Intersubunit Communication via Changes in Hemoglobin Quaternary

Sep 25, 2013 - Kaustuv Sahoo , Sriharsha Karumuri , Rangika S. Hikkaduwa Koralege , Nicholas H. Flynn , Steve Hartson , Jing Liu , Joshua D. Ramsey , ...
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Intersubunit Communication via Changes in Hemoglobin Quaternary Structures Revealed by Time-Resolved Resonance Raman Spectroscopy: Direct Observation of the Perutz Mechanism Kenta Yamada,† Haruto Ishikawa,† Misao Mizuno,† Naoya Shibayama,‡ and Yasuhisa Mizutani*,† †

Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498, Japan

‡

S Supporting Information *

ABSTRACT: Time-resolved resonance Raman spectroscopy was used to investigate intersubunit communication of hemoglobin using hybrid hemoglobin in which nickel was substituted for the heme iron in the β subunits. Changes in the resonance Raman spectra of the α heme and the β Ni−heme groups in the hybrid hemoglobin were observed upon CO photolysis in the α subunit using a probe pulse of 436 and 418 nm, respectively. Temporal evolution of the frequencies of the ν(Fe−His) and the γ7 band of α heme was similar to that of unsubstituted hemoglobin, suggesting that substitution with Ni− heme did not perturb the allosteric dynamics of the hemoglobin. In the β subunits, no structural change in the Ni−heme was observed until 1 μs. In the microsecond regime, temporal evolution of the frequencies of the ν(Ni−His) and the γ7 band of β Ni−heme was observed concomitant with an R → T quaternary change at about 20 μs. The changes in the ν(Fe−His) and ν(Ni−His) frequencies of the α and β subunits with the common time constant of ∼20 μs indicated that the proximal tension imposed on the bond between the heme and the proximal histidine strengthened upon the quaternary changes in both the α and the β subunits concertedly. This observation is consistent with the Perutz mechanism for allosteric control of oxygen binding in hemoglobin and, thus, is the first real-time observation of the mechanism. Protein dynamics and allostery based on the observed time-resolved spectra also are discussed.

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INTRODUCTION Upon binding of a ligand, a protein molecule often undergoes conformational changes that modify its properties at a distant site. This phenomenon is widely known as allostery1,2 and is responsible for dynamic regulation of biological functions. The molecular mechanism of cooperativity in oxygen binding of hemoglobin (Hb) is of long-standing interest in allostery. Human adult hemoglobin A (HbA) has a tetrameric structure, consisting of two α and two β subunits. HbA exhibits positive cooperativity in oxygen binding, which is a reversible transition between the two quaternary states upon partial ligation of the four hemes.3,4 X-ray crystallographic data5 demonstrated the presence of two distinct quaternary structures that correspond to the low-affinity (T or tense) and high-affinity (R or relaxed) states. In HbA, the completely unliganded (deoxy) structure typically adopts the T state, while the fully liganded form adopts the R state. The largest structural differences between the T and R states are located in the α1−β2 subunit interface, where rearrangements of hydrogen bonds and the cleavage of salt bridges occur upon ligation.6 In HbA, the Fe2+ ion of the heme is bound to the proximal histidine (His) as the only covalent link to the protein. The heme iron binds diatomic molecules, such as O2, CO, and NO, © 2013 American Chemical Society

at the opposite side of the proximal His. The binding of ligands to the hemes in HbA induces a sequence of propagating structural events that culminates in a change of quaternary structure, proceeding from the T state to the R state. Perutz presented his model of allosteric control of oxygen affinity as follows. Ligand binding is accompanied by the displacement of the iron atom toward the heme plane, which pulls the proximal histidine and results in a shift of the helix. The tertiary changes in the ligated subunit then are propagated to the other subunits of the α2β2 tetramer through the α−β dimer interface. As a result, the hydrogen bonds and salt bridges characterizing the deoxy structure at this interface are broken to allow the change in the quaternary structure. This change in the quaternary structure affects the structure of the heme pocket and controls oxygen affinity of the heme. Thus, the protein dynamics inducing the change in the quaternary structure must be elucidated to fully understand the allosteric mechanism of Hb. Time-resolved resonance Raman (RR) spectroscopy provides information on the dynamics of the tertiary and Received: August 2, 2013 Revised: September 19, 2013 Published: September 25, 2013 12461

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Perutz mechanism for allosteric control of oxygen binding in HbA.

