It was confirmed that the choice of the two pixels did not affect the results on the calculated moments. The temporal changes of the Raman band intensities observed for Mb are plotted in
Ultrafast Dynamics of Myoglobin
Figure 2. TR3 Stokes spectra of photodissociated MbCO in the 150850 cm-1 region. The Stokes spectra of the equilibrium deoxyMb and MbCO are depicted at the bottom for comparison. An example of drawing the baseline is shown with the 220 cm-1 band of deoxyMb spectrum. The baselines for all the TR3 spectra were drawn in the same way as that of the deoxyMb spectrum (see text). Experimental conditions are the same as those of Figure 1.
Figures 3 (ν7 and ν4 bands) and 4 (γ7 and ν(Fe-His) bands31). Temporal behaviors shown demonstrate that the band intensities of heme in-plane (ν7 and ν4 bands) and out-of-plane vibrations (γ7 band) develop within the instrument response time and remain constant. In contrast thereafter, the band intensity of ν(Fe-His) shows a gradual increase up to a 20 ps delay in addition to an instantaneous rise upon photodissociation. The fraction of the slow rise component is 8 ( 1%. Because there is no intensity change in the heme in-plane and out-of-plane vibrational modes in a time range of 3-50 ps, the gradual intensity increase in the ν(Fe-His) band is ascribed to the structural changes in the Fe-His linkage. The incomplete development of the ν(Fe-His) intensity in 1 ps was also reported by Franzen et al.32 The same measurements were carried out for a model system without a protein matrix, which is a 2-methylimidazole complex of iron protoporphyrin-IX, (FeIIPPIX)-2MeIm, in a 0.1% CTAB (hexadecyltrimethylammonium bromide) aqueous solution. The results are shown in Figure 5. Temporal behaviors similar to those for the corresponding modes of Mb were observed for the ν(Fe-Im) and the porphyrin bands of the complex, indicating that the structural change seen for Mb is not associated with protein relaxation and that it is a characteristic of the heme-histidine (or imidazole) unit. The peak positions of the observed five bands of Mb and the (FeIIPPIX)-2MeIm complex are plotted against time in Figure 6. For Mb, the ν(Fe-His) band showed a small downshift in a
J. Phys. Chem. B, Vol. 105, No. 44, 2001 10995
Figure 3. Temporal change of the Raman intensity of the Stokes ν4 (squares) and ν7 bands (diamonds) of photodissociated MbCO. The lines are calculated for a step function (assuming an instantaneous rise and very slow decay) convoluted with a Gaussian instrument response function for which the cross correlation time is 2.3 ps. The lower panel shows a closeup of the curve in the early time region.
100 ps time range but the other two bands showed no shift in this range (Figure 6 parts A and B). In contrast, neither the ν(Fe-Im) nor γ7 band of the (FeIIPPIX)-2MeIm complex showed a frequency shift as the delay time increased (Figure 6C). Therefore, the downshift of the ν(Fe-His) band of Mb should be attributed to some change in the ligation state of the proximal His caused by a structural change of globin. The downshift of the ν(Fe-His) band of Mb can be well described by single-exponential kinetics providing a time constant of ca. 100 ps, although the kinetics would involve several steps of relaxation. The protein relaxation of Mb upon CO dissociation was previously thought to complete within 30 ps, because previous TR3 measurements with a 30 ps wide pulse could not detect any frequency difference between the initial 30 ps spectrum and the equilibrium spectrum.9 However, the highprecision measurements in this study revealed the presence of the relaxation process of Mb in the 100 ps time range. IV. Discussion Change of Core Size. Most Raman modes of hemes in the high-frequency region (1450-1650 cm-1) have been shown to be sensitive to the core size of the porphyrin.18-21,33 Especially, the ν2 band with the strongest intensity among these has been used to study the core size of porphyrin. The photoproduct of MbCO generated at 4 K exhibited the ν2 band at 4 cm-1 lower than that of the deoxy preparation.34 Such a difference was not detected in the photoproduct generated at room temperature with 10 ns pulses.9 This suggests that a structural change of the surrounding protein is necessary for the photodissociated heme in Mb to be fully relaxed. With higher time resolution, structural
10996 J. Phys. Chem. B, Vol. 105, No. 44, 2001
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Figure 4. Temporal change of the Raman intensity of the Stokes ν(Fe-His) (circles) and γ7 bands (triangles) of photodissociated MbCO. The solid line for the ν(Fe-His) mode is a fit using an exponential function of the form, A[1 - B exp(- t/τrise)] (a rise containing an instantaneous and slow phases and very slow decay) convoluted with the instrument response function, as shown in the caption of Figure 3. The line for the ν(Fe-His) mode shown in this figure was obtained with the parameters of τrise ) 6.5 ( 2.5 ps and B ) 0.08 ( 0.01. The solid line for the γ7 mode is a fit using a step function convoluted with the instrument response function. The lower panel shows a closeup of the curve in the early time region.
