structural and oxygen binding properties of dimeric horse myoglobin
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Cite this: DOI: 10.1039/c2dt30893b
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Structural and oxygen binding properties of dimeric horse myoglobin†
Satoshi Nagao,a Hisao Osuka,‡a Takuya Yamada,a Takeshi Uni,a Yasuhito Shomura,b,c Kiyohiro Imai,d
Yoshiki Higuchi*b,c and Shun Hirota*a
Received 24th April 2012, Accepted 24th July 2012DOI: 10.1039/c2dt30893b
Myoglobin (Mb) stores dioxygen in muscles, and is a fundamental model protein widely used inmolecular design. The presence of dimeric Mb has been known for more than forty years, but itsstructural and oxygen binding properties remain unknown. From an X-ray crystallographic analysis at1.05 Å resolution, we found that dimeric metMb exhibits a domain-swapped structure with two extendedα-helices. Each new long α-helix is formed by the E and F helices and the EF-loop of the originalmonomer, and as a result the proximal and distal histidines of the heme originate from differentprotomers. The heme orientation in the dimer was in the normal mode as in the monomer, but regulatedfaster from the reverse to normal orientation. The dimer possessed the oxygen binding property, althoughit exhibited a slightly higher oxygen binding affinity (∼1.4 fold) compared to the monomer and showedno cooperativity for oxygen binding. The oxygen binding rate constant (kon) of the dimer ((14.0 ± 0.7) ×106 M−1 s−1) was similar to that of the monomer, whereas the oxygen dissociation rate constant (koff ) ofthe dimer (8 ± 1 s−1) was smaller than that of the monomer (12 ± 1 s−1). We attribute the similar konvalues to their active site structures being similar, whereas the faster regulation of the heme orientationand the smaller koff in the dimer are presumably due to the slight change in the active site structure and/ormore rigid structure compared to the monomer. These results show that domain swapping may be a newtool for protein engineering.
Introduction
Self-aggregation of proteins is a common mechanism for anumber of important neurodegenerative diseases.1 Due to thisconnection to neurodegenerative diseases, protein structuralchange has been studied intensively,2,3 and oligomeric proteinshave gained interest as initial intermediates.4–7 The number ofproteins identified with domain-swapped structures,8–10 includ-ing heme proteins,11–13 has been increasing. A domain-swappeddimer has been detected in diphtheria toxin.14 The domain-swapped dimeric structure of serpin, a family of proteins which
form large stable multimers leading to intracellular accretion anddisease, has been reported.15 We have recently shown that oligo-merization occurs via domain swapping in cytochrome c (cyt c),breaking its heme–Met coordination bond and increasing its per-oxidase activity.16,17
Myoglobin (Mb) is a monomeric oxygen storage hemeprotein.18 Mb was the first protein to have its three-dimensionalstructure revealed,19 and has been used as a model protein forprotein engineering studies.20–25 It has also been shown to formamyloid fibrils.26 Horse Mb consists of 153 amino acids witheight α-helices (A to H helices) and seven non-helical seg-ments.27,28 The heme of Mb is coordinated by His93, and His64creates a hydrogen bond with the bound oxygen.29 Mb canpossess two interconvertible heme orientations (normal andreverse), which rotate 180° between each other against the α–γmeso carbon axis of the heme.30 In 90% of native Mb, theheme is bound to the protein in the normal orientation – theremainder being in reverse orientation.30 Dimeric Mb hasbeen detected during purification of monomeric Mb andremoved to obtain pure monomeric Mb, but its structuraland oxygen binding properties have remained unknown formore than forty years.31 In this study, we show that dimeric Mbforms a unique domain-swapped structure with active sites con-structed by two different protomers. The dimer shows a slightincrease in oxygen binding affinity, where the kon value of thedimer is similar but koff value is smaller compared to themonomer.
