Article pubs.acs.org/biochemistry
Interrelationship among Fe−His Bond Strengths, Oxygen Affinities, and Intersubunit Hydrogen Bonding Changes upon Ligand Binding in the β Subunit of Human Hemoglobin: The Alkaline Bohr Effect Shigenori Nagatomo,*,† Miki Okumura,† Kazuya Saito,† Takashi Ogura,‡ Teizo Kitagawa,‡ and Masako Nagai§,∥ †
Department of Chemistry, Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan Picobiology Institute, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamigori, Ako-gun, Hyogo 678-1297, Japan § Research Center for Micro-Nano Technology, Hosei University, Koganei, Tokyo 184-0003, Japan ∥ School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Ishikawa 920-0942, Japan ‡
S Supporting Information *
ABSTRACT: Regulation of the oxygen affinity of human adult hemoglobin (Hb A) at high pH, known as the alkaline Bohr effect, is essential for its physiological function. In this study, structural mechanisms of the alkaline Bohr effect and pH-dependent O2 affinity changes were investigated via 1H nuclear magnetic resonance and visible and UV resonance Raman spectra of mutant Hbs, Hb M Iwate (αH87Y) and Hb M Boston (αH58Y). It was found that even though the binding of O2 to the α subunits is forbidden in the mutant Hbs, the O2 affinity was higher at alkaline pH than at neutral pH, and concomitantly, the Fe−His stretching frequency of the β subunits was shifted to higher values. Thus, it was confirmed for the β subunits that the stronger the Fe−His bond, the higher the O2 affinity. It was found in this study that the quaternary structure of α(Fe3+)β(Fe2+-CO) of the mutant Hb is closer to T than to the ordinary R at neutral pH. The retained Aspβ94−Hisβ146 hydrogen bond makes the extent of proton release smaller upon ligand binding from Hisβ146, known as one of residues contributing to the alkaline Bohr effect. For these T structures, the Aspα94−Trpβ37 hydrogen bond in the hinge region and the Tyrα42−Aspβ99 hydrogen bond in the switch region of the α1−β2 interface are maintained but elongated at alkaline pH. Thus, a decrease in tension in the Fe−His bond of the β subunits at alkaline pH causes a substantial increase in the change in global structure upon binding of CO to the β subunit.
H
binding in Hb A have been advanced on the basis of mechanistic elucidations of the quaternary structure change between typical T and R. Perutz interpreted the origins of cooperativity of Hb A as follows.4 O2 binds to a pyramidal heme of deoxyHb A in the T structure; the heme iron moves toward the plane of the porphyrin ring, and concomitantly, the bond between iron and the proximal histidine (Fe−His bond) moves. This movement of the Fe−His bond induces perturbation of helix F, including the proximal histidine, and brings about substantial changes in the interactions between subunits α1 and β2, and finally in the quaternary structure of R (fully oxygen binding form) from T (deoxy form). Thus, a substantial increase in O2 affinity during oxygenation, which is called cooperativity, is caused by the quaternary structure change. Thus far, this Perutz mechanism
uman adult hemoglobin (Hb A) exhibits cooperative oxygen binding1 and has been investigated extensively as a model of general allosteric proteins.2,3 Hb A is composed of two α and two β subunits, forming an α2β2 tetramer. The α and β subunits, which have 141 and 146 amino acid residues, respectively, have one Fe-protoporphyrin-IX, called protoheme, bound coordinatively to a proximal histidine of helix F (HisF8). X-ray crystallographic studies of Hb A have already demonstrated the presence of two distinct quaternary structures, T (tense) and R (relaxed), corresponding to the low-affinity and high-affinity states.4−6 However, it has been reported in recent studies that an R structure with a low affinity and a T structure with high affinities are also present.7−10 In addition, recent X-ray studies have clarified the existence of other quaternary structures, including R2, RR2, R3, and TR.11−13 Thus, an appreciable diversity of quaternary structures has to be recognized. However, the structures for the unliganded form (deoxyHb A) and oxygen- or CO-bound form (oxyHb A or COHb A) can be treated as typical T and R structures, respectively.4−6 In fact, studies that aim to improve our understanding of the mechanism of cooperative oxygen © XXXX American Chemical Society
Received: November 2, 2016 Revised: February 4, 2017 Published: February 15, 2017 A
DOI: 10.1021/acs.biochem.6b01118 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry
Figure 1. Hemes and axial ligands of the α subunits in Hb A (left), Hb M Iwate (αH87Y) (center), and Hb M Boston (αH58Y) (right). Hb A is in the oxy form, but Hb M Iwate and Hb M Boston are half-met forms, α(Fe3+)β(Fe2+-deoxy/O2). Hemes of the α subunits of Hb M Iwate and Hb M Boston in the half-met forms have ferric irons (Fe3+), and therefore, the hemes cannot bind ligands such as O2 and CO.