Figure 5. Temporal change of the Raman intensity of the Stokes ν(Fe-Im) (circles) and γ7 bands (triangles) of the photoproduct of the CO-bound (FeIIPPIX)-2MeIm complex. The solid line for the ν(FeIm) mode is a fit using an exponential function of the form, A[1 - B exp(- t/τrise)] (a rise containing an instantaneous and slow phases and very slow decay) convoluted with the instrument response function, as shown in the caption of Figure 3. The line for the ν(Fe-Im) mode shown in this figure was obtained with the parameters of τrise ) 7.8 ( 2.7 ps and B ) 0.11 ( 0.02. The solid line for the γ7 mode is a fit using a step function convoluted with the instrument response function. The lower panel shows a closeup of the curve in the early time region.
relaxation of the heme might be observed at room temperature if the structural change of the protein necessary for full relaxation of the heme is slow. Even with picosecond resolution, however, the frequency shift of the ν2 mode was not observed in the present study. This suggests that both the structural change of the porphyrin ring and that of the protein necessary for full relaxation of the porphyrin ring are completed within the instrument response time (∼2 ps). Miller and co-workers observed ultrafast change of phase anisotropy in heterodynedetected transient grating signals following CO dissociation and ascribed it to a change of protein shape of the photodissociated Mb.35 Such a change in the protein shape may include the structural change of the protein necessary for full relaxation of the heme. Slow Intensity Rise of the ν(Fe-His) RR Band. To understand the intensity behavior of the Fe-His stretching mode, we follow the analysis of Bangcharoenpaurpong et al.36 The Fe-His configuration is defined by (1) the out-of-plane displacement of the Fe atom caused by ligand photodissociation (defined as the distance from the average plane of the porphyrin ring), (2) the tilt angle of the Fe-His bond (relative to heme normal), and (3) the azimuthal angle (about heme normal). Bangcharoenpaurpong et al. found that the Fe-His stretching vibration is relatively strongly coupled to the Soret transition.36 Their hypothesis is that the origin of the RR intensity of the Fe-His vibrational mode results from the orbital overlap between the σ* orbital of the Fe-His bond (σ*Fe-NHis) and the
π* orbital of the porphyrin ring (π*por), which is small in the planar structure but becomes large in the nonplanar structure. For nearly zero degree of the azimuthal angle, the orbital mixing of σ*Fe-NHis-π*por may occur. The electron-electron repulsive interaction between the single electron in σ*Fe-NHis and the filled πpor electron density on the heme nitrogens can lead to σ*Fe-NHis f p*por electron donation. The πpor-π*por excitation can result in direct harmonic coupling of the Fe-NHis mode via the orbital mixing of π*por and σ*Fe-NHis (the πpor-π*por transition gives rise to the Soret band). The stronger this coupling, the higher the resonance Raman intensity of the Fe-His stretching mode upon Raman excitation into the Soret band. The band intensity of the ν(Fe-His) mode increases with increasing out-of-plane displacement of the iron, because of the increased orbital overlap between the σ*Fe-NHis and π*por (which is zero by symmetry for the in-plane iron). Just as the azimuthal rotation affects the coupling, the tilt angle also affects it: the greater the tilt angle, the stronger the coupling. The theoretical model by Stavrov also pointed out that out-of-plane displacement of the iron gives rise to intensity and frequency changes of the ν(Fe-His) band because of the orbital mixing of π*por with σ*Fe-NHis.37 We now consider the origin of the slow phase of the ν(FeHis) intensity change that was observed both for myoglobin and the model compound. A high-resolution X-ray study5 indicates that the iron out-of-plane displacement is about 0 Å in the CO-bound form but increases to about 0.3 Å in the deoxy
Ultrafast Dynamics of Myoglobin
J. Phys. Chem. B, Vol. 105, No. 44, 2001 10997
Figure 7. Superposition of temporal change of the Raman intensity of the ν(Fe-His) band (solid circles), the out-of-plane motion of iron calculated in molecular dynamics simulation (solid line, from ref 40), and the spectral shift of band III (open circles, from ref 41). The data for the ν(Fe-His) RR intensity and the position of band III were scaled to minimize the deviation between the experimental points and solid curve.