†Electronic supplementary information (ESI) available: FPLC elutioncurves, crystal structure data and Hill plots for Mb. See DOI:10.1039/c2dt30893b‡Present address: Department of Life Science, University of Hyogo,3-2-1 koto, Kamigori-cho, Ako-gun, Hyogo 678-1297, Japan.
aGraduate School of Materials Science, Nara Institute of Science andTechnology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.E-mail: [email protected]; Fax: +81-743-72-6119;Tel: +81-743-72-6110bDepartment of Life Science, Graduate School of Life Science,University of Hyogo, 3-2-1 Koto, Kamigori-cho, Ako-gun, Hyogo678-1297, Japan. E-mail: [email protected] SPring-8 Center, 1-1-1 Koto, Sayo-cho, Sayo-gun, Hyogo679-5148, JapandDepartment of Frontier Bioscience, Faculty of Bioscience and AppliedChemistry, Hosei University, 3-7-2 Kajino-cho, Koganei, Tokyo184-8584, Japan
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Experimental
Preparation of dimeric Mb and reconstituted monomeric anddimeric Mbs
Dimeric Mb was prepared by dissolving horse muscle Mb(1 mM) (Nacalai Tesque, Japan) in water followed by addition of5% (v/v) ethanol. Ethanol was added to obtain a larger amountof dimeric Mb. The Mb solution was lyophilized, and theobtained precipitate was dissolved in 10 mL of 50 mM potass-ium phosphate buffer, pH 7.0. The obtained metMb solution wasfiltrated and the produced dimeric metMb was purified by gelchromatography (HiLoad 26/60 Superdex75, GE Healthcare)using a fast protein liquid chromatography (FPLC) system (Bio-Logic DuoFlow 10, Bio-Rad, CA) several times with the samebuffer. The concentrations of the proteins were calculated fromthe absorbance at 409 nm and adjusted to desired concentrations.
Apomyoglobin (apoMb) was obtained by the method pre-viously reported.32 The heme (Tokyo Chemical Industry Co.,Ltd, Japan) was dissolved in a minimal amount of 1 M NaOH,and the obtained solution was diluted with distilled water to aheme concentration of ∼10 mg ml−1. ApoMb was reconstitutedwith a stoichiometric amount of heme by titration using theoptical absorption spectra. The reconstituted Mb was purified bypassing it through a DE-52 column (GE Healthcare) with50 mM potassium phosphate buffer, pH 7.0.
To obtain dimeric apoMb, monomeric apoMb was treatedwith ethanol and lyophilization by the same procedure as that toobtain dimeric holo Mb from monomeric holo Mb. However, wecould not purify dimeric apoMb before heme reconstitution,since the apo dimer dissociated to monomers very quickly at4 °C (the half-life was shorter than 5 h). Therefore, we reconsti-tuted the apoMb dimer in the presence of the apoMb monomeras soon as monomeric and dimeric apoMbs were preparedtogether, and subsequently purified the holo forms. The mixtureof monomeric and dimeric apoMbs were dissolved in 50 mMpotassium phosphate buffer, pH 7.0 after lyophilization andreconstituted together with a stoichiometric amount of heme bytitration. The solution containing reconstituted monomeric anddimeric Mbs was purified by passing it through a DE-52 column(GE healthcare) with 50 mM potassium phosphate buffer,pH 7.0. Dimeric Mb was separated from monomeric Mb by gelchromatography (HiLoad 26/60 Superdex75, GE healthcare)using the FPLC system with the same buffer.
Optical absorption and CD measurements
Absorption spectra were measured with a UV-2450 spectro-photometer (Shimadzu, Japan) using a 1 cm path-length quartzcell. CD spectra were measured with a J-725 circular dichroismspectropolarimeter (Jasco, Japan) using a 0.1 cm path-lengthquartz cell.
X-ray crystallography
Crystallization was carried out at 277 K using the sitting dropvapor diffusion method with crystal plates (CrystalClear DStrips, Douglas Instruments, Hampton Research, CA, USA). Theprotein concentration was adjusted to 27 mg ml−1 in 100 mM
Tris-HCl buffer, pH 7.0. The droplets prepared by mixing 2 μlof the protein solution with 2 μl reservoir solution wereequilibrated. The best reservoir solution was found to be 10%PEG 6000, 200 mM CH3COONa and 0.1 M Tris-HCl buffer,pH 7.0.
