Báo cáo Y học: The a1b1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen Further evidence from the iron valency hybrids potx

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Báo cáo Y học: The a1b1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen Further evidence from the iron valency hybrids potx

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The a1b1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen Further evidence from the iron valency hybrids Jun pei Yasuda 1 , Takayuki Ichikawa 1 , Mie Tsuruga 1 , Ariki Matsuoka 2 , Yoshiaki Sugawara 3 and Keiji Shikama 1,4 1 Biological Institute, Graduate School of Science, Tohoku University, Sendai, Japan; 2 Fukushima Medical University, Fukushima, Japan; 3 Hiroshima Prefectural Women's University, Hiroshima, Japan; 4 PHP Laboratory for Molecular Biology, Sendai, Japan When the a and b chains were separated from human oxyhemoglobin (HbO 2 ), each individual chain was o xidized easily to the ferric form, their rates being a lmost the same with a very strong acid-catalysis. In the HbO 2 tetramer, o n the other hand, both chains become considerably resistant to autoxidation over a wide range of pH values (pH 5±11). Moreover, HbA showed a biphasic autoxidation curve containing the t wo rate co nstants, i.e. k f for the fast ox idation due to the a chains, and k s for t he slow oxidation to the b chains. The k f /k s ratio increased from 3.2 at pH 7.5±7.3 at pH 5.8, but became 1 : 1 at pH values higher than 8.5. In the present work, we used the valency hybrid tetramers such a s (a 3+ ) 2 (bO 2 ) 2 and (aO 2 ) 2 (b 3+ ) 2 , and demonstrated that the autoxidation rate of either the a or b chains (when O 2 - ligated) is independent of the valency state of the corre- sponding counterpart chains. From these results, we have concluded that the formation of the a1b1ora2b2 contact suppresses remarkably the au toxidation rate o f the b chain and thus plays a key role in stabilizing t he HbO 2 tetramer. Its mechanistic details were also given in terms of a nucleophilic displacement of O 2 ± from the FeO 2 center, and the emphasis was placed on the proton-catalyzed process p erformed by the distal histidine residue. Keywords: Hb oxidation; chain nonequivalence; valency hybrids; a1b1 contact; a cid-catalysis. The reversible and stable binding of molecular oxygen to the heme iron(II) is the b asis of hemoglobin function. However, the oxygenated form of hemoglobin, as well as of myoglo- bin, is known to be oxidized easily to the ferric met-form, which cannot bind dioxygen and is therefore physiologically inactive, with generation of the superoxide anion [1±7]. In this reaction, human oxyhemoglobin (HbO 2 )showsa biphasic autoxidation curve containing the two rate con- stants, a fast one due to the a chains and a slow one for the b chains, respectively [3]. Such chain heterogeneity could be maintained even in the low concentrations of hemoglobin corresponding to appreciable dissociation into a1b1and a2b2 dimers [5]. When the a and b chains are separated from the HbO 2 tetramer, both chains were oxidized much more rapidly than those in the tetrameric parent, and become freed from their rate differences 1 overthewiderange of pH 5±11 [8]. These recent new ®ndings h ave led us to conclude that the formation of the a1b1ora2b2 contact produces a conformational constraint in the b chain where- by the distal (E7) histidine at position 63 is tilted slightly away from the boun d dioxygen, so as to prevent the proton- catalyzed displacement of O 2 ± from the FeO 2 center by an entering water molecule. The b chain s have thus acquired a remarkably d elayed oxidation rate in the HbO 2 tetramer, and this is the origin of such chain heterogeneity found in the hemoglobin autoxidation at acidic pH [8]. To further characterize the nature of the a1b1ora2b2 interface in stabilizing the heme-bound dioxygen, we have constructed iron valency hybrid hemoglobins, and studied their autoxidation behavior at several different pH values as compared with the native or reconstructed HbO 2 .Such examinations seem to be of primary importance, not only for a full understanding of the molecular mechanism of hemoglobin autoxidation, but also for planning new molecular designs for synthetic oxygen carriers that are highly resistant against t he heme oxidation under physio- logical conditions. Finally, we will revisit the hemoglobin function as seen from the two different types of the ab contact, and try to reconcile the cooperative oxygen binding with the stabilization of the bound dioxygen. With respect to this, we will also give possible implications for the unstable hemoglobin mutants leading to the