Figure 6. Temporal changes of the Raman frequencies of some Stokes Raman bands in the low-frequency region. (A) The time dependence of the position of the Stokes ν(Fe-His) (circles) and γ7 (triangles) bands of photodissociated MbCO. The temporal shift of the ν(Fe-His) band was fitted using a single-exponential function, yielding time constants of 106 ( 14 ps (solid line). (B) The time dependence of the position of the Stokes δ(CβCcCd) band (diamonds) of photodissociated MbCO. (C) The time dependence of the position of the Stokes ν(Fe-Im) (circles) and γ7 (triangles) bands of the photoproduct of the CO-bound (FeIIPPIX)-2MeIm complex. The band positions were obtained as the first moment of the observed bands. The broken lines show the band positions of the equilibrium deligated form (i.e., the deoxy form).
structure. The tilt angle is slightly altered by CO dissociation, 0° in the CO-bound form38 and 10° in the deoxy form,39 but the azimuthal angle remains unaltered at 19° for both the deoxy and CO-bound forms.38,39 Accordingly, among the three factors mentioned above, the iron out-of-plane displacement and the tilt angle are possible structural parameters from which the slow phase of the ν(Fe-His) intensity change originated. The molecular dynamics (MD) simulation by Kuczera et al.40 pursued the time-dependent out-of-plane displacement of iron after ligand dissociation. The trajectory indicated that 80% of the out-of-plane displacement occurs within 50 fs along the repulsive part of the iron-porphyrin potential energy surface, but the remaining 20% takes place over time scales ranging from picosecond to tens of picoseconds. The bandshift in the near-IR absorption (band III) observed in the ultrafast transient absorption spectra of Mb at an ambient temperature was interpreted successfully along this line.41 A comparison of the band III shift with the trajectory of the MD simulations by Kuczera et al.40 indicated that the temporal behaviors correlate with one another over the first 100 ps of relaxation. Figure 7 shows a superposition of temporal change of the Raman intensity of the ν(Fe-His) band (solid circles), the out-of-plane motion of iron calculated in molecular dynamics simulation40 (solid line), and the spectral shift of band III41 (open circles). The temporal behavior of ν(Fe-His) intensity closely resembles those of the out-of-plane displacement of Fe in the MD
simulation and the position of band III. These results led us to conclude that the slow rise of ν(Fe-His) intensity primarily originates from the slow phase of the iron out-of-plane displacement. However, there are contrasting MD simulations42,43 concluding that the heme doming is completed within one picosecond. In the latter analysis, the slow rise of ν(Fe-His) Raman intensity would result from a change in the tilt angle. At the present stage, it is difficult to attribute the slow rise of ν(Fe-His) intensity exclusively to either structural parameter. In either case, the structural change responsible for the slow rise does not involve global conformational changes of the protein, as it was similarly observed for Mb and the (FeIIPPIX)2MeIm complex. Frequency Changes of the ν(Fe-His) Mode. Previous Raman studies on equilibrium deoxyHbs44-46 and photoinduced ligand-free transient Hbs47-51 have demonstrated that the frequency of the Fe-His stretching mode is responsive to a quaternary structure. In addition, studies on the transients47-49 have revealed that this frequency is sensitive to ligand-induced changes in the tertiary structure within both the R and T quaternary states. The general trend is that the frequency of this mode increases in going from the T to R states and that the changes within a given quaternary structure caused by ligand binding push the frequency still higher. Picosecond studies show that the frequency in a nanosecond time scale is already fully developed within 30 ps following photolysis.9 Similar to the equilibrium species, each of the T and R states has a frequency range: 216-222 cm-1 and 222-232 cm-1 for the photodissociated T and R transients (e10 ns), respectively. The difference between the ν(Fe-His) frequencies of T and R forms was attributed to a difference in the azimuthal angle for Hb.36 Recently, Peterson et al. measured the ν(Fe-His) frequencies of several mutants of sperm whale Mb and examined a correlation between the ν(Fe-His) frequency and the geometry of heme-proximal His moiety.52 They attributed changes in the ν(Fe-His) frequency to changes in the azimuthal angle of the His imidazole ring driven by a change in steric factors. Same conclusion was obtained for human Mb studied by Shiro et al.53 However, a deoxyMb/MbCO comparison of the crystallographic data indicated that the azimuthal angle does not change in Mb as described above.38,39 Stavrov pointed out that the iron outof-plane displacement is the parameter which mainly controls the ν(Fe-His) frequency.37 If the frequency shift originated from a change in the iron out-of-plane displacement and/or the histidine tilt angle, the shift would accompany an appreciable