The diffraction data were collected at the BL44XU beam lineat SPring-8, Japan. The crystal was mounted on a cryo-loop andflash-frozen at 100 K in a nitrogen cryo system. The detectorwas a MX225HE (Rayonix). Two data sets were collected forthe same crystal: large (about 5.0 to 1.05 Å) and small (about50 to 3.0 Å) angle regions. For the large angle data, the crystal-to-detector distance was 95 mm and the wavelength was 0.8 Å.The oscillation angle was 0.25° and exposure time was 1 s perframe. The total number of frames was 480. The beam powerwas attenuated using an Al plate to collect the small angle data.To obtain the small angle data, the crystal-to-detector distancewas 233 mm and the wavelength was 0.8 Å. The oscillationangle was 1° and exposure time was 1 s per frame. The totalnumber of frames was 120. The two diffraction data weremerged and processed using the programs DENZO and SCALE-PACK.33 The preliminary structure was obtained by a molecularreplacement method (MOLREP) using the atomic coordinates ofthe monomer structure of horse Mb (PDB code: 1WLA) as astarting model. The structure refinement was performed usingthe programs REFMAC and SHELX.34 The molecular model wasmanually corrected, and water molecules were picked up in theelectron density map using the program COOT. The data collec-tion and refinement statistics are summarized in Table S1.† TheX-ray dose absorbed by the crystal was calculated byRADDOSE as 22.3 MGy,35 showing that the iron site was pre-sumably in the FeII–OH/H2O state.36
NMR measurements
The NMR measurements of reconstituted dimeric Mb (hemeconcentration, 0.5 mM) were performed about 18 h after recon-stitution due to purification of the protein, where the sample waskept at 4 °C until the measurement. However, we could notmeasure the kinetic parameter of the heme orientation in thedimer, since the heme reorientation reaction was too fast andno reverse heme orientation was observed in the NMRspectrum after quenching the reorientation reaction by additionof potassium cyanide immediately after reconstitution of theheme.37 Dissociation of dimeric Mb to monomers was per-formed by incubation of the dimer (heme concentration,0.5 mM) in 50 mM potassium phosphate buffer, pH 5.0, for15 min. After the dissociation, the pH of the solution wasadjusted immediately to 7.0, and the buffer was exchanged toD2O buffer within 4 h. The 1H NMR spectra of monomericand dimeric metMbs (heme concentration, 0.5–1 mM) in 50 mMpotassium phosphate buffer, pD 7.0, were recorded at 25 °Cwith a JNM-ECA600 spectrometer (JEOL, Japan) operated atthe 1H frequency of 600 MHz. The 1H NMR spectra wereobtained by collecting 8192 transients using 8192 data pointsover 200 ppm bandwidths. The water signal was suppressed bypresaturation of the signal. All chemical shifts were referencedto the chemical shifts of external 2,2-dimethyl-2-silapentane-5-sulfonate (DSS).
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Oxygen equilibrium curves
Oxygen equilibrium curves (OECs) of monomeric and dimericMbs (heme concentration, 60 μM) were measured in 50 mMpotassium phosphate buffer, pH 7.0, at 20 °C, using thepreviously described automatic oxygenation apparatus.38 P50
(partial pressure of oxygen at half saturation) and nmax
(maximum slope of Hill plot) values were determined by least-squares fitting to the obtained OECs with the Hill equation.
Kinetic measurements
Purified dimeric metMb (heme concentration, 200 μM) in50 mM potassium phosphate buffer, pH 7.0, was degassed witha vacuum line and subsequently flushed with nitrogen gas, fol-lowed by anaerobic addition of dithionite (final concentration,4 mM) in order to reduce the heme. Dithionite was removedfrom the solution by a PD-10 column (GE Healthcare, Buckin-ghamshire), and dimeric Mb was obtained in the oxy form.Dimeric oxyMb was stable enough for kinetic measurements.Monomeric oxyMb was prepared by the same procedure.
The concentration of monomeric and dimeric oxyMb wasadjusted to 20 μM (heme unit) and the sample was equilibratedunder an atmosphere of oxygen plus nitrogen, prepared using agas generator (MX-3S, Crown, Tokyo), such that the partialpressure of oxygen varied from 20 to 100%. The sample solutionwas transferred into a sealed quartz cell, which was filled withthe same gas mixture. To obtain the O2 binding rate constant,flash photolysis of dimeric oxyMb samples was accomplishedusing the second harmonic (532 nm) of a Nd:YAG laser(Surelight I-10, Continuum, Santa Clara; pulse energy, 15 mJ;pulse width, 5 ns; pulse frequency 10 Hz) for excitation. Time-resolved absorbance changes at 435 nm were measured at 20 °Cwith illumination from a Xe lamp orthogonal to the laserpulse and were recorded on a digital oscilloscope (TDS 3012B,Tektronix, Tokyo), which received voltage signals from thephotomultiplier attached to a monochromator (RSP-601-03,Unisoku, Osaka). The traces were obtained as averages of64 pulses.