structures of general oligomer proteins. Therefore, in this study, we decided to investigate more concretely the difference in roles between the α and β subunits in the α2β2 tetramer, following the previous study,10 in which we clarified the roles of the α and β subunits in cooperative oxygen binding of Hb A. In practice, we study the role of β subunits thoroughly using naturally occurring valency hybrid mutant Hbs, α(Fe3+)β(Fe2+), Hb M Iwate (Hisα87 → Tyr), and Hb M Boston (Hisα58 → Tyr), while we examine the other type of combination, α(Fe2+)β(Fe3+), in the future. We call α(Fe3+)β(Fe2+) a half-met form hereafter. The α subunits of the halfmet Hb M Iwate and Hb M Boston mutants have coordination bonds between the heme iron and the proximal or distal tyrosine, which stabilize the ferric (Fe3+) hemes of α subunits without binding O2. Characteristic differences among α subunits of Hb A, Hb M Iwate, and Hb M Boston are illustrated in Figure 1. It has been clarified that a heme (Fe3+) of an α abnormal subunit in Hb M Boston takes pentacoordination from X-ray crystallographical analysis,39 1H NMR,40 and resonance Raman spectroscopy41 except for a solution at higher pH with binding of a ligand to the β subunit.38 These Hb Ms (Hb M Iwate and Hb M Boston) have many advantages for the investigation of the relationship between function and structure, and many studies have used not only a valency hybrid form, α(Fe3+)β(Fe2+), but also fully met, α(Fe3+)β(Fe3+), and fully reduced, α(Fe2+)β(Fe2+), forms.38,41−47 Using these Hb Ms, we can observe directly both the Fe−His frequency changes of β subunits as the O2 affinity increases or decreases and the effects of quaternary structure changes upon binding of a ligand to the β subunit. Although it is known that the half-met forms, α(Fe3+)β(Fe2+), in Hb M Iwate and Hb M Boston exhibit only a small Bohr effect, these Hbs show the alkaline Bohr effect between pH 7 and 9 as shown below. The three likely sites that contribute to the alkaline Bohr effect are (1) the imidazole of Hisβ146, (2) the N-terminal amino group of the α subunit, and (3) the imidazole of Hisα122. A study utilizing deuterium exchange has confirmed that under physiological conditions, Hisβ146 contributes mainly to the alkaline Bohr effect.48,49 However, how the movement of Fe2+ upon O2 binding is
has been experimentally proven via time-resolved resonance Raman spectroscopy.14−18 The Fe−His coordination bond is a sole chemical connection between globin and heme and is, therefore, very important for the regulation of O2 affinity.4−6,8−10,14−31 In 1985, Matsukawa et al. found a correlation between the Fe−His frequencies and O2 affinity (strictly, a dissociation constant, K1, for the first O2 molecule).20 They showed that the higher the affinity of Hbs, the stronger the Fe−His bonds.20 Therefore, the O2 affinity of Hb A is regulated by two mechanisms: (1) the Perutz mechanism (by quaternary structure change concomitant with O2 binding) and (2) tertiary structure changes accompanied by Fe−His bond strength. It should be noted that the α and β subunits have not yet been distinguished. In contrast, the cooperative oxygen binding of Hb A does occur in neither the homodimer, α2, nor the homotetramer, β4, but needs the α2β2 heterotetramer.1,32 In a previous paper, we demonstrated that the lack of the Fe−His bond in β subunits in the α2β2 tetramer increased the oxygen affinity of the α subunits and reconfirmed that the lack of the Fe−His bond of α subunits inhibited the quaternary structure change.10,33,34 This means that in deoxyHb A the Fe−His bond of β subunits decreases the O2 affinity of α subunits, but when O2 binds to one α subunit of deoxy Hb A, its binding causes a quaternary structure change and increases the O2 affinity of both remaining α and β subunits.10 This would yield a sigmoidal O2 binding curve. On the other hand, the difference between