10998 J. Phys. Chem. B, Vol. 105, No. 44, 2001 intensity change in the ν(Fe-His) RR band. Figure 4 shows, however, that there is no change in ν(Fe-His) intensity in the 100 ps time range. Therefore it is unlikely that the observed frequency shift of the ν(Fe-His) mode is caused by a change of these structural parameters. Changes in the basicity of an imidazole ring of His residue are known to change the frequency of the ν(Fe-His) mode.6 An extreme example is found in cytochrome c peroxidase (CCP),54 in which the carboxyl group of Asp235 is hydrogenbonded to the proximal His at NδH. The ν(Fe-His) band in CCP contains two components: one at 233 cm-1, assigned to a conformation with a very strong hydrogen bond to the NδH of the imidazole ring, and the other at 246 cm-1, assigned to a conformation in which a complete proton transfer from the imidazole to the Asp carboxylate group has occurred. The changes in the iron out-of-plane displacement and/or the histidine tilt and azimuthal angles are expected to result in a change in the coupling of the Fe-NHis mode to the πpor-π*por transition via the orbital mixing of π*por with σ*Fe-NHis and thereby in an appreciable change in ν(Fe-His) intensity. In contrast, the change in the hydrogen bond gives rise to a change in the extent of the σ*Fe-NHis f π*por electron donation and would little affect the orbital mixing of π*por with σ*Fe-NHis. Therefore, the change in the hydrogen bond would not necessarily accompany a change in the ν(Fe-His) Raman intensity. In fact, the Nδ proton of proximal His in Mb is hydrogen-bonded to the backbone carbonyl of Leu89 and also to the Oγ atom of Ser92. Accordingly, the most probable origin of the ν(Fe-His) downshift of the photodissociated Mb is a change in the hydrogen bond of the proximal His to the surrounding. Shiro et al. reported an upshift of the ν(Fe-His) band by 3 cm-1 upon mutation of Ser92 to Ala.53 This mutant does not have the hydrogen bond between proximal His and Ala92. The lack of the hydrogen bond is responsible for the observed change in the ν(Fe-His) frequency. Nonetheless, the observed upshift cannot be explained solely by the disruption of the hydrogen bond in the proximal His, because the upshift of ν(Fe-His) band implies strengthening of hydrogen bond, that is opposite to what is expected upon disruption of the hydrogen bond. The expected relation is also observed for a model Fe(II)-porphyrin 2methyl-imidazole (2MeIm) complex, for which the Fe(II)(2MeIm) stretching frequency becomes higher when a hydrogen bond between axial 2MeIm and solvent become stronger.55 Some factors in addition to the hydrogen bond (e.g., change in the azimuthal angle) should affect the frequency change of the ν(Fe-His) mode upon the mutation of Ser92 to Ala. Previous RR study reported that the ν(Fe-His) frequency of photodissociated MbCO in aqueous solution at room temperature did not shift significantly between 30 ps and 10 ns and that the ν(Fe-His) band at only a few picoseconds is already very close to the equilibrium deoxy frequency.9 However, cryogenic studies below ∼180 K and room-temperature studies in high-viscosity solvents56 revealed an increase in the ν(Fe-His) frequency by several wavenumbers in the 10 ns CO photoproduct, indicating that the photoproduct structure is midway between the CO and deoxy structures because the protein relaxation is slowed under these conditions. Sage et al. suggested from their low-temperature experiments using viscous solvent that RR intensity of the ν(Fe-His) band is related with the iron out-of-plane displacement, whereas the ν(Fe-His) frequency is connected to slower tertiary relaxation.56 This is consistent with the interpretation of the present study. The present study demonstrated the presence of such a relaxation of Mb in the picosecond time domain in a physiological condition.