formation of Heinz bodies in red blood cells, resulting in hemolytic anemia. MATERIALS AND METHODS Chemicals Sodium p-hydroxymercuribenzoate (p-MB) was from Sig- ma. Mes, Mops, Hepes, Tris and Caps for buffer systems, 2-mercaptoethanol, and all other chemicals were of reagent grade from Wako Pure Chemicals, Osaka. Solutions were made with deionized and glass-distilled water. Correspondence to K. Shikama, PHP Laboratory for Molecular Biology, Nakayama-Yoshinari 1-16-8, Sendai 989±3203, Japan. Fax: + 81 22 278 6180, E-mail: shikama@mail.cc.tohoku.ac.jp Abbreviations: p-MB, sodium p-hydroxymercuribenzoate. (Received 22 August 2001, revised 23 October 2001, accepted 29 October 2001) Eur. J. Biochem. 269, 202±211 (2002) Ó FEBS 2002 Preparation of human oxyhemoglobin Human hemoglobin A was prepared from freshly drawn blood (30 mL each t ime) by the method of Williams & Tsay [9], with our previous speci®cations [5,8]. The major band of HbA, which was developed on a DEAE-cellulose column (3.5 ´ 12 cm), was eluted out completely wi th 20 m M Hepes buffer at pH 7.9. The HbO 2 solution was then concentrated by centrifugation in a Centriprep-10 tube (Amicon), and kept at low temperature (4 °C) until use. The concentration of hemoglobin was determined as heme, after conversion into cyanomet form, using the absorption coef®cient of 10.4 m M )1 ácm )1 at 540 nm. This value was obtained on the basis of t he pyridine hemo- chromogen method [10]. Isolation of mercuribenzoated a and b chains All separations were carried out with fresh HbO 2 solutions at low temperature (0±4 °C) by a two-column method. The procedure was essentially the same as described by Geraci et al . [11] and by Turci & McDonald [12], with our previous speci®cations [8]. Each time, p-MB (100 mg) was dissolved in 2 mL of 0.1 M NaOH and neutralized with 1 M CH 3 COOH. This was react ed with 10 mL of HbO 2 solution (4±7 m M as heme) in 50 m M phosphate buffer, pH 6.0, and in the presence of 0.1 M NaCl. After passing through a Sephadex G -25 column ( 2.5 ´ 40 cm), the mercurated HbO 2 solution was applied on a DEAE-cellulose column (3.5 ´ 12 cm) to elute out the a p-Mb chains, or on a CM-cellulose column (3.5 ´ 12 cm) for the b p-Mb chains. In each case the counterpart chain had remained on the top of the column. Regeneration of SH groups from mercuribenzoated a and b chains To recover sulfhydryl groups from the mercuribenzoated protein, 75 mL of the a p-Mb or b p-Mb solution ( % 200 l M as heme) were treated with 20 m M 2-mercaptoethanol for 10minin10m M phosphate buffer at 0 °C, as described previously [8]. The mixture (% 150 mL) was placed on a CM-cellulose column (2.5 ´ 6cm)forthea chain, or on a DEAE-cellulose column (5 ´ 6cm) for the b chain, to remove excess amounts o f the reagent. After washing each column with a large volume of the b uffer alone, the regenerat ed a or b chains were eluted out complete ly as the oxy-form by changing the buffer, and kept stably in liquid nitrogen until use. The concentration of each separated chain was determined, after conversion into cyanomet- form, using the following absorption coef®cients at 540 nm: 10.5 m M )1 ácm )1 for the a chain and 11.2 m M )1 ácm )1 for the b chain. These values were obtained on the basis of the pyridine hemochromogen method [10]. Titration of SH groups According to the method of Boyer [13], free sulfhydryl groups of the regenerated a or b chains were tit rated spectrophotometrically at 250 nm with p-hydroxy- mercuribenzoate in 0.1 M Mops buffer, pH 7.0. The result- ing contents were 1.0 (1.05  0.08) for the a chain and 2.0 (2.01  0.08) for the b chain, respectively, as might be expected from the number of cysteines located at positions a104(G11), b93 (F9) and b112(G14) for HbA. Preparation of valency hybrid hemoglobins from separated a and b chains Reconstructed HbA ( a O 2 ) 2 ( b O 2 ) 2 . A 2-mL solution of the oxygenated a chain (% 300 l M ) was mixed with an equal volume of the b chain (% 300 l M )in10m M phos- phate buffer a t pH 6.8. The mixed solution was then applied to a C M-cellulose column (2.5 ´ 3 cm) equilibrated with the same buffer. After a small peak of unassociated bO 2 chains passed through the column, the buffer was changed to 20 m M Hepes ( pH 7.9) to completely elute out the major peak of the reconstructed HbO 2 . Under this condition, a small quantity of unassociated aO 2 chains remained on the top of the column . Valency hybrids ( a 3+ ) 2 ( b O 2 ) 2 and ( a O 2 ) 2 ( b 3+ ) 2 .For conversion of the separated a or b chains from the oxy form to the ferric met-form, 2.5 m L of each solution (% 0.5 m M as heme) were oxidized with 1.5 m M potassium ferricyanide in 0.1 M phosphate