the α and β subunits also provides a difference in the level of contribution to the Bohr effect. It had been pointed out from kinetic experiments by Olson and Gibson that binding of a ligand to the α subunit causes 80% proton release but binding of a ligand to the β subunit 20% proton release.35,36 Some hemoglobins with O2 binding sites only in the β subunits such as [α(Fe3+)β(Fe2+-O2)] exhibit increases in the O2 affinity of the β subunit at alkaline pH.37,38 This suggests another role for the Fe−His bonds in the β subunits in addition to the decrease in the O2 affinity of α subunits. Such detailed analyses for the discrimination of each subunit are expected to provide some basic information for understanding the role of higher-order B
DOI: 10.1021/acs.biochem.6b01118 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry
Figure 2. RR spectra of deoxyHb A and the deoxy heme of half-met forms of Hb M Iwate and Hb M Boston, α(Fe3+)β(Fe2+-deoxy), excited at 441.6 nm at pH 7.00, 6.41, and 6.53, respectively. Difference spectra of deoxyHb A minus the deoxy heme of half-met Hb M Iwate and deoxyHb A minus the deoxy heme of half-met Hb M Boston are colored red and green, respectively. Spectra were observed between 180 and 530 cm−1.
communicated to the α1−β2 subunit interface through the Fe− His bond and causes the alkaline Bohr effect is still to be clarified. Therefore, in this study, we focus on the role of the Fe−His bonds of β subunits in O2 binding properties and the quaternary/tertiary structure changes upon ligand binding and their pH dependencies. The overall goal of our series of works is to elucidate structurally the communication pathways for producing cooperativity and the Bohr effect.
deoxy) to the fully reduced form with sodium dithionite is slow, taking ∼1 day at room temperature.52 All measurements were taken at room temperature with a spinning cell (1800 rpm). The laser power at the scattering point was 4.0 mW. Raman shifts were calibrated with indene and carbon tetrachloride as a frequency standard, and the frequency accuracy was ±0.4 cm−1 for well-defined Raman bands. The integrity of samples after visible Raman measurement was carefully confirmed by the visible absorption spectra. Visible absorption spectra were recorded with a Hitachi U-3310 spectrophotometer. UVRR Measurements. The excitation light at 229 nm, which was obtained from an intracavity frequency-doubled Ar+ ion laser (Coherent, Innova 300C FRED), was introduced onto a sample from the lower front side of the spinning cell, which was a quartz NMR tube (Wilmad-LabGlass, 535-PP-9SUP, diameter of 5 mm). A 135° backscattering geometry was adopted. Other details of the apparatus in UVRR measurements have been described previously.10 The hemoglobin concentration was 200 μM (in heme) in a 0.05 M phosphate buffer (pH 6.1) and Tris buffer (pH 9.1) containing 0.2 M SO42− as the internal intensity standard.53 The deoxy and CO forms of Hb A and Hb M Iwate were prepared by adding sodium dithionite (1 mg/mL) to oxy Hb A and Hb M Iwate, α(Fe3+)β(Fe2+-O2), after the replacement of the air inside the sample tube with N2 and CO, respectively. After UVRR measurement, the integrity of α(Fe3+)β(Fe2+deoxy) was confirmed by absorption spectra, similar to the case in the visible RR experiment. The presented spectra are an average of two scans, each of which is the sum of 180 exposures, each exposure allowing accumulation of data for 10 s in Hb A, and an average 8−12 scans of each of which is the sum of 60 exposures, each exposure allowing accumulation of data for 10 s in Hb M Iwate. An accumulation time of one sample was made short to prevent the formation of fully reduced forms, α(Fe2+-deoxy)β(Fe2+-deoxy) for half-met Hb M Iwate, α(Fe3+)β(Fe2+-deoxy). The laser power applied to samples was between 0.65 and 1.0 mW. Heme concentrations were 200 μM for all samples, containing 0.2 M Na2SO4.