The O2 dissociation rate constant was determined usingstopped-flow apparatus (RSP-601, Unisoku, Osaka). Monomericand dimeric oxyMbs (10 μM, heme unit) in 50 mM potassiumphosphate buffer, pH 7.0, were mixed with an equal volume of10 mM sodium dithionite in deoxygenated 50 mM potassiumphosphate buffer, pH 7.0, at 20 °C. The absorbance changeswere followed at 435 nm for at least 3 independent experimentsfor each sample. Least-squares exponential fits were performedfor all the time-resolved absorption data using Igor Pro ver. 6.0software package (WaveMetrics, Portland).
Results and discussion
Dimeric horse metMb was produced by an addition of 5% (v/v)ethanol to monomeric horse metMb, subsequent lyophilization,and dissolution of the obtained precipitates with buffer andpurification using gel chromatography (Fig. S1†). Although highorder oligomers were produced in the case of cyt c,16 monomericand dimeric proteins were the main products for Mb with a smallamount of the trimeric protein. These results suggest that the
structure of dimeric Mb inhibits formation of high order oligo-mers. Dimeric Mb was stable in 50 mM potassium phosphatebuffer, pH 7.0, at 4 °C in the absence of ethanol, although it con-verted to monomers when heated at 70 °C for 5 min or incubatedat relatively low pH (pH 5.0) at 4 °C (Fig. S1†). We have pre-viously shown that domain-swapped oligomers of cyt c dis-sociate to monomers when heated at 70 °C for 5 min.16 RNaseA oligomers have also been shown to dissociate to monomerswhen heated at 65 °C for 10 min.39
The maximum wavelength of the Soret band at 409 nm in theoptical absorption spectrum did not change upon dimerization ofMb (Fig. 1). However, the intensities of the 208 and 222 nmα-helix-related negative bands in the CD spectrum increasedslightly by dimerization (Fig. 2). These results indicate that theactive site structure is similar between monomeric and dimericMbs but the secondary structure is slightly different.
To elucidate the detailed structure of dimeric horse Mb, wesolved its X-ray crystal structure (PDB ID: 3VM9) (Fig. 3). Thestructure of the dimeric protein at 1.05 Å resolution exhibited adomain-swapped structure, where the distance between the twoirons was 39.8 Å and new extended α-helices (Glu59–Thr95)were formed by the original E (Glu59–Lys77) and F(Leu86–Thr95) helices and the EF-loop (Lys78–Glu85) of the
Fig. 1 Optical absorption spectra of monomeric (red) and dimeric(blue) horse metMbs: (a) 250–750 nm range and (b) expansion of theSoret and Q-band regions. The monomeric protein was obtained byincubation of the solution at 70 °C for 5 min. Measurement conditions:sample concentration (heme unit): 6.6 μM; buffer: 50 mM potassiumphosphate; pH: 7.0; room temperature.
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monomers. Therefore, the slight increase in the intensities of theα-helical bands in the CD spectrum by dimerization can beattributed to the extension of the α-helix at the EF-loop in thedimer.
Trifluoroethanol has been shown to promote helical formationin the amino acid sequences that have a tendency to be helical insolution.40,41 In fact, the EF-loop (Lys78–Glu85) of monomericMb converted to an α-helix, and formed an extended newα-helix by connecting the E and F helices in dimeric Mb. Asimilar loop-to-helix transition in a hinge loop, which links thehelical domains, has recently been reported for the Mycobac-terium tuberculosis Rv3019c-Rv3020 ESX complex.42
We have calculated the root-mean-square deviation (rmsd)values of the N-terminus to E helix (Gly1–Lys77) and F helix toC-terminus (Leu86–Gly153) regions (excluding the EF-loop)between the structures of the monomer and each independentprotomer of the dimer (Table S2†). The rmsd values of bothregions were less than 1.22 Å. These results indicate that thestructures of both regions in the monomer and protomers of thedimer are similar. The E and F helices construct the heme pocketin the monomer. The heme is coordinated with His93 and awater molecule in the monomer, where the coordinated water ishydrogen bonded to His64. In the dimer, the heme pocket isformed by the same amino acids as the monomer but the proxi-mal and distal sites originate from different protomers; i.e.,His64 and His93 belong to different protomers. It is noteworthythat the positions of the amino acid side chains of the dimer arewell overlapped with those of the monomer (Fig. 4), althoughthe Fe–OH2, Fe–His64 and Fe–His93 distances were slightlydifferent between the dimer and monomer (Table 1).