quaternary structures of heme proteins. Along with studies of Gibson,7 ligand photolysis techniques have been applied to time-resolved RR studies of Hb. Structural changes in the heme and heme pocket have been investigated by observing changes in the RR spectra of the heme groups upon CO photolysis.8−10 These studies have revealed the tertiary structural change of Hb on a nanosecond time scale. Also, structural changes in the α−β subunit interface have been investigated by observing changes in the RR spectra of the tyrosine and tryptophan residues in the subunit interface upon CO photolysis.11−13 These studies showed the quaternary structural changes in HbA on a microsecond time scale. However, how the heme of one subunit responds to ligand dissociation of a heme on a neighboring subunit has not been fully elucidated, despite the importance of understanding this intersubunit communication to clarify the molecular mechanism of Hb cooperativity. Discriminating between the α and β subunits spectroscopically is key because both subunits have identical heme structures. To investigate the properties of each individual subunit, a variety of metal-substituted Hbs including hybrids were prepared using Ni, Co, Cu, Cr, Zn, Mn, and Mg.14−24 The Co-substituted Hb could bind O2 cooperatively, but could not bind CO. The subunit substituted with metal species other than Co could not even bind O2. Hybrid Hbs containing Fe and another metal formed a half-liganded intermediate with two ligands on the Fe subunits. A quaternary structure of the halfliganded Hb depended on the metal species hybridized. Physicochemical properties of Ni−Fe hybrid Hb varied depending on whether Ni was incorporated into the α or the β subunit.20,21 Hybrid hemoglobin containing nickel protoporphyrin IX (Ni−heme) in the α subunits and heme (ferrous protoporphyrin IX) in the β subunits [α2(Ni)β2(Fe)] exhibited noncooperativity in oxygen binding; the coordinated nickel protoporphyrin IX was dominant in the α subunits. In contrast, α2(Fe)β2(Ni), in which five-coordinate Ni−heme was dominant in the β subunits, exhibited positive cooperativity for oxygen binding. For α2(Fe)β2(Ni), the deoxy structure typically adopted the T state, while the half-liganded form adopted the R state. The present strategy to investigate intersubunit communication through the α−β subunit interface involved observation of changes in the RR spectra of the Ni−heme in the β subunit upon CO photolysis in the α subunit using carbonmonoxy α2(Fe)β2(Ni) [α2(Fe−CO)β2(Ni)]. Hybrid Hb α2(Fe−CO)β2(Ni) complexes possess two characteristics that make them extremely useful for investigating individual subunit properties in time-resolved experiments. First, resonance enhancement enables the selective observation of the Fe−heme and Ni− heme spectra in the hybrid Hb because the peak wavelengths of the Soret band are different for the deoxy Hb and Nisubstituted Hb. Second, no photochemical reaction occurs in the Ni−heme upon irradiation because it does not bind a ligand. Thus, the RR spectrum of the Ni−heme is a good spectroscopic probe for investigating intersubunit communication. In this study, intersubunit communication was observed by examining the changes in the RR spectra of the Ni−-heme in the β subunits upon CO photolysis in the α subunits using α2(Fe−CO)β2(Ni). Concerted changes in the ν(Fe−His) and ν(Ni−His) frequencies of the α and β subunits of ∼20 μs indicated that the proximal tension imposed on the bond between the heme and proximal histidine strengthened after the quaternary changes. This observation is consistent with the