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EXPERIMENTAL PROCEDURES Materials. Preparation and Purification of Hemoglobins. Hb A was purified from human hemolysate by preparative isoelectric focusing.50 Human hemolysate was prepared from concentrated red blood cells provided by the Japanese Red Cross Kanto-Koshinetsu Block Blood Center. Hb M Iwate and Hb M Boston were purified from a patient’s hemolysate via Amberlite CG-50 column chromatography.51 Methods. Visible Resonance Raman (RR) Measurements. Visible RR spectra were excited at 441.6 nm with a He/Cd laser (Kinmon Koha, model CD4805R), dispersed with a 1 m single polychromator (Ritsu Oyo Kogaku, model MC-100DG, grating of 1200 grooves/mm) and second-order diffraction, and detected with a UV-coated, liquid nitrogen-cooled CCD detector (Roper Scientific, LN/CCD-1100-PB/VISAR/1). The hemoglobin concentration was 200 μM (in heme) in a 0.05 M phosphate buffer (from pH 5.6 to 8.3) and a 0.05 M Tris buffer (from pH 8.8 to 9.4). DeoxyHb A was prepared by adding sodium dithionite (1 mg/mL) to the oxy form after the replacement of the air inside the sample tube with N2. The deoxy form of Hb M Iwate, α(Fe3+)β(Fe2+-deoxy), also was prepared by adding sodium dithionite (1 mg/mL) to the oxy form, α(Fe3+)β(Fe2+-O2), after the replacement of the air inside the sample tube with N2. Deoxygenation of α(Fe3+)β(Fe2+-O2) to α(Fe3+)β(Fe2+-deoxy) was confirmed by absorption spectra given in Figure S1. Absorption bands at both 570 and 600 nm were carefully examined, because those bands served as an indicator of β(Fe2+-deoxy) and α(Fe3+) in α(Fe3+)β(Fe2+deoxy), respectively. We measured the α(Fe3+)β(Fe2+-deoxy) sample immediately after adding dithionite to α(Fe3+)β(Fe2+O2), because it is known that the conversion of α(Fe3+)β(Fe2+C
DOI: 10.1021/acs.biochem.6b01118 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry
crystallographic analysis, the mean 8-vinyl δ(CβCaCb) angles of α and β hemes are 125.3° and 128.8°, respectively.55 This difference in the δ(CβCaCb) angles of the 8-vinyl between α and β hemes seems to be reflected in the RR spectra shown in Figure 2. Another difference is observed in the γ7 (299.3 cm−1) bands. It is reported that the intensity of the γ7 band is larger in the α heme than in the β heme, because of the greater distortion of methane carbons in the α heme than in the β heme.56 Although Podstawka et al.56 reported the γ7 bands of α and β hemes at 299 and 304 cm−1, respectively, γ7 bands observed for Hb M Iwate and Hb M Boston, α(Fe3+)β(Fe2+-deoxy), do not have a peak at 304 cm−1; the band observed at 299.3 cm−1 seems to be the γ7 band of the β heme. This suggests that the environments of heme pockets of the β heme in Hb M Iwate and Hb M Boston, α(Fe3+)β(Fe2+-deoxy), are different from those of deoxyHb A, α(Fe2+-deoxy)β(Fe2+-deoxy). In