Hydrogen bonds and intimate hydrophobic interactions areimportant for stabilization of protein structures. There are threekey hydrogen bonds (<3.0 Å between heavy atoms) (Gly1/Lys133, His24/His119 and Lys42/Lys98) between the N-termi-nus to E helix (Gly1–Lys77) and the F helix to C-terminus(Leu86–Gly153) regions in monomeric horse metMb (PDB:1WLA) (Fig. 3). Interestingly, the number of key hydrogenbonds between these regions increases in the dimer (Glu6A/Lys133B, Asn12A/Asp122B, His24A/His119B, Glu27A/Lys118B,
Lys42A/Lys98B, Glu6B/Lys133A, His24B/His119A, Glu27B/Lys118A, Lys42B/His97A and Lys42B/Lys98A) (A and B rep-resent each protomer) (Fig. 3). However, some hydrogen bondsin the dimer are perturbed. Since the EF-loop of the monomerbecomes part of the extended α-helix by dimerization, the intra-molecular hydrogen bonds between His82 and Asp141 seen inthe monomer are broken in the dimer and a new intermolecularhydrogen bond between Lys79 of one protomer with Asp141 ofanother protomer is formed (Fig. 3). Including this hydrogenbond, two key hydrogen bonds (Asp4/Lys79 and His82/Asp141)exist between the hinge loop and the rest of the protein in mono-meric Mb, whereas three key bonds (Lys78A/Glu85B, Lys79A/Asp141B and Asp141A/Lys79B) are detected in the dimer. Thesenew hydrogen bonds play important roles in stabilizing thedimeric structure.
It has been shown that the heme orientation in monomeric Mbis determined by the interaction between the heme and aminoacid residues, which define the heme pocket structure.37 Thesimilarity in the optical absorption spectra of monomeric anddimeric Mbs can be attributed to the similarity in the hemepocket structures, although proximal and distal sites are con-structed by different protomers in the dimer. Since the hemepocket is formed by different protomers, it has to be opened toform a dimer. However, the electron density distributions of theheme methyl and vinyl groups of the dimer were well resolvedin single orientations in the normal orientation (Fig. S2†),showing that the heme orientation of the dimer exhibits thesingle native-like normal orientation.
The heme orientation of dimeric Mb was investigated in detailusing NMR measurements to elucidate whether the heme dis-sociates from the protein during the dimerization (Fig. 5). Theproton signals of the heme side chains in metMb shift largelytoward the low magnetic field due to the unpaired electrons ofthe heme iron (high-spin, S = 5/2). The chemical shifts of thesedimeric metMb protons were similar to those of monomericmetMb (Fig. 5a and b). These results show that the hemeenvironment and spin state are similar between monomeric anddimeric metMbs in solution, consistent with the similarities inthe absorption spectra and X-ray crystal structures. The simi-larities in the chemical shifts also show that most of the heme inthe dimer exhibits the normal orientation.
In the monomeric metMb, one tenth of the heme has beenfound to be in the reverse form (a typical chemical shift at about65 ppm) (Fig. 5a).30 The heme is initially inserted in the normaland reverse orientations in about equal amounts when reconsti-tuted from the monomeric apo protein,45 and the normal orien-tation to reverse orientation ratio changes to 7 : 3 after incubationat 4 °C for 18 h (Fig. 5c). Conversion of the heme orientationfrom the reverse to the normal mode takes 30 h (half-life) under10 °C at pH 8.4 for the monomer.46 Interestingly, the hemeorientation of the dimer was in a single normal orientation afterpurification at 4 °C for 18 h when produced from the reconsti-tuted monomer, suggesting a relatively fast regulation of theheme orientation during or after dimerization (Fig. 5d). To evalu-ate further, dimeric Mb was reconstituted from the dimeric apoprotein, where no reverse heme orientation was observed afterpurification (Fig. 5e). However, the monomeric protein exhibiteda mixed heme orientation in the initial state when the monomericprotein was produced from the dimer by dissociation at pH 5.0
Fig. 2 CD spectra of monomeric (red) and dimeric (blue) horsemetMbs. Each concentration of the protein was adjusted according to theintensity of its Soret band. Measurement conditions were the same asthose in Fig. 1, except for the sample concentration (heme unit) of5 μM.