buffer, pH 6.8, and in the presence of 10% (v/v) glycerol. To remove the residual oxidizing agent, the resultant met-species was immediately passed through a Sephadex G-25 column (2.5 ´ 10 cm) equilibrated with 10 m M phosphate buffer, pH 6.8. All these procedures were carried out at low t emperature (0±4 °C) to avoid hemi- chrome formation as w ell as protein denaturation. In preparing the valency hybrid (a 3+ ) 2 (bO 2 ) 2 ,a1.8-mL solution of ferric a chains (% 150 l M )wasmixedwith 360 lLofbO 2 solution (% 750 l M ). The resultant mixture was then applied to a CM-cellulose column (2.5 ´ 3cm) equilibrated with 1 0 m M phosphate buffer, pH 6.8. After a small quantity of unassociated bO 2 chains passed through the column, the buffer was changed to 50 m M Hepes (pH 7.9) to elute out the major peak of the hybrid tetramer (a 3+ ) 2 (bO 2 ) 2 . Under this condition, unassociated a 3+ chains had r emained on the top of the column. E ssentially the same p rocedure can be used for the preparation of another hybrid (aO 2 ) 2 (b 3+ ) 2 . In this case, a small qu antity of unassociated b 3+ chains passed through a CM-cellulose column (2.5 ´ 3cm)with10m M phosphate buffer, pH 6.8. The h ybrid t etramer was then e luted out completely by changing the buffer to 20 m M Hepes, pH 7.9. Autoxidation rate measurements According to our previous procedures [5,8], the rate of autoxidation of HbA and its derivatives was measured at 35 °Cin0.1 M buffer containing 1 m M EDTA. To meet various hemoglobin concentrations required, a 1-cm cell was used for a 3-mL sample containing 10±50 l M heme, while a 1-mm cell was employed for a 0.5-mL sample containing 300 l M heme. For spectrophotometry, the reaction mixture was quickly transferred to a quartz cell held at 35  0.1 °C, and changes in the absorption spectrum from 450 to 700 nm were recorded on the same chart at measured intervals of time. For separated a and b chains, the rate measurement w as usually carried out with 10 l M protein (as heme) and in the presence of 20% (v/v) glycerol. As the ®nal state of each run, the hemoglobin was completely converted to t he ferric met-form by the addition Ó FEBS 2002 The a1b1 contact in HbO 2 autoxidation (Eur. J. Biochem. 269) 203 of potassium ferricyanide. The buffers used were Mes, maleate, Mops, and Caps. The pH of the reaction mixture was carefully checked, before and after the run, with a Hitachi±Horiba pH meter (Model F-22). Spectrophotometric measurements Absorption spectra w ere recorded in a Hitachi two-wave- length doub le-beam spectrophotometer (model 557, U-3210 or U-3300) or in a B eckman spectrophotometer (model DU-650), each being equipped with a thermostatically controlled (within  0.1 °C) cell holder. Curve ®ttings Biphasic autoxidation curves were analyzed by an iterative least-squares method on a computer (NEC PC-9821 V12) with graphic display, according to our previous speci®ca- tions [5,8]. RESULTS Biphasic nature of the autoxidation reaction for human HbO 2 In air-saturated buffers, the oxygenated form of HbA is oxidized easily to the ferric met-form (metHb) with generation of the superoxide anion [1,2], HbO 2 3 k obs metHb  O À 2 1 where k obs represents the ®rst-order rate constant observed at a given pH in terms of the constituent chains. This autoxidation reaction can be monitored by the spectral changes with time, after fresh HbO 2 wasplacedin0.1 M buffer c ontaining 1 m M EDTA at 35 °C. The spectra evolved to the ®nal state, which was identi®ed as the usual ferric met-form, with a s et of isosbestic points. Consequent- ly, the process was followed by a plot of experimental data as ±ln([HbO 2 ] t /[HbO 2 ] 0 )vs.timet, where the ratio of HbO 2 concentration a fter time t to that at time t  0canbe obtained by the absorbance changes at 576 nm for the a-peak of human HbO 2 . Figure 1 shows such examples of the ®rst-order plot for the autoxidation reaction of human HbO 2 at two different pH values. At pH 6.2, HbA showed a biphasic curve that can be described completely by the ®rst-order kinetics containing two rate constants as follows: HbO 2  t HbO 2  0  P Á expÀk f Á t 1 À PÁexpÀk s Á t2 In this equation, a fast ® rst-order rate constant k f is attributed to the a chains and a slow rate constant k s is for the b chains in the H bO 2 tetramer. P is the molar fraction of the rapidly reacting hemes. This conclusion is based on the rapid chain separation experiment of partially (30%) oxidized HbO 2 on a 7.5% polyacrylamide gel [8], this being in good agreement with that of Mansouri & Winterhalter [3]. By iterative least-squares procedures inserting various values for k f and k s into Eqn (2), the best ®t to the experimental