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EXPERIMENTAL SECTION Preparation of Hb(WT) and Hb(βD99N). Recombinant human Hb(WT) and a mutant (βD99N) were expressed in E. coli SGE1661 and purified according to a previously described procedure.10,25,26 The pSGE1702 expression vector contains one copy each of the α-globin and β-globin genes, in which the valine residues at the N-terminal are replaced by methionine. Because oxygen affinity of the α(V1M)/β(V1M) mutant Hb is identical to that of native HbA, the double mutant Hb is referred to as WT (wild type) rHb (recombinant hemoglobin).10,25,26 Site-directed mutagenesis was performed using the PrimeSTAR max DNA polymerase (TaKaRa Bio) and mutagenic primers (5′-CACGTTAACCCGGAAAACTTCCGTCTG-3′ and 5′-TTCCGGGTTAACGTGCAGTTTGTCGCA-3′) using a standard protocol. Mutated sites in the primer sequences are underlined. Introduced mutations were confirmed by DNA sequencing. Preparation of Ni-Substituted Hbs. To obtain heme-free chain globins, a purified rHb(WT) solution was mixed with 0.1 M sodium fluoride, and the pH of the solution was adjusted to 3.9 by addition of 0.1 M HCl. The heme-free chain globins were prepared from rHb using the ethyl methyl ketone method.27 After dialysis against 10 mM sodium hydrogen carbonate solution, the sample was dialyzed against potassium phosphate buffer at pH 6.8. The apo-Hb solution was mixed with a 1.2-fold amount of Ni−heme, which was dissolved in a minimal amount of 20 mM NaOH. The mixture was gently stirred at 4 °C for 2 h, and then diluted 10-fold and passed through a CM-agarose column (GE Healthcare, HiTrap CM Fast Flow) equilibrated with 10 mM sodium phosphate buffer at pH 6.8. The Ni-substituted WT Hb [NiHb(WT)] was eluted with 100 mM sodium phosphate buffer at pH 7.5. The NiHb(βD99N) was prepared using the same procedure as for NiHb(WT). Preparation of α2(Fe−CO)β2(Ni). Isolated chains of rHb were prepared in CO-bound forms as described previously.10,25 Heme-free chain globins were prepared from the isolated β chain using the acid acetone method.20,27 The apo-β-chain was dissolved in 20 mM borate/NaOH buffer at pH 11, followed by adjustment of the pH to 9.3 by addition of 0.2 M HCl. The apo-β-chain solution was mixed with 0.75 mol equiv of α chain and then with a 1.2-fold amount of Ni−heme, which was dissolved in a minimal amount of DMF. The mixture was gently stirred at 4 °C for 2 h, and then dialyzed against 20 mM Tris-HCl buffer at pH 7.2. The α2(Fe−CO)β2(Ni) solution was applied to a DEAE-agarose column (GE Healthcare, HiTrap DEAE Fast Flow) followed by application to a CM-agarose column (GE Healthcare, HiTrap CM Fast Flow), both of which were equilibrated with the same 20 mM Tris-HCl buffer. The fraction that passed through both the DEAE- and the CMagarose columns was collected. Time-Resolved and Steady-State Resonance Raman Measurements. Nanosecond time-resolved RR measurements were carried out with two nanosecond pulse lasers operating at 1 kHz. The probe pulses were the second harmonics of the output of an Nd:YLF-pumped Ti:sapphire laser (Photonics Industries, TU-L). Probe pulses at 436 and at 418 nm were used to obtain RR spectra of the α heme and β Ni−heme, respectively, in α2(Fe−CO)β2(Ni). Probe power was set as low as possible (1.0 μJ/pulse) to avoid ligand photolysis by the 12462

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assigned to ν8, a metal−pyrrole stretch and substituents bend. The bands at 364 and 403 cm−1 are substituent modes, δ(CβCcCd), involving deformation of the propionate methylene groups, and δ(CβCaCb), involving deformation of the vinyl groups, respectively. A similar RR spectrum was observed for α2(Fe)β2(Ni) measured with a probe pulse of 436 nm (trace b). In the 418-nm excited RR spectrum of NiHb(WT) (trace d), a band was observed at 235 cm−1, due to the stretching mode of the covalent bond between the nickel of the substituted heme and the Nε of the proximal His, ν(Ni− His).31 A weak band around 275 cm−1 was not assigned in the previous study.31 A similar RR spectrum was observed for α2(Fe)β2(Ni) measured with a probe pulse of 418 nm (trace c); no ν(Fe−His) band was observed. These data demonstrate selective observation of the heme and Ni−heme in RR spectra using a probe pulse of 436 and 418 nm, respectively. Detailed data for the excitation wavelength dependence in the RR spectra of a 1:1 mixture of the deoxy forms of rHb and Nisubstituted Hb are provided in Figure S1 of the Supporting Information. Figure 2 shows time-resolved RR spectra of the α heme in α2(Fe−CO)β2(Ni) following CO photolysis, measured with a

probe pulse. Pump pulses at 532 nm were generated with a diode-pumped Nd:YAG laser (Megaopto, LR-SHG), with the power adjusted to 185 μJ/pulse. The pump and probe beams were directed collinearly using a dichroic mirror and focused onto the sample cell with spherical and cylindrical lenses. Pulse widths of the pump and probe pulses were 20 and 25 ns, respectively. Timing between the pump and probe pulses was adjusted with a computer-controlled pulse generator (Stanford Research Systems, DG 535) via a GPIB interface. Jitters in the delay time were within ±5 ns. Time-resolved RR data acquisition was conducted as described previously.28 The time delay of the probe pulse with respect to the pump pulse was determined by detecting the two pulses with a photodiode (Electro-Optics Technology, ET-2000) just before the sample point, and monitored with an oscilloscope (Iwatsu, Waverunner DS-4262). The sample solution was contained in an airtight NMR tube (10-mm diameter) and spun with a spinning cell device designed to minimize off-center deviation during rotation. Raman scattered light was detected with a liquid nitrogen-cooled charge-coupled device camera (Roper Scientific, Spec-10:400B/LN) attached to a custom-made prism prefilter (Bunkoukeiki) equipped with a single spectrograph (Horiba, iHR550). Spectra were calibrated using standard Raman spectra of cyclohexane and carbon tetrachloride. Steady-state RR measurements were performed using a Nd:YLF-pumped Ti:sapphire laser operating at 1 kHz. Probe pulses at 410, 415, 418, 420, 430, and 436 nm were the second harmonics of the output of the laser. Energy of the probe pulse was set as low as possible (1.0 μJ/pulse) to avoid photodamage of the sample due to the probe pulse. The sample solution contained 0.1 M NaClO4 as an internal intensity standard.