addition, difference spectra of deoxyHb A minus Hb M Iwate, α(Fe3+)β(Fe2+-deoxy), and deoxyHb A minus Hb M Boston, α(Fe3+)β(Fe2+-deoxy), show interesting features. The Fe−His bands of these two difference spectra show two components, though one appears as a shoulder. Assuming that Fe−His bands of the β subunit in Hb M Iwate and Hb M Boston of α(Fe3+)β(Fe2+-deoxy) are the same as that in deoxyHb A, the Fe−His bands of both difference spectra arise from Fe−His bands of the α subunit in deoxyHb A. Therefore, Fe−His bands of the α subunit in deoxyHb A may be composed of two kinds of Fe−His bands, different from those of the β subunit in Hb M Iwate and Hb M Boston, α(Fe3+)β(Fe2+-deoxy). It is interesting to note that two components of the Fe−His band are reported for metal hybrid Hb, α(Fe2+-deoxy)β(Co2+).57 The pH dependencies of RR spectra of Hb M Iwate and Hb M Boston excited at 441.6 nm are shown in Figures 3 and 4,
Raman shifts were calibrated with cyclohexane as a frequency standard, and the frequency accuracy was ±1 cm−1 for welldefined Raman bands. The difference spectra were recorded so that the Raman band of SO42− (981 cm−1) was abolished. The integrity of the sample after exposure to the UV laser light was carefully confirmed by the visible absorption spectra measured before and after the UVRR measurements. When spectral changes were recognized, the Raman spectra were discarded. Visible absorption spectra were recorded with a Hitachi U-3310 spectrophotometer. 1 H NMR Measurements. The 1H NMR spectra were recorded with a Bruker AVANCE 600 FT NMR spectrometer operating at a 1H frequency of 600 MHz at the OPEN FACILITY of the Research Facility Center for Science and Technology of the University of Tsukuba. The hemoglobin concentrations of Hb A (deoxy and CO forms), Hb M Iwate, α(Fe3+)β(Fe2+-deoxy) at pH 7, and α(Fe3+)β(Fe2+-CO) at pH 7 and 9 were 1 mM on a heme basis in 0.05 M phosphate buffer (pH 7) and Tris buffer (pH 9). The deoxyHb A and COHb A forms were prepared by adding sodium dithionite (1 mg/mL) to the oxy form after replacement of the air inside the sample tube with N2 and CO, respectively. Hb M Iwate, α(Fe3+)β(Fe2+-CO), at pH 7 and 9 were prepared by adding CO gas to α(Fe3+)β(Fe2+-O2). Hb M Iwate, α(Fe3+)β(Fe2+-deoxy), at pH 7 was prepared by repeatedly removing and adding nitrogen gas. The spectra were recorded by the water suppression method by presaturation with approximately 2k−4k scans, a spectral width of 36 kHz (60 ppm), 32k data points, a 90° pulse of 12.0 μs, and recycle times of 0.5 s for deoxyHb A, 1 s for COHb A, 0.5 s for Hb M Iwate, α(Fe3+)β(Fe2+-deoxy), and 0.5−1 s for Hb M Iwate, α(Fe3+)β(Fe2+-CO). Chemical shifts are given in parts per million downfield from sodium 2,2dimethyl-2-silapentane-5-sulfonate, with residual H2HO as an internal reference.