data was obtained as a function of time t.In these computations, the initial value for each of the rate constants was taken f rom the corresponding slo pe of a biphasic curve (as delineated in Fig. 1 by two dotted lines), and was re®ned by the step sizes of 0.01±0.001 h )1 to ®nd out the best values of k f and k s , according to our previous speci®cations [5]. The value of P was a lso allowed to vary a large range (from 0.40 to 0.60) in all cases. In this way, the following param eters were established a t pH 6.2; k f  0.82  0.03 ´ 10 )1 h )1 , k s  0.13  0.01 ´ 10 )1 h )1 , and P  0.52  0.04 i n 0.1 M Mes buffer at 35 °C. At pH 9.2, on the other hand, the reaction could be described completely by a single ®rst-order rate constant of 0.99  0.02 ´ 10 )2 h )1 (i.e. k f  k s with P  0.50) in 0.1 M Caps buffer at 35 °C. Table 1 represents such examples for a pair of the ®rst- order rate constants deduced from each autoxidation curve at different values of pH. From the k f /k s ratios, one can easily realize the biphasic nature emerged in the autoxida- tion of HbA. Moreover, we have found that such chain heterogeneity can be kept even in very diluted concentra- tions of hemoglobin from 1.0 ´ 10 )3 M to 3.2 ´ 10 )6 M as heme [5]. When the HbO 2 sample is diluted 2 , the tetrameric species is known to dissociate into ab dimers along the a1b2 or a2b1 interface, so that the dimers formed are of the a1b1 or a2b2 type [14,15]. From these results, we can unequiv- ocally conclude that the remarkable stability of the b chain 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 - ln { [ HbO 2 ] t / [HbO 2 ] 0 } 0 10203040506070 Time (h) pH 9.2 pH 6.2 k f k s HbA(α 2 β 2 ) Fig. 1. First-order plots for the autoxidation reaction of human HbO 2 in 0.1 M buer at 35 °C. Theratemeasurementswerecarriedoutwith 75 l M HbO 2 (300 l M as hem e) i n t he presence of 1 m M EDTA. E ach curve (±±) was obtained by a least-squares ®tting to the experimental points (s), based on Eqn (2). At pH 6.2, HbA showed a b iphasic autoxidation curve containing two rate constants, k f and k s , respec- tively. At pH 9.2, however, the reaction was monophasic. The buer used was Mes for pH 6.2 and Caps for pH 9.2. 204 J. p. Yasuda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 against the acidic autoxidation must have been produced by the formation of the a1b1ora2b2 c ontact. To see more quantitatively the effect of the a1b1ora2b2 contact on the autoxidation reaction, our next step was to construct the iron valency hybrid tetramers containing either the a or b chains in the ferric state, and to examine for their stability properties as compared with the native H bO 2 and its separated chains. Preparation of the valency hybrid hemoglobins and their autoxidation behavior By mixing equivalent amounts of the separated a and b chains whose sulfhydryl groups were completely recovered, we have prepared the reconstructed HbO 2 and its valency hybrid tetramers. Figure 2 shows such an example for the chromatographic separation of the hybrid tetramer (a 3+ ) 2 (bO 2 ) 2 from unassociated chains. In this case, the mixed chain solution (2.2 mL) was applied to a CM- cellulose column (2.5 ´ 3 cm) that had been equilibrated with 10 m M phosphate b uffer, pH 6.8. After a small band of the unassociated bO 2 passed through t he column, t he buffer was changed to 50 m M Hepes, pH 7.9, to obtain the major peak of the hybrid tetramer. When the iron valency hybrids are placed in air-saturated buffers, the oxygenated chains o f e ach tetramer are oxidized easily to the ferric met-form. Figure 3 shows such an example of the spectral changes with time for the autoxi- dation reaction of hybrid Hb (a 3+ ) 2 (bO 2 ) 2 in 0.1 M Mes buffer pH 6.2, and in the presence of 1 m M EDTA at 35 °C. In this tetramer, even if freshly prepared, the a-peak (at 577 nm) was always lower than the b-peak (at 541 nm) with an absorbance ratio of a/b  0.90, this being in contrast to a value of 1.06 for the native or reconstructed HbO 2 .The Table 1. Comparison of the two rate constants involved in the autoxidation reaction of human HbO 2 at various pH values and 35 °C. pH k obs (h )1 ) k f /k s Concentration (l M as heme)k f k s 5.8 0.24 0.33 ´ 10 )1 7.3 300 6.2 0.82 ´ 10 )1 0.13 ´ 10 )1 6.3 300 6.5 0.56 ´ 10 )1 0.90 ´ 10 )2 6.2 300 7.5 0.16 ´ 10 )1 0.50 ´ 10 )2 3.2 300 9.0 0.48 ´ 10 )2 0.48 ´ 10 )2 1.0 300 9.2 0.99 ´ 10 )2 0.99 ´ 10 )2 1.0 300 9.6 0.25 ´ 10 )1 0.25 ´ 10 )1 1.0 50 0 2 4 6 8 10 12 0102030 Fraction Number (4 ml / tube) 0 2 4 6 8 10 12 A 280 ( ) CM-cellulose Valency hybrid 22 ( ) 3+ ( O 2 A 415 ( ) 3+ O 2 ) Fig. 2. CM-cellulose chromatography of the valency hyb rid (a 3+ ) 2 (bO 2 ) 2 tetramer. The e quim olar mixture (2.2 mL) of the a 3+ and bO 2 chains was applied to a CM-cellulose column (2.5 ´ 3cm) equilibrated with 10 m M phosphate buer, pH 6.8. A small band of unassociated bO 2 chains passed through the column with the same buer. To elute out the major peak o f the hybrid tetramer, the b uer was changed to 50 m M Hepes (pH 7.9) at the point indicated by the ®rst arrow. The unassociated a 3+ chains coul d be remov ed by the addition of 1 M NaCl as indicated by the second arrow. The protein and the heme p rotein leve ls were mon itored by th e abso rbances at 280 nm (s) and 415 nm (d), respectively. 