Mizutani and Kitagawa Frequency Change of the Propionate Mode. The carboxylate group of the heme-7-propionate is hydrogen-bonded to the His97 imidazole ring and the hydroxyl group of Ser92. The frequency of the δ(CβCcCd) band correlates with the state of the hydrogen bond to the propionates in the proximal heme pocket. If the propionates are involved in hydrogen bonding with other residues in the pocket as seen in Mb and dimeric Hb from the blood clam Scaphrca inaequiValVis,57 the δ(CβCcCd) mode tends to appear at higher frequencies, but this frequency decreases if the propionates are solvent-exposed as in human Hb. In addition, the frequency in a given protein increases as the number of hydrogen bonds to the propionates increases, although there is no fixed relationship between its frequency and the number of hydrogen bonds. Unlike the ν(Fe-His) band, the δ(CβCcCd) band did not exhibit any frequency shift (Figure 6B), although the δ(CβCcCd) band for the CO-bound form appears at a frequency 9 cm-1 higher than that for the deoxy form. This result suggests that the change in a hydrogen bond of the propionates upon CO dissociation is small and/or takes place within the instrument response time. Structural Relaxations Probed with Different Techniques. Several experiments have been performed to study the protein response upon CO dissociation, and their results indicated that the protein motion of Mb spans on different time scales. The differences among all of the measurements may be due to the differences in what was probed. Miller and co-workers found in the femtosecond heterodyne-detected four-wave-mixing measurements that protein effectively changed its global shape within 500 fs.35 The observed fast response is consistent with the collective motion mechanism, in which a protein vibration along the reaction coordinate is directly excited by photodissociation of CO. Picosecond time-resolved IR (TRIR) spectroscopy on the amide I band of MbCO indicated that the conformation change of a polypeptide skeleton occurs in a 6-8 ps time scale.10 Therefore, changes in the tertiary structure probed by transient grating or picosecond TRIR spectroscopy do not affect the ν(Fe-His) mode. In contrast, Xie and Simon reported that relaxation of the transient circular dichroism (CD) signal of the N band (probed at 355 nm) of the photodissociated MbCO to the equilibrium deoxy spectrum requires 300 ps.12 They attributed the CD change to a change in the orientations of the transition dipole moments of the heme relative to those of the aromatic side chains locating closely to the heme. The observed temporal behavior of the ν(Fe-His) frequency resembles that of the transient CD signal. The transient absorption measurements of band III showed a highly nonexponential relaxation to its equilibrium position with time constants of 3.5, 83, and 3300 ps.11 The time constant of the second phase is close to that of the ν(Fe-His) downshift. The latter two experiments probed relatively local changes of protein, that is, changes in the proximal heme pocket. Therefore, there may be relatively slow structural rearrangements around the heme, including a change in hydrogen bonding and the orientational changes of aromatic side chains in the proximal heme pocket. V. Conclusion The ultrafast structural change at the heme site perturbs the protein structure nearly in a step-function manner. Therefore, photodissociation of CO in Mb provides a good opportunity to study the protein response function for the reaction at the heme. This study demonstrated that the protein response gives rise to the frequency shift of the ν(Fe-His) mode with the time constant of 100 ps, which was not detected in the previous study.9 Ultraviolet resonance Raman spectra are expected to