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RESULTS Figure 1 shows RR spectra of rHb, NiHb(WT), and α2(Fe)β2(Ni) in the 180−430 cm−1 region measured with

Figure 2. Time-resolved RR spectra in the 180−850 cm−1 region for α2(Fe−CO)β2(Ni) following CO photolysis measured with a probe pulse of 436 nm. Spectra of the equilibrium states of the deoxy and CO-bound forms are depicted at the bottom for comparison. Timeresolved difference spectra were generated by subtracting the probeonly spectrum from the pump−probe spectra at each delay time. Accumulation time for each spectrum was 48 min.

Figure 1. RR spectra of (a) rHb (probe pulse 436 nm), (b) α2(Fe)β2(Ni) (probe pulse 436 nm), (c) α2(Fe)β2(Ni) (probe pulse 418 nm), and (d) NiHb(WT) (probe pulse 418 nm) in the 180−430 cm−1 region.

probe pulses of 418 and 436 nm. In the 436-nm excited RR spectrum of rHb (trace a), the bands at 218, 301, 341, 364, and 403 cm−1 were assigned to vibrations of the heme. The RR band at 218 cm−1 arises from the stretching mode of the covalent bond between the heme iron and Nε of the proximal His, ν(Fe−His).29 The band at 301 cm−1 is an out-of-plane mode (γ7; methane wagging).30 The band at 341 cm−1 is

probe pulse of 436 nm. In these spectra, the contribution from unreacted species has been subtracted.32 The band intensities in the spectra at 20 μs are weaker than those in the other timeresolved spectra. This is due to population changes in the photolyzed species resulting from geminate ligand recombination. The spectrum for a 50-ns delay contained bands arising from vibrations of the heme at 223 [ν(Fe−His)], 302 (γ7), 341 12463

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(ν8), 364 [δ(CβCcCd)], 403 [δ(CβCaCb)], and 673 cm−1 (ν7). The ν7 band is due to a breathing-like mode of the porphyrin inner ring. The time-resolved RR spectra at 50 ns differed from the spectrum of the deoxy form. The frequencies of the ν(Fe− His) and γ7 bands in the 50-ns time-resolved RR spectra were higher than those in the spectrum of the deoxy form. The spectra exhibited a frequency shift of the ν(Fe−His) and γ7 bands as the delay time increased. The ν(Fe−His) band was observed at 223 cm−1 at 50 ns and downshifted to 220 cm−1 at 20 μs. The γ7 frequency of 302 cm−1 at 50 ns shifted to 301 cm−1 at 20 μs. Similar temporal changes in the RR spectra for the dissociated form have been reported for HbA12 and rHb.10 The frequency of the ν(Fe−His) band in the α subunits of deoxy α2(Fe)β2(Ni) was different from those reported for the deoxy forms in hybrid Hbs prepared from HbA.21,24 In the hybrid Hbs prepared from HbA, the ν(Fe−His) band of the α subunits was a broad doublet at 200−205 and 210−215 cm−1. In the present study, the ν(Fe−His) band of the α subunit was a singlet at 218 cm−1. The molecular structure of rHb and HbA may be slightly different, although rHb displays a similar degree of cooperativity in oxygen binding as compared to HbA.26 The time-resolved RR spectra of β Ni−heme in α2(Fe− CO)β2(Ni) as measured with a probe pulse of 418 nm is shown in Figure 3. The contribution of unreacted species in these spectra has been subtracted. The weaker band intensities at 20 μs due to geminate recombination were observed, similar to the time-resolved spectra shown in Figure 2. The spectrum involving a 50-ns delay contains bands arising from vibrations of the heme at 236 [ν(Ni−His)], 310 (γ7), 341 (ν8), 367 [δ(CβCcCd)], 404 [δ(CβCaCb)], and 673 cm−1 (ν7). The time-