0 0.1 0.2 0.3 0.4 0.5 Absorbance 450 500 550 600 650 700 Wavelength (nm) 22 pH 6.2 Valency hybrid Start Finish (α 3+ )(β O 2 ) Fig. 3. Spectral changes with time for the autoxidation of valency hybrid Hb (a 3+ ) 2 (bO 2 ) 2 in 0.1 M Mes buer at pH 6.2 and 35 °C. Sc ans were made at 270-min intervals in the presence of 1 m M EDTA. The ®nal spectrum was for the acidic metHb with a s et of isosb estic points at 526 and 592 nm. Hb concentration: 50 l M as heme. Ó FEBS 2002 The a1b1 contact in HbO 2 autoxidation (Eur. J. Biochem. 269) 205 hybrid tetramer also exhibited very intensive charge-transfer bands at 501 nm as well as 631 nm. All these features seemed to be produced by a spectral overlapping of ferric a 3+ chains. Furthermore, the reaction spectra evolved to the ®nal state of a run, which was identi®ed as the usual acidic (or aquo) metHb. If the contribution o f ferric a 3+ chains could be subtracted from the oxidation spectra o f the (a 3+ ) 2 (bO 2 ) 2 tetramer on a computer, we may have the spectral changes that can b e ascribed to the autoxidation of the b chains alone. Such computations have disclosed that the reaction started from the fully oxygenated b chains with an absor- bance ratio of a/b  1.05, and that the oxidation proceeded to the usual acidic met-form with a set of isosbestic points at 526 and 592 nm, as depicted in Fig. 4. This process was therefore followed by absorbance changes at 578 nm for the a-peak o f the b chain, an d could b e described completely by a single ®rst-order rate constant of k obs  0.19 ´ 10 )1 h )1 . This oxidation rate is e ssentially the same with that of t he b chains in the HbO 2 tetramer (see Fig. 1). In separated chain solutions, the p rotein is known to exist in a n equilibrium of a ÀÀB AÀÀ a 2 or b ÀÀB AÀÀ b 4 , respec- tively. Under our experimental conditions (10±25 l M as heme), the monomeric form (87%) was predominant in the a chain, while the tetrameric form (99%) was for the b chain. This estimation was made on the basis of the results by McDonald et al. [16]. In a previous paper [8], we have reported that the separated a and b chains are both oxidized much more rapidly than those in parent HbO 2 tetramer over the wide range of pH 5±10. Figure 5 shows such spectral changes with t ime f or the autoxidation of separated bO 2 chains in 0.1 M maleate buffer, pH 6.2, and inthepresenceof1m M EDTA plus 20% (v/v) glycerol at 35 °C. The oxidation began with an absorbance ratio of a/ b  1.04, and proceeded very rapidly with a ®rst-order rate constant of k obs  0.10 h )1 . T his rate is several times higher than the c orresponding k s value for the b chains either in the hybrid tetramer (a 3+ ) 2 (bO 2 ) 2 or reconstructed HbO 2 . Moreover, the ®nal state of the run was not for the usual acidic met-form but for an admixture with hemi- chrome. For the oxidation product of separated b chains, we have already carried out 8K EPR a nalysis i n 10 m M maleat e buffer at pH 6.2 [8]. In addition to a high spin EPR spectrum attributed to the usual aqua-met species with g values of 5.86 and 1.99, the b chains exhibited a low spin spectrum with g 1  2.77, g 2  2.27, and g 3  1.68, which differentiates this species f rom that o f the hydroxid e-type complex. According to Rifkind et al.[17],suchlowspin complexes characterized by the h ighest g value in the range of 2.83 ±2.75 and the lowest g value i n t he range of 1.69±1.63 have been designated as complex B, indicating crystal ®eld parameters of the reversible hemichrome. They also suggest that the bis-histidine complex B may s till have a w ater molecule retained in the heme pocket, and therefore in solution it is in rapid equilibrium with the h igh spin a quo- 0 0.1 0.2 0.3 0.4 Absorbance 450 500 550 600 650 700 Wavelength (nm) 2 in pH 6.2 Start Finish 2 2 (β O 2 ) (α 3+ ) 2 (β O 2 ) Fig. 4. Spectral changes w ith time for the autoxidation of the bch ains of valencyhybridHb(a 3+ ) 2 (bO 2 ) 2 in 0.1 M Mes buer at pH 6.2 and 35 °C. Spectral subtraction o f the (a 3+ ) 2 part from the hybrid Hb w as made at 270-min intervals on a computer. The bc hains were found to oxidize from the fully o xygenated form to the usual acidic met-form. Heme concentration: 25 l M . 