Ultrafast Dynamics of Myoglobin provide rich information on structure and environments of aromatic amino acid residues. Extension of the probe wavelength to ultraviolet region in TR3 measurements will bring us more detailed view of protein response upon the ligand dissociation in hemeproteins. Acknowledgment. Stimulating discussions with Prof. Paul M. Champion of Northeastern University are greatly acknowledged. This research was supported in part by a Grant-in-Aid for Encouragement Young Scientists (No. 11740339) given to Y.M. from the Japan Society for the Promotion of Science and by a Grant-in-Aid for Scientific Research on Priority Area (Nos. 11175100 and 12045264) given to T.K. from the Ministry of Education, Science, Sports and Culture, Japan. References and Notes (1) Dickerson, R. E.; Geiss, I. Hemoglobin: Structure, Function, EVolution, and Pathology; Benjamin Cummings: Menlo Park, CA, 1983. (2) Greene, B. I.; Hochstrasser, R. M.; Weisman, R. B.; Eaton, W. A. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 5255. (3) Petrich, J. W.; Poyart, C.; Martin, J. L. Biochemistry 1988, 27, 4049. (4) Gibson, Q. H.; Olson, J. S.; McKinnie, R. E.; Rohlfs, R. J. J. Biol. Chem. 1986, 261, 10228. (5) Kachalova, G. S.; Pepov, A. N.; Bartunik, H. D. Science 1999, 284, 473. (6) Kitagawa, T. The Heme Protein Structure and the Iron Histidine Stretching Mode. In Biological Applications of Raman Spectroscopy; Spiro, T. G., Ed.; John Wiley & Sons: New York, 1987; Vol. III, p 97. (7) Rousseau, D. L.; Friedman, J. M. Transient and Cryogenic Studies of Photodissociated Hemoglobin and Myoglobin. In Biological Applications of Raman Spectroscopy; Spiro, T. G., Ed.; John Wiley & Sons: New York, 1987; Vol. III, p 133. (8) Scott, T. W.; Friedman, J. M. J. Am. Chem. Soc. 1984, 106, 5677. (9) Findsen, E. W.; Scott, T. W.; Chance, M. R.; Friedman, J. M.; Ondrias, M. R. J. Am. Chem. Soc. 1985, 107, 3355. (10) Causgrove, T. P.; Dyer, R. B. J. Phys. Chem. 1996, 100, 3273. (11) Jackson, T. A.; Lim, M.; Anfinrud, P. A. Chem. Phys. 1994, 180, 131. (12) Xie, X.; Simon, J. D. Biochemistry 1991, 30, 3682. (13) Mizutani, Y.; Kitagawa, T. Chem. Record 2001, 1, 258. (14) Kaminaka, S.; Kitagawa, T. Appl. Spectrosc. 1995, 49, 685. (15) Mizutani, Y. J. Chem. Phys. 1998, 109, 9197. (16) Abe, M.; Kitagawa, T.; Kyogoku, Y. J. Chem. Phys. 1978, 69, 4526. (17) Li, X.-Y.; Czernucszewicz, R. S.; Kincaid, J. R.; Stein, P.; Spiro, T. G. J. Phys. Chem. 1990, 94, 47. (18) Spaulding, L. D.; Chang, C. C.; Yu, N.-T.; Felton, R. H. J. Am. Chem. Soc. 1975, 97, 2517. (19) Spiro, T. G.; Stong, J. D.; Stein, P. J. Am. Chem. Soc. 1979, 101, 2648. (20) Choi, S.; Spiro, T. G.; Langry, K. C.; Smith, K. M.; Budd, D. L.; La Mar, G. N. J. Am. Chem. Soc. 1982, 104, 4345. (21) Oertling, W. A.; Salehi, A.; Chung, Y. C.; Leroi, G. E.; Chang, C. K.; Babcock, G. T. J. Phys. Chem. 1987, 91, 5887. (22) Dasgupta, S.; Spiro, T. G.; Johnson, C. K.; Dalickas, G. A.; Hochstrasser, R. M. Biochemistry 1985, 24, 5295. (23) Lim, M.; Jackson, T. A.; Anfinrud, P. A. J. Phys. Chem. 1996, 100, 12043. (24) Petrich, J. W.; Martin, J. L. Chem. Phys. 1989, 131, 31. (25) Cornelius, P. A.; Steele, A. W.; Chernoff, D. A.; Hochstrasser, R. M. Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 7526.