resolved RR spectra at 50 ns differed from the spectra of the deoxy form. The frequencies of the ν(Ni−His) and γ7 bands in the 50-ns time-resolved RR spectra were higher and lower, respectively, than those in the spectrum of the deoxy form. The spectra exhibited a frequency shift in the ν(Ni−His) and γ7 bands as delay time increased. The ν(Ni−His) band was observed at 236 cm−1 at 50 ns and downshifted to 235 cm−1 at 20 μs. The γ7 frequency of 310 cm−1 at 50 ns shifted to 311 cm−1 at 20 μs. These small frequency shifts were confirmed in the magnified view of the time-resolved spectra and difference spectra between the time-resolved spectra at 50 ns and 20 μs (Figure S2 in the Supporting Information). It should be noted that, for the two end-point structures, the ν(Ni−His) band was observed at 237 and 235 cm−1 for α2(Fe−CO)β2(Ni) and α2(Fe)β2(Ni), respectively. Thus, the ν(Ni−His) frequency decreased monotonously from that of α2(Fe−CO)β2(Ni) to that of α2(Fe)β2(Ni). Figure 4 shows the 418-nm excited RR

Figure 4. RR spectra of the Ni-substituted Hbs (a) WT and (b) βD99N mutant, in the 180−430 cm−1 region. The spectra were obtained using a 418-nm pulse.

spectra of NiHb(WT) and NiHb(βD99N) in the 180−430 cm−1 region. Frequencies of the bands for NiHb(βD99N) are the same as those for NiHb(WT), except for the ν(Ni−His), γ7, and δ(CβCaCb) bands. The ν(Ni−His) band was observed at 238 cm−1 for NiHb(βD99N), while it was observed at 235 cm−1 for NiHb(WT). The γ7 band was observed at 310 cm−1 for NiHb(βD99N), while it was observed at 312 cm−1 for NiHb(WT). Similar differences between WT and the βD99N mutant of HbA were observed for the ν(Fe−His) bands, while the γ7 band appeared at the same position in both WT and βD99N mutant 33 (see Figure S3 in the Supporting Information). The deoxy form of Hb Kempsey, which is an abnormal HbA with a βD99N mutation, adopts the R state, while the deoxy form of WT adopts the T state. These data showed that the frequencies of the ν(Ni−His) and γ7 bands were different between the R and T states. Thus, frequency shifts observed in the time-resolved spectra for α2(Fe− CO)β2(Ni) reflect a quaternary structural transition from the R to the T structure. Figure 5 compares the temporal evolution of the frequencies of the ν(Fe−His) and ν(Ni−His) and γ7 bands of α heme and β Ni−heme of α2(Fe)β2(Ni) following CO dissociation. Frequencies of the bands were calculated in terms of the first moments of the bands.28,34 The frequency shifts of the ν(Fe− His) and γ7 bands of α heme in α2(Fe)β2(Ni) were fitted using triple-exponential functions and single-exponential functions,

Figure 3. Time-resolved RR spectra at 180−850 cm−1 for α2(Fe− CO)β2(Ni) following CO photolysis measured with a probe pulse of 418 nm. Spectra of the equilibrium states of the deoxy and CO-bound forms are shown at the bottom for comparison. Time-resolved difference spectra were generated by subtracting the probe-only spectrum from the pump−probe spectra at each delay time. Accumulation time for each spectrum was 56 min. 12464

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ν(Ni−His) and γ7 bands of β heme were 18.0 and 18.9 μs, respectively, demonstrating that ∼20 μs is required for the heme of the β subunits to respond to ligand dissociation of the α subunits. In addition, note that the 20-μs component was commonly observed both in ν(Fe−His) of the α subunit and in ν(Ni−His) of the β subunit, meaning that the bonds between heme and the proximal histidine change concertedly in both subunits.