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for 15 min (Fig. 5f and Fig. S1†). These results show that theheme–protein bond is cleaved during dimerization and the hemeorientation is regulated faster to the normal orientation in thedimer than in the monomer.
Oxygen binding properties of monomeric and dimeric Mbswere compared with their OECs. The P50 values of the monomerand dimer were 0.53 and 0.39 mmHg, respectively, at pH 7.0and 20 °C (Fig. 6). These results show that the dimerization ofMb decreases the P50 value by a factor of ∼1.4. However, theHill coefficient nmax values were 1.01 for monomeric anddimeric Mbs (Fig. S3†), showing that the dimer possesses no
cooperativity for oxygen binding despite the presence of twooxygen binding sites.
To investigate the difference in the oxygen binding affinitiesbetween monomeric and dimeric Mbs in detail, oxygen re-binding kinetic experiments were performed as a function ofoxygen concentration, yet exploring a range of values that ensurefull saturation of Mb with oxygen before photolysis in each case.Only about 2% of the oxygenated proteins reacted and producedthe deoxy protein according to the initial absorbance changes.The observed rate constant (kobs) values increased linearly asa function of oxygen concentration as expected for a simple
Fig. 3 Crystal structures of monomeric and dimeric horse Mbs in stereo views. Protein structures of monomeric Mb reported previously (PDB:1WLA) (gray) and dimeric Mb (pink and blue). The Lys78–Glu85 residues (hinge loop of the monomer) are depicted in red and dark blue in thedimer and dark gray in the monomer. Key hydrogen bonds are shown in blue lines. The N- and C-termini and the E and F helices are labeled as N, C,E, and F, respectively. Hemes are shown as yellow stick models. Side-chain atoms of His64, His93 and other amino acids in the heme pocket areshown as stick models with labels. The water molecules which are coordinated to the heme irons are depicted as light blue balls. The numbers (1–14)denote the key hydrogen bonds: Dimer, 1: Glu6A(Oε2)–Lys133B(Nζ), 2: Asn12A(Nδ2)–Asp122B(Oδ1), 3: His24A(Nε2)–His119B(Nε2),4: Glu27A(Oε1)–Lys118B(Nζ), 5: Lys42A(Nζ)–Lys98B(O), 6: Lys78A(Nζ)–Glu85B(Oε2), 7: His64A(Nε2)–Wat155B(O), 8: Ser92B(Oγ)–HemeB(O2A),9: His97B(Nε2)–HemeB(O1A), 10: Tyr146B(Oη)–Ile99B(O), 11: Ile101B(N)–G153B(OT1), 12: Ser108B(Oγ)–Arg139B(Nη1), 13: Glu18A(Oε2)–Lys77A(Nζ), 14: Lys79A(Nζ)–Asp141B(Oδ2); Monomer, 1: Glu1(O)–Lys133(Nζ), 2: Asp4(Oδ2)–Lys79(Nζ), 3: His24(Nε2)–His119(Nε2), 4: His116(Nε2)–Gln128(Nε2), 5: Lys42(Nζ)–Lys98(O), 6: His82(Nε2)–Asp141(Oδ2), 7: His64(Nε2)–Wat156(O), 8: Ser92(Oγ)–Heme(O2A), 9: His97(Nε2)–Heme(O1A), 10: Tyr146(Oη)–Ile99(O). The hydrogen bonds stabilizing the dimeric form (1–7 and 14 in the dimeric form) are labeled in red, other-wise in black.