J. Phys. Chem. B, Vol. 105, No. 44, 2001 10999 (26) Li, P.; Sage, J. T.; Champion, P. M. J. Chem. Phys. 1992, 97, 3214. (27) Rosca, F.; Kumar, A. T. N.; Ye, X.; Sjodin, T.; Demidov, A. A.; Champion, P. M. J. Phys. Chem. A 2000, 104, 4280. (28) Gibson, Q. H.; Olson, J. S.; McKinnie, R. E.; Rohlfs, R. J. J. Biol. Chem. 1986, 261, 10228. (29) Kitagawa, T.; Nagai, K.; Tsubaki, M. FEBS Lett. 1979, 104, 376. (30) Hu, S.; Smith, K. M.; Spiro, T. G. J. Am. Chem. Soc. 1996, 118, 12638. (31) The shoulder on the high-frequency side of the ν(Fe-His) band has been assigned to ν9, the pyrrole tilting mode. The ν9 band is not resolved from the ν(Fe-His) band and was included in the moment analysis. However, the contribution of the ν9 band to temporal changes of the zeroth and first moments observed in the present study is supposed to be negligible because of the following reason. Porphyrin RR bands other than the ν9 band showed no temporal changes in the 10-100 ps time range. Therefore, it is reasonable to assume that the ν9 band showed no temporal changes in the 10-100 ps time range the same as the other porphyrin RR bands. Especially, a strong correlation between the positions of the ν9 and the δ(CβCcCd) bands was reported in ref 52. No temporal change in the δ(CβCcCd) frequency suggests that the ν9 band would show no temporal changes in the 10-100 ps time range. Therefore, the contribution of the ν9 band to temporal changes of the zeroth and first moments observed in the present study is supposed to be negligible. (32) Franzen, S.; Bohn, B.; Poyart, C.; Martin, J. L. Biochemistry 1995, 34, 1224. (33) Felton, R. H.; Yu, N. T.; O’Shea, D. C.; Schelnutt, J. A. J. Am. Chem. Soc. 1974, 96, 3675. (34) Rousseau, D. L.; Argade, P. V. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 1310. (35) Goodno, G. D.; Astinov, V.; Miller, R. J. D. J. Phys. Chem. A 1999, 103, 10630. (36) Bangcharoenpaurpong, O.; Schomacker, K. T.; Champion, P. M. J. Am. Chem. Soc. 1984, 106, 5688. (37) Stavrov, S. S. Biophys. J. 1993, 65, 1942. (38) Takano, T. J. Mol. Biol. 1977, 110, 537. (39) Takano, T. J. Mol. Biol. 1977, 110, 569. (40) Kuczera, K.; Lambry, J.-C.; Martin, J.-L.; Karplus, M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5805. (41) Lim, M.; Jackson, T. A.; Anfinrud, P. A. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5801. (42) Schaad, O.; Zhou, H.-X.; Szabo, A.; Eaton, W. A.; Henry, E. R. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9547. (43) Li, H.; Elber, R.; Straub, J. E. J. Biol. Chem. 1993, 268, 17908. (44) Nagai, K.; Kitagawa, T. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 2033. (45) Nagai, K.; Kitagawa, T.; H., M. J. Mol. Biol. 1980, 136, 271. (46) Ondrias, M. R.; Rousseau, D. L.; Shelnutt, J. A.; Simon, S. R. Biochemistry 1985, 21, 3428. (47) Friedman, J. M.; Rousseau, D. L.; Ondrias, M. R.; Stepnoski, R. A. Science 1982, 218, 1244. (48) Friedman, J. M.; Stepnoski, R. A.; Nobel, R. W. FEBS Lett. 1982, 146, 278. (49) Friedman, J. M.; Rousseau, D. L.; Ondrias, M. R. Annu. ReV. Phys. Chem. 1982, 33, 471. (50) Irwin, M. J.; Atkinson, G. H. Nature (London) 1981, 293, 317. (51) Stein, P.; Terner, J.; Spiro, T. G. J. Phys. Chem. 1982, 86, 168. (52) Peterson, E. S.; Friedman, J. M.; Chien, E. Y. T.; Sligar, S. G. Biochemistry 1998, 37, 12301. (53) Shiro, Y.; Iizuka, T.; Marubayashi, K.; Ogura, T.; Kitagawa, T.; Balasubramanian, S.; Boxer, S. G. Biochemistry 1994, 33, 14986. (54) Smulevich, G.; Mauro, J. M.; Fishel, L. A.; English, A. M.; Kraut, J.; Spiro, T. G. Biochemistry 1988, 27, 5477. (55) Teraoka, J.; Kitagawa, T. J. Biol. Chem. 1981, 256, 3639. (56) Sage, J. T.; Schomacker, K. T.; Champion, P. M. J. Phys. Chem. 1995, 99, 3394. (57) Royer, W. E., Jr. J. Mol. Biol. 1994, 235, 657.