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DISCUSSION In the present study, the frequency shift of the ν(Fe−His) and γ7 bands of α heme and of the ν(Ni−His) and γ7 bands of β Ni−heme unambiguously showed intersubunit communication in α2(Fe)β2(Ni) following CO dissociation. This is the first RR observation of structural changes induced by the neighboring subunits in Hb. Structural changes based on the observed spectral changes were examined. ν(Fe−His) Band of α Heme. Kitagawa and co-workers29,35 showed that RR spectra of deoxy forms of heme proteins with an axial ligand of His contain a band in the region between 200 and 250 cm−1, assignable to the ν(Fe−His) mode. The Fe−His bond in Hb is the sole covalent linkage between the heme and the protein. Consequently, this mode is a good indicator of heme protein tertiary and quaternary structures.36,37 The ν(Fe−His) band was observed only in the unliganded ferrous form of heme proteins. This is due to the origin of the intensity of the ν(Fe−His) mode resulting from orbital overlap between the σ*-orbital of the Fe−His bond and the π*-orbital of the porphyrin ring, which is small in the planar structure but becomes large in the domed structure.38 The proximal Fe−His linkage in heme proteins plays a pivotal role in communicating protein structural changes to the functional heme group, thus affecting its biological properties.39 The deoxy form within the quaternary T state has been reported to have a band frequency of 216 cm−1, whereas the R state of the modified deoxy NESdes(Arg141α)-Hb exhibited a Raman band at a frequency of 221 cm−1.40 Temporal changes in the ν(Fe−His) band of HbA were investigated using time-resolved RR spectroscopy over a wide time range. In the low-frequency region of the RR spectrum, the ν(Fe−His) RR band appears at a shifted position of 223 cm−1 in the photolyzed (R state) HbA as measured at 3 ps,41 which then relaxes to the equilibrium (T state) deoxy position of 210−216 cm−1 on a 100 ns to microsecond time scale.8 Hence, the ν(Fe−His) band is a prominent marker band for investigation of the relationship between the structure and function of Hb. The frequency shift in the ν(Fe−His) band of α heme in α2(Fe)β2(Ni) was fitted using triple-exponential functions, similar to the shift of rHb.10 The three time constants of the

Figure 5. Logarithmic time plots of frequencies of (A) ν(Fe−His) and ν(Ni−His) and (B) γ7 band of α heme and β Ni−heme of α2(Fe)β2(Ni) following CO dissociation. Red ● and blue ▲ represent frequencies of RR bands of α heme and β Ni−heme, respectively. Solid lines indicate the best fit obtained using an exponential function or a sum of exponential functions. The red and blue broken lines show values of α heme and β Ni−heme, respectively, in deoxy α2(Fe)β2(Ni).

respectively. For β Ni−heme, no temporal changes in the ν(Ni−His) and γ7 bands of β Ni−heme were observed earlier than 1 μs, which is consistent with the lack of reaction of Ni− heme upon excitation by the pump pulse. In the microsecond regime, frequency shifts were observed, indicating structural changes in the β subunit induced by the structural changes in the α subunits. Temporal changes in the ν(Ni−His) and γ7 bands of the β Ni−heme in α2(Fe)β2(Ni) were fitted using a single-exponential function. The time constants of the evolutions are summarized in Table 1. The temporal evolution of the frequencies of ν(Fe−His) of the α heme in α2(Fe)β2(Ni) was similar to that of α and β heme in rHb.10 This supports a structural change in the α subunit of α2(Fe)β2(Ni) that is similar to that of rHb, and indicates that replacement of heme with Ni−heme in the β subunit does not influence the allosteric dynamics of rHb. Time constants of the evolutions of the

Table 1. Time Constants of Temporal Evolution of Raman Bands of α2(Fe)β2(Ni)a and rHbb α subunit in α2(Fe)β2(Ni) β subunit in α2(Fe)β2(Ni) rHbb

ν(Fe−His) γ7 ν(Ni−His) γ7 ν(Fe−His) γ7

τ1/ns

τ2/μs

τ3/μs

20.2 ± 10.1 (24 ± 5%)c 48.3 ± 1.9

0.688 ± 0.207 (19 ± 2%)

19.5 ± 1.6 (57 ± 2%)

0.631 ± 0.243 (61 ± 2%)

18.0 18.9 17.3 (16

20.0 ± 7.1 (23 ± 4%) 43.4 ± 5.2

± ± ± ±

0.7 1.5 1.3 2%)

The temporal evolution is fitted to an exponential function or a sum of exponential functions. bFrom ref 10. cNumbers in parentheses represent relative contribution of the component.