0 0.1 0.2 0.3 0.4 Absorbance 450 500 550 600 650 700 Wavelength (nm) 4 pH 6.2 Finish Start (β O 2 ) Fig. 5. Spectral changes with time for the autoxidation of separated b chains in 0.1 M maleate buer at pH 6.2 and 35 °C. Sc ans wer e made at 70-min intervals in the presence of 1 m M EDTA and 20% (v/v) glyc- erol. The ®nal spectrum was not for the acidic met-form , b ut an ad- mixture with hemichrome having a peak at 53 0 nm an d a s houlde r near 560 nm. Heme concen tration: 25 l M . 206 J. p. Yasuda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 complex [18]. As shown in Fig. 5, the molar fraction of t he hemichrome (complex B) was estimated to be 75% at pH 6.2. Furthermore, Borgstahl et al.[19]reportedthe 1.8 A Ê structure of carbonmonoxy-b 4 (COb 4 )tetramerof human hemoglobin, and compared subunit±subunit con- tacts between three t ypes of interfaces (a1b1, a1b2and a1a2) of HbO 2 and the corresponding COb 4 interfaces. As a result, they found that, in contrast to the stable b1b4 interface, the b1b2 interface of the COb 4 tetramer is less stable and mo re loosely p acked than its a1b1 counterpart in HbO 2 . At all rates, the present spectral examinations clearly indicate that the formation of the a1b1ora2b2 contact suppresses remarkably the a cidic a utoxidation o f the b chain, and prevents its hemichrome formation as well. This is true no matter which valency state the partner a chains may take, the oxy-form or the ferric met-form. Unlike separated b chains, the spontaneous formation of hemichrome was at variance with separated a chains in the pH range 5±10. T herefore, in another valency hybrid (aO 2 ) 2 (b 3+ ) 2 the oxidation of the a ch ains was found to start with an absorbance ratio of a/b  1.07 and to p roceed as usual a s in the HbO 2 tetramer. Under our experimental conditions, the valency hybrid hemoglobins were suf®ciently stable for the rate measurements over a long period of time. In separated a and b chains, on the other hand, the addition of 20% (v/v) glycerol was most effective in preventing occasional precipitations. Kinetic analysis of the autoxidation reaction of valency hybrid hemoglobins Figure 6 represents ®rst-order plots to show wide differences in the autoxidation rate of the b chain , when it exists as the separa ted (bO 2 ) 4 , valency hybrid (a 3+ ) 2 (bO 2 ) 2 , and reco n- structed HbO 2 tetramers in 0.1 M Mes buffer at pH 6 .2 and 35 °C. In this way, all the spectrophotometric data were subjected to ® rst-order kinetics using Eqn (2). The resulting rate constants f or the native, separated, reconstructed, and valency hybrid hemoglobins are summarized in Tables 2±4 at three different values of pH. At pH 6.2, for example, the HbO 2 tetramer exhibited a biphasic autoxidation curve with the rate constants of k f  0.82 ´ 10 )1 h )1 and k s  0.13 ´ 10 )1 h )1 . Almost the same oxidation rates were obtained for the reconstructed HbO 2 giving a value of k f /k s  6.1. Among those, the most r emarkable effect was found on the b chain. Separated b chains undergo quite rapid oxidation with a rate constant of k obs  0.10 h )1 ,but this inherent rate was dramatically suppressed i n the reconstructed as w ell as the native HbO 2 . More importantly, such a retarded k s value could be kept almost completely in the valency hybrid (a 3+ ) 2 (bO 2 ) 2 tetramer, too. All these features were essentially the same at other pH values as seen in Tables 3 a nd 4. Certainly, the biphasic nature o f the autoxidation rate became much less steep at pH 7.5, and even disappeared at pH 9.0. Nevertheless, the r ate of oxidation of the separated b chain was markedly reduced by up to 15-fold at pH 7.5 and up to 23-fold at pH 9.0 in the tetrameric hemoglobin, either it is native or reconstructed or valency hybrid species. The similar situation was also found in the a chain, but its effect on the HbO 2 tetramer was much less crucial than the b chain. At pH 9.0, the rate of oxidation of the separated a chain was reduced by up to 16-fold in the HbO 2 tetramer, but such a rate suppression was decreased with increasing hydrogen ion concentration. This is due to the fact that the a chain exhibits a very strong proton-catalysis not only in the separated chain but also in the HbO 2 tetramer. Among those, an unexpected re sult was found at pH 6.2. At present, we do not know exactly why the oxidation rate of the a chains was m ore suppressed in the hybrid tetramer (aO 2 ) 2 (b 3+ ) 2 than in the native HbO 2 . The most probable case was in the hemichrome formation that might