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re-binding phenomenon.47 From the plots of kobs versus [O2](Fig. 7a), oxygen binding rate constant, kon can be calculatedusing eqn (1).
kobs ¼ kon ð½deoxy heme� þ ½O2�Þ þ koff ð1Þ[deoxy heme] and [O2] represent the concentrations of deoxyheme and oxygen at the final equilibrium, respectively. Underour experimental conditions, oxygen is in large excess withrespect to Mb (at 20 °C, [O2] = 220 μM, [Mb]total (heme unit) =10 μM), hence the term [deoxy heme] can be neglected in eqn(1) and the observed re-binding rate depends linearly on theoxygen concentration. The kon value of dimeric Mb was obtainedas (14.0 ± 0.7) × 106 M−1 s−1. This value was very close to thatreported for monomeric horse Mb (12 × 106 M−1 s−1)48 andsperm whale Mb ((14 ± 3) × 106 M−1 s−1)47 at the same pH andtemperature (pH 7.0 and 20 °C) (Table 2).
The O2 dissociation rate constant (koff ) was obtained bymeasuring the absorption changes of monomeric and dimericoxygenated Mbs (oxyMbs) at 435 nm in the presence of dithio-nite (Fig. 7b). The koff values of monomeric and dimeric Mbswere obtained as 12 ± 1 and 8 ± 1 s−1, respectively, where thekoff value of monomeric Mb was similar to that reported forhorse Mb (11 s−1)49 and sperm whale Mb (12 ± 2 s−1) at thesame pH and temperature (pH 7.0 and 20 °C) (Table 2).47
According to the NMR measurements, the heme orientationwas regulated faster in the dimer than in the monomer. Thefaster regulation in the dimer can be attributed to the rigid struc-ture of the dimer due to the new extended α-helix, which pre-vents the reverse heme orientation. The oxygen binding affinitywas slightly higher in the dimer compared to the monomer
according to the OEC analyses. This difference can be mainlyattributed to the difference in the koff values. The kon values ofmonomeric and dimeric Mbs were similar, whereas the koff valueof the dimer was smaller than that of the monomer. We attributethe similar kon values to their active site structures being similar,even though the proximal and distal sites originate from differentprotomers. A slightly lower koff value in the dimer can also be
Fig. 4 Active site structures of monomeric (gray) and dimeric (pinkand blue) Mbs in stereo view. The heme and water molecule of mono-meric and dimeric Mbs are shown in the same way as in Fig. 3.
Table 1 Fe–OH2, Fe–His64 and Fe–His93 distances in dimeric andmonomeric Mbs
Fe–OH2 (Å) Fe–His64 (Å) Fe–His93 (Å)
Dimer 2.10 4.25 (His64A–FeB) 2.052.09 4.23 (His64B–FeA) 2.03
Monomera 2.17 4.41 2.12
a PDB: 1WLA.
Fig. 5 1H NMR spectra of monomeric and dimeric metMbs. (a) Nativemonomeric metMb; (b) dimeric metMb produced from monomericmetMb; (c) monomeric metMb reconstituted from monomeric apoMb;(d) dimeric metMb produced from reconstituted monomeric Mb withmixed heme orientations; (e) dimeric metMb reconstituted from dimericapoMb; (f ) monomeric metMb produced by dissociation of dimeric Mb.Samples for spectra (c) and (d) were prepared together and separated bygel chromatography using a FPLC column at the final stage. The peaksA–K in spectrum (a) have been assigned as A: 8-CH3, B: 5-CH3, C: 7-Hα, D: 3-CH3, E: 6-Hα, F: 1-CH3, G: 4,6-Hα, H: heme meso protons, I:2,7-Hα, and J, K: 6,7-Hβ.
43,44 Measurement conditions: sample concen-tration (heme unit): 0.5–1 mM; buffer: 50 mM potassium phosphate;pD: 7.0; temperature: 25 °C.
Fig. 6 Oxygen equilibrium curves of monomeric and dimeric horseMbs. Best-fitted curves using the Hill equation are indicated by brokenlines for monomeric (red) and dimeric (blue) Mbs. Measurement con-ditions: Mb concentration (heme unit): 60 μM; buffer: 50 mM potassiumphosphate; pH: 7.0; temperature: 20 °C.