a

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ν(Fe−His) shift observed for rHb are close to those observed in time-resolved ultraviolet RR spectroscopy, which were interpreted as follows.12 The first component is due to the step in which the heme structure changes and proximal strain is released via the postulated rotation of the EF helical section. In the step of the second component, interhelical H-bonds are restored following motion of the A and H helices. The third component is assigned to the frequency shift due to the quaternary change. Time constants of α2(Fe)β2(Ni) that were similar to those of rHb indicate that, upon CO photolysis in the α heme, the α subunits in α2(Fe)β2(Ni) undergo structural changes in a manner similar to those of rHb. γ7 Band of α Heme. The heme γ7 band is sensitive to the out-of-plane distortion of the heme; the frequency becomes lower as heme distortion increases.42 The frequency shift of the γ7 band of α heme in α2(Fe)β2(Ni) was fitted using a singleexponential function with a time constant of 48.3 ns. A frequency of the γ7 mode higher than that of the deoxy form and frequency shift of 43.4 ± 5.2 ns were observed for rHb.10 The present experimental data indicate that heme distortion increases in the α subunits of α2(Fe)β2(Ni) at a rate similar to those of HbA and rHb. ν(Ni−His) Band of β Heme. The Ni−His bond in NiHb is the sole covalent linkage between the Ni−heme and the protein. Consequently, this mode can be a good indicator of heme protein tertiary and quaternary structures as well as ν(Fe−His). The ν(Ni−His) band was observed at 238 cm−1 for the deoxy form of NiHb(βD99N), but at 235 cm−1 for that of NiHb(WT), as shown in Figure 4. The deoxy form of Hb Kempsey (βD99N mutant) adopts the R state, while that of WT adopts the T state. These results showed that the ν(Ni− His) band is a good marker of quaternary structure in Nisubstituted Hb as well as the ν(Fe−His) band in HbA. This interpretation also is supported by the experimental evidence that the frequency of the ν(Ni−His) band of NiHb(WT) is lower than that of Ni-substituted myoglobin.31 The higher ν(Ni−His) frequency in the R state as compared to the T state is consistent with Perutz’s strain model, in which strain imposed on the Ni−His bond is stronger in the T state than in the R state similarly to ν(Fe−His) frequency. The frequency of the ν(Ni−His) band of β Ni−heme in α2(Fe)β2(Ni) shifted at ∼20 μs, which suggests that the strain imposed on the Ni−His bond in the β subunit strengthens at ∼20 μs changing from the R to T state upon CO photolysis in the α subunit. γ7 Band of β Heme. The frequency of the γ7 band of the β Ni−heme in α2(Fe)β2(Ni) upshifted with a time constant of ∼20 μs. The direction of the frequency shift of the γ7 band was consistent with the experimental observation of a higher frequency of the γ7 band of the NiHb(WT) as compared to that of NiHb(βD99N): the γ7 band of β Ni−heme was observed at 310 cm−1 in the RR spectra of NiHb(βD99N), but at 312 cm−1 in the RR spectra of NiHb(WT). This suggests that the β Ni− heme distortion changes in ∼20 μs from the R to the T state upon CO photolysis of the α subunit. Intersubunit Communication through α−β Subunit Interface. Structural changes in the heme, heme pocket, and α−β subunit interface have been investigated for allosteric dynamics of HbA. However, very few studies have examined propagation of structural changes from one subunit to a neighboring one through the α−β subunit interface. Eaton and co-workers studied the speed of intersubunit communication using transient absorption measurements on Fe−Co hybrid Hb.43 Later, they also studied the speed in unsubstituted HbA

by partial photolysis experiments.44 Both of these studies suggested that a structural change in one subunit is transmitted to the neighboring subunit in ∼20 μs. The similar time constant of ∼20 μs suggests that the structural changes observed by transient absorption studies are the same as those observed by the present time-resolved RR study. The present data not only show that a structural change in the heme of the neighboring subunit occurs in ∼20 μs, but also that the changes include the proximal tension, which is a key factor controlling the oxygen affinity of HbA. Moreover, the present study demonstrated long-distance propagation of structural changes spanning a distance >20 Å in the protein. Two critical contact regions in the α1−β2 subunit interface were observed from the quaternary structural change in Hb (Figure 6): structural change in the hinge region, involving

Figure 6. Crystallographic structure around the α−β subunit of deoxy HbA (PDB ID: 2HHB). Blue and red atoms indicate nitrogen and oxygen, respectively. Yellow dashed lines indicate hydrogen bonds. Aspα94 and Tyrα42 are hydrogen-bonded to Trpβ37 and Aspβ99, respectively. The region between the FG corner and the C terminus of the α subunit and the C helix of the β subunit is referred to as the hinge region. The region between the FG corner and the C terminus of the β subunit and the C helix of the α subunit is referred to as the switch region.