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RESULTS pH Dependence of Fe−His Frequencies of Hb M Iwate and Hb M Boston. Resonance Raman (RR) spectra of deoxyHb A and deoxy forms of Hb M Iwate and Hb M Boston, α(Fe3+)β(Fe2+-deoxy), excited at 441.6 nm are shown in Figure 2. The RR bands observed for the three Hbs are derived from hemes of the Fe2+-deoxy form, because Fe3+ high-spin hemes have a resonance effect weaker than that of Fe2+-deoxy heme upon excitation at 441.6 nm. Actually, absorption maxima (λmax) in the Soret band of α(Fe3+) and β(Fe2+-deoxy) of Hb M Iwate (Figure S1) and Boston,41 α(Fe3+)β(Fe2+-deoxy), are observed at 407 and 428 nm, respectively. Although the RR spectrum of deoxyHb A contains vibrations of the α and β hemes, the spectra of Hb M Iwate and Hb M Boston, α(Fe3+)β(Fe2+-deoxy), contain the vibration of β heme alone.41 Therefore, band widths of Fe−His stretching of Hb M Iwate and Hb M Boston are narrower than that of Hb A, because of the lack of a contribution from the Fe−His bond of the α subunit.19 While peak positions of other observed bands of Hb M Iwate and Hb M Boston are almost the same as those of deoxyHb A, there are some differences. The 8-vinyl δ(CβCaCb) band of deoxyHb A is observed at a wavenumber lower by 2.3 cm−1 than those of Hb M Iwate and Hb M Boston. This means that a δ(CβCaCb) band of the 8-vinyl of the α heme in deoxyHb A is lower than that of the β heme in deoxyHb A54 and, thus, that the bending force constant against the 8-vinyl δ(CβCaCb) angle is smaller in the α heme than in the β heme. In X-ray
Figure 3. pH dependence of resonance Raman spectra of the half-met form of Hb M Iwate, α(Fe3+)β(Fe2+-deoxy), excited at 441.6 nm. Spectra A−F were observed between 180 and 530 cm−1 at pH 5.62, 6.41, 7.46, 7.63, 8.88, and 9.25, respectively.
respectively. As mentioned previously, the observed spectra reflect vibrations of the heme of the β subunit of Hb M Iwate and Hb M Boston, α(Fe3+)β(Fe2+-deoxy), because the Raman intensity of the β(Fe2+-deoxy) subunit in α(Fe3+)β(Fe2+-deoxy) is more enhanced than that of α(Fe3+).41 It is noticed that in Figure 3 the Fe−His frequencies are shifted to higher wavenumbers as the pH increases, while the other bands D
DOI: 10.1021/acs.biochem.6b01118 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry
Table 1. Oxygen Binding Function and Structure of Hemoglobins Lacking an Fe−His Bond in Their α Subunits P50 (mmHg)
Hill’s n
α1−β2b
Nαa
Hb M Iwate, αFe βFe 1.0 5 T (Trp, ∼T; Tyr, T) 1.0 5 T (Trp, R; Tyr, T) Hb M Boston, αFe3+βFe2+ 1.2 5 T (Trp, ∼T; Tyr, T) T (Trp, R; Tyr, 1.4 5(M), T) 6(m)e αNiβFe-Hb, αNi2+βFe2+ 1.0 4 T (Trp, ∼T; Tyr, T) 1.0 5 T (Trp, R; Tyr, ∼T) α(F8Gly)β(F8His)Hbc 0.45 6 T (Trp, ∼T; Tyr, T) 3+
Figure 4. pH dependence of resonance Raman spectra of the half-met form of Hb M Boston, α(Fe3+)β(Fe2+-deoxy), excited at 441.6 nm. Spectra A−G were observed between 180 and 530 cm−1 at pH 5.60, 6.53, 7.52, 7.67, 8.27, 8.81, and 9.38, respectively.
pH 6
50
pH 9
35
pH 7
27.5
pH 9
21.5
pH 6.5
106
pH 8.5
16.2
pH 7
5.6/60(β)d
ref
2+
37, this study 37, this study 38 38
22, 33 22, 33
10
Coordination number of the α heme upon binding of a ligand (CO) to the β subunit. bα1−β2 subunit contact (quaternary structure) upon binding of a ligand (CO) to the β subunit as judged by ultraviolet resonance Raman (UVRR) spectroscopy. T and R described in Trp and Tyr show that signal changes of Trp and Tyr upon ligand binding are absent and are as much as those (maximal changes) of ligand binding in deoxy Hb A. An approximate symbol shows a change that is