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attributed to the more rigid structure in the dimer, where thebound oxygen molecule may be stabilized by a rigid hydrogenbond with the protein surrounding. However, the slight deviationof the distal pocket may also lower the koff value, since theshorter distance of ∼0.2 Å between the heme iron and His64 in
the dimer compared to the monomer (Table 1) may provide astronger hydrogen bond. These results show that the active sitestructure is similar between the monomer and dimer, governingthe oxygen binding property, but the regulation of the hemeorientation and oxygen dissociation may be affected not only bythe active structure but also by the decreased fluctuation of theprotein moiety around the active site. However, dimeric Mbexhibited no cooperativity for oxygen binding, despite the pres-ence of two oxygen binding sites in one dimer.
The domain-swapped dimeric structure of serpin has beensolved, and runaway domain swapping (successive domainswapping) of two β-strands for polymerization has beensuggested from the dimeric structure.15,50 Higher order oligomershave been detected in RNase A, and runaway domain swappinghas been suggested as a mechanism for the formation offibrils of RNase A (3D domain-swapped zipper-spine model).51
In dimeric and trimeric cyt c, the heme–Met coordination iscleaved and cyt c forms polymers by successively swapping itsC-terminal domain.16 Although the heme–His bond of Mb iscleaved during dimerization, the new extended α-helix indimeric Mb needs to partially unwind for formation of trimersand higher order oligomers, which may make it difficult for Mbto form domain-swapped polymers.
Conclusions
Dimeric Mb with a domain-swapped structure is formed from itsmonomer by cleaving the heme–protein bond and constructingthe proximal and distal sites of the heme active sites by differentprotomers. The dimer exhibited a slightly higher oxygen bindingaffinity than that of the monomer, but showing no cooperativityfor oxygen binding. The oxygen binding rate constant is main-tained in the dimer due to the similar active site structures in themonomer and dimer, although the heme regulation and oxygendissociation velocities differed from those of the monomer. Thepresent results show that domain swapping is a common charac-ter for many proteins, including heme proteins, and could berelated to the initial aggregation step of proteins. Domain swap-ping may also be used to design protein oligomers, since oxygenbinding was observed in domain-swapped dimeric Mb.
Acknowledgements
We thank Mrs Koji Aoki and Makoto Amagai for preliminaryexperiments. We are also grateful to Mr Leigh McDowell forhis advice during manuscript preparation. Diffraction datawere collected at the Osaka University beamline BL44XU atSPring-8 equipped with MX225HE (Rayonix), which is finan-cially supported by Academia Sinica and National SynchrotronRadiation Research Center (Taiwan, ROC). This work was par-tially supported by Grants-in-Aid for Scientific Research fromMEXT (Priority Areas, No. 23107723 (S.H.); GCOE Program(Y.H.)) and JSPS (Category B No. 21350095 (S.H.); 18GS0207(Y.H.)). This study was also supported by JST (S.H.), SankyoFoundation of Life Science (S.H.), Toray Science Foundation(S.H.), and the Japanese Aerospace Exploration AgencyProject (Y.H.).
Fig. 7 Kinetic measurements of horse oxyMb. (a) Flash-photolysis ofdimeric oxyMb. Absorbance changes at 435 nm by 532 nm pulseirradiation under various oxygen concentrations are shown. Single expo-nential best-fitted curves are indicated by red broken lines. (Inset) Plotsof kobs vs. [O2] for the reaction of the heme with oxygen, together withthe least-squares-fitted line according to eqn (1); (b) stopped-flowmeasurements of monomeric and dimeric oxyMbs. Absorbance changesat 435 nm after mixture with dithionite are shown. Single exponentialbest-fitted curves are indicated by broken lines for monomeric (red) anddimeric (blue) Mbs. Experimental conditions: sample concentration: 20(a) and 5 μM (heme unit) (b); buffer: 50 mM potassium phosphate; pH:7.0; temperature: 20 °C; laser pulse power: 15 mJ; laser pulse frequency:10 Hz; [O2]: 0.28, 0.56, 0.70, 0.91, and 1.39 mM (remaining gas is N2).
Table 2 Oxygen binding (kon) and dissociation (koff) rate constants ofmonomeric and dimeric Mbsa
Species kon × 10−6 (M–1 s−1) koff (s–1)
Monomer Horse 12b 12 ± 1Sperm whale 14 ± 3c 12 ± 2
Dimer Horse 14.0 ± 0.7 8 ± 1
aAt pH 7.0 and 20 °C. bRef. 48. cRef. 47.
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