rotation about the contacts between the C helix of the β subunit and the FG corner of the α subunit, reorients the residue side chains, as well as those of the switch region, which involve contacts between the C helix of the α subunit and the FG corner of the β subunit. The time-resolved ultraviolet RR spectroscopy showed that T state quaternary contacts in the subunit interface are formed in two well-separated steps, with time constants of 3 and 20 μs.13 The first step involves the hinge region contacts, as monitored by the Trpβ37···Aspα94 H-bond, while the second involves the switch region, as monitored by the Tyrα42···Aspβ99 H-bond. A time-resolved wide-angle X-ray scattering study on Hb showed that the relative rotation of one αβ dimer with respect to the other occurred at 1−3 μs.45 This suggests that the change in the hinge region is involved in the relative rotation from the R to the T state. The time constant of the change in the switch region coincides with that of structural changes in the β subunit through the α−β subunit interface observed in this study. This suggests that proximal tension is imposed synchronously with the change in the α−β subunit interface in ∼20 μs. Thus, dimer rotation occurring at 3 μs can induce a change in proximal 12466

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photolysis, RR spectra of rHbs, WT, and the βD99N mutant, and curve-fitting analysis on the γ7 band of β Ni−heme of α2(Fe)β2(Ni). This material is available free of charge via the Internet at http://pubs.acs.org.

tension of the α and β subunits associated with the change in the switch region. Perutz’s Strain Model. Perutz put the allosteric mechanism of HbA, proposed by Monod, Wyman, and Changeux,1 on a stereochemical basis.3,5 He interpreted their two-state model in terms of an equilibrium between two alternative structures, a tense one with low oxygen affinity constrained by salt bridges between the C-termini of the four subunits (T structure), and a relaxed one lacking these bridges (R structure). The equilibrium was considered to be governed primarily by the positions of the iron atoms relative to the porphyrin: out-ofplane in the deoxy form and in-plane in the oxy form. In turn, the tension exerted by the salt bridges in the T structure was transmitted to the Fe−His bond and restrained the movement of iron atoms into the porphyrin plane necessary for oxygen binding. Thus, the heme and the subunit interface are bidirectionally associated with each other. Matsukawa et al. studied various abnormal Hbs that had a replacement amino acid residue in the subunit interface. They observed a correlation between the ν(Fe−His) frequency and the first Adair constant K1, which is related to the Gibbs energy of the first oxygenation step.46 This finding fits well with Perutz’s strain model.3,5 Common time constants of ∼20 μs observed for frequency shifts of the ν(Fe−His) (α subunits) and ν(Ni−His) bands (β subunits) also are consistent with Perutz’s strain model; that is, the structural change in the subunit interface from the R to T state induces tension in the Fe−His bonds (this model suggests that the tension on the Fe−His bonds of both the α and the β subunits changes synchronously). Accordingly, the observed synchronous changes of the ν(Fe−His) and ν(Ni−His) bands upon the R−T transition can be regarded as real-time observations of Perutz mechanism. No previous RR observations of the structural dynamics of heme induced by quaternary structural changes in HbA have been reported because ligand photolysis can occur all four hemes in HbA and observed spectral changes cannot be definitely attributed to a heme structural change induced by quaternary change. In the present study using hybrid Hb, ligand photolysis resulted in a quaternary change, which then induced structural changes in the heme with no ligand photolysis, Ni− heme. Selective observation of Ni−heme demonstrated that a structural change in the proximal histidine of ∼20 μs was induced by a quaternary structural change in Hb.

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Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor John S. Olson and Dr. Eileen Singleton for providing the pSGE1702 plasmid and E. coli SGE1661 and for instructions on the procedures for protein expression purification. We also thank Professor Masako Nagai of Hosei University for her valuable advice on the preparation of the isolated chains. This work was supported in part by a Grant-inAid for Scientific Research on the Priority Area “Molecular Science for Supra Functional Systems” (Grant No. 19056013) to Y.M. from the Ministry of Education, Science, Sports, and Culture of Japan.

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REFERENCES

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CONCLUSIONS In the present study, the allosteric protein dynamics of Fe−Ni hybrid Hb was examined using time-resolved RR spectroscopy. Selective observation by resonance enhancement revealed that the structural changes starting at the photodissociated heme in the α subunits was transmitted to the heme in neighboring β subunits through the α−β subunit interface in ∼20 μs. Results demonstrated that the R→T quaternary change induces a change in the proximal tension of the α and β subunits. These are the first real-time observations that proximal tensions in the α and β subunits change synchronously, which support Perutz’s mechanism.

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AUTHOR INFORMATION

ASSOCIATED CONTENT

S Supporting Information *

Excitation wavelength dependence on RR spectra of a 1:1 mixture of deoxy forms of rHb and Ni-substituted Hb, detailed time-resolved RR spectra for α2(Fe−CO)β2(Ni) following CO 12467

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