have occurredinparttotheb 3+ chains when preparing the corresponding valency hybrid. However, it should be noted that such a v alency state can never occur in the autoxidation reaction of the HbO 2 tetramer, because k f ³ k s at any physiological pH. DISCUSSION In hemoglobin research, the central problem is understand- ing the cooperative binding of m olecular oxygen to the a 2 b 2 tetramer. For human HbA, the a and b chains contain 141 and 146 amino-acid residues, respectively, and a r epresen- tative set of the s uccessive oxygen-binding constants is g iven in terms of mmáHg )1 as follows: K 1  0.0188, K 2  0.0566, 0 0.2 0.4 0.6 0.8 1.0 1.2 - ln { [ HbO 2 ] t / [HbO 2 ] 0 } 010203040 Time (h) in Valency hybrid pH 6.2 2 2 (β O 2 ) (α 3+ ) 2 (β O 2 ) 4 (β O 2 ) 2 (α O 2 ) 2 (β O 2 ) Reconstructed k f k s Fig. 6. First-order plots to show dierent autoxidation rates of the b chain between three dierent hemoglobin derivatives in 0.1 M maleate buer at pH 6.2 an d 35 °C. Each curve (±±) was obtained by a least- squares ®tting to the experimental points, based on Eqn (2). The oxidation of separated b chains could be described by a single rate constant of k obs  0.10 h )1 in the p resence of 20% (v/v) glycerol. This inherent rate was dramatically suppressed not only in reconstructed HbO 2 but also in valency hybrid (a 3+ ) 2 (bO 2 ) 2 as well. Heme co ncen - tration: 25 l M for separated b chains, 300 l M for reconstructed HbO 2 , and 50 l M for valency hybrid Hb. Ó FEBS 2002 The a1b1 contact in HbO 2 autoxidation (Eur. J. Biochem. 269) 207 K 3  0.407 and K 4  4.28 in 0.1 M Bis/Tris buffer con- taining 0.1 M KCl at p H 7.4 an d 25 °C [20]. In this reaction, major d ifferences have been de®ned between deoxyhemo- globin and o xyhemoglobin by c omparing their X -ray crystal structures. These include a movement of the iron atom into the heme plane with a simultaneous change in the orienta- tion of the proximal (F8) histidine, a rotation of the a1b1 dimer relative to the other a2b2 dimer about an axis P by 12±15 degrees, and a translation of one dimer relative to th e other along the P axis by % 1A Ê . The latter two changes are accompanied with sequential breaking of the so-called salt bridges by C -terminal residues [21±25]. Therefore, the two types of the ab contact a re de®ned in the molecule. One is the a1b1(ora2b2) contact involving B, G, and H helices and the GH corner, and other is the a1b2(ora2b1) contact involving m ainly helices C and G and the FG corner [19,24]. When HbA goes from the deoxy to the oxy con®guration, the a1b2anda2b1 contacts undergo the principal changes associated with the cooperative oxygen binding, so that these are named the sliding contacts. At the a1b1anda2b2 interfaces, on the other hand, negligible changes are found insofar as the crystal structure was examined. Consequently, these are called simply the packing contacts, and their role in hemoglobin function was not clear for a very long period of time. To these packing contacts, we have recently assigned a key role in stabilizing the HbO 2 tetramer, as the formation of the a1b1ora2b2 contact greatly suppresses t he autoxidation rate, particularly of the b chains [8]. In the autoxidation reaction of HbO 2 ,aswellasMbO 2 , pH can affect the rate in many d ifferent ways. Recent kinetic and thermodynamic studies of the stability of mammalian oxymyoglobins have shown that the autoxidation reaction is not a simple, dissociative loss of O 2 ± from MbO 2 but is due to a nucleophilic displacement of O 2 ± from MbO 2 by a water molecule or a hydroxyl ion that can enter the heme pocket from the surrounding solvent. The iron is thus converted to the ferric met-form, and the water molecule or the hydroxyl ion remains bou nd to the Fe(III) at the sixth Table 2. Comparison of the autoxidation rate constants between the whole, separated, reconstructed, and hybrid hemoglobins in 0.1 M buer at pH 6 .2 and 35 °C. Hb Sample k obs (h )1 ) Concentration (l M as heme) k f k s Whole HbO 2 0.82 ( 0.03) ´ 10 )1 0.13 ( 0.01) ´ 10 )1 300 Separated chains (aO 2 ) 1 0.89 ( 0.03) ´ 10 )1 ±10 (bO 2 ) 4 ± 0.10 ( 0.01) 10±25 Reconstructed (aO 2 ) 2 (bO 2 ) 2 0.85 ( 0.06) ´ 10 )1 0.14 ( 0.04) ´ 10 )1 300 Hybrid (a 3+ ) 2 (bO 2 ) 2 ± 0.19 ( 0.02) ´ 10 )1 50 (aO 2 ) 2 (b 3+ ) 2 0.77 ( 0.03) ´ 10 )1 ±50 Table 4. Comparison of t he autoxidation rate constants between the whole, separated, reconstructed, and hybrid hemoglobins in 0.1 M buer at pH 9.0 and 35 °C. Hb Sample k obs (h )1 ) Concentration (l M as heme) k f k s Whole HbO 2 0.48 ´ 10 )2 0.48 ´ 10 )2 300 Separated chains (aO 2 ) 1 0.78 ´ 10 )1 ±10 (bO 2 ) 4 ± 0.11 10 Reconstructed (aO 2 ) 2 (bO 2 ) 2 0.67 ´ 10 )2 0.67 ´ 10 )2 50 Hybrid (a 3+ ) 2 (bO 2 ) 2 ± 0.62 ´ 10 )2 50 (aO 2 ) 2 (b 3+ ) 2 0.61 ´ 10 )2 ±50 Table 3. Comparison of t he autoxidation rate constants between the whole, separated, reconstructed, and hybrid hemoglobins in 0.1 M buer at pH 7.5 and 35 °C. Hb Sample k obs (h )1 ) Concentration (l M as heme) k f k s Whole HbO 2 0.16 ´ 10 )1 0.50 ´ 10 )2 300 Separated chains (aO 2 ) 1 0.35 ´ 10 )1 ±10 (bO 2 ) 4 ± 0.75 ´ 10 )1 10 Reconstructed (aO 2 ) 2 (bO 2 ) 2 0.23 ´ 10 )1 0.50 ´ 10 )2 50 Hybrid (a 3+ ) 2 (bO 2 ) 2 ± 0.63 ´ 10 )2 50 (aO 2 ) 2 (b 3+ ) 2 0.63 ´ 10 )2 ±50 208 J. p. Yasuda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 coordinate position so as to form aqua- or hydroxide- metMb. Even the complicated pH-dependence for the autoxidation rate can thereby be explained primarily in terms of the following three types of displacement process [6,7,26±28]: MbIIO 2 H 2 O 3 k 0 MbIIIOH 2 O À 2 3 MbIIO 2 H 2 OH  3 k H MbIIIOH 2 HO 2 4 MbIIO 2 OH À 3 k OH MbIIIOH À O À 2 5 In these e quations, k 0 is the rate c onstant for the basal displacement by H 2 O, k H istherateconstantfortheproton- catalyzed displacement by H 2 O, and k OH is the rate constant for the displacement by O H ± . The extent of contribution of these elementary p rocesses to t he observed o r overall autoxidation rate, k obs in Eqn (1), can vary with the concentrations of H + or OH ± ion. Consequently, the autoxidation rate exhibits a very strong parabolic depen- dence on pH. The reductive displacement of the bound dioxygen as O 2 ± by H 2 O can proceed without any proto- nation, but it has b een clearly shown that the rate is greatly accelerated with the proton assistance by a factor of more than 10 6 mol )1 , as formulated by Eqn (4). In this proton catalysis, the distal histidine, which forms a hydrogen bond to the bound dioxyge n [29], appears t o facilitate the effec tive movement of a c atalytic proton from the solvent to the bound, polarized dioxygen via its imidazole ring and by a proton-relay mechanism [6,7]. In our previous paper [8], such a nucleophilic displace- ment mechanism w as successfully applied to d etailed pH-dependence studies of the k f and k s values, both for the HbO 2 tetramer and its separated chains, at more than 70 different values o f pH from 5 to 11 in 0.1 M buffer at 35 °C. When the a and b chains were separated from the HbO 2 tetramer, e ach individual chain was oxidized much more rapidly than in the p arent HbO 2 , exhibiting a proton- catalyzed displacement process performed by its o wn distal histidine residue with pK a  6.1. At the same time, the oxidation rates of both chains were essentially the same over the wide r ange of pH 5±11, so that their p H- dependences could be formulated in terms of an Ôacid- catalyzed two-state modelÕ. However, this is not the case with the HbO 2 tetramer. The value of k f increased very rapidly with increasing hydrogen ion concentration, in- volving a proton-catalysis by the distal (a58) histidine with pK a  6.2, as with the separated chains. The value of k s also increased with increasing hydrogen ion concentration but much less so than for k f .Rather,thek s value showed a rate saturation behavior with pK a  5.1 on the acidic side. This pH-pro®le was therefore explained as a single dissociation process f or the d istal histidine at position b63, and described in terms of a Ôtwo -state modelÕ without any proton catalysis. Such a unique stability of the HbO 2 tetramer was found to remain even in the low concentra- tions of hemoglobin corresponding to appreciable dissoci- ation into a1b1ora2b2dimers[5]. We have recently proposed that the distal histidine residue can play a dual role in the nucleophilic displace- ment of O 2 ± from MbO 2 or HbO 2 [30]. One is in a proton-relay mechanism via its imidazole ring, as random and undirected access of a proton to the bound dioxygen cannot yield such an enzyme-like, catalytic e ffect on the autoxidation rate of MbO 2 or HbO 2 . Insofar as we have examined for more than a dozen of myoglobins, such a proton-catalyzed process could never be observed in t he autoxidation of myoglobins lacking the usual distal histidine r esidue, no matter what the protein i s, the naturally occurring or the distal His mutant as well [30]. The other role is in the maximum protection of the FeO 2 center against a water molecule or a hydroxyl ion that can enter the heme pocket from the su rrounding solvent. The latter case may be in the b chains of the HbO 2 tetramer. To investigate more exactly the effect of the a1b1ora2b2 contact on t he stability of human HbO 2 ,wehaveusedthis time the v alency hybrid tetramers. As a r esult, the b chain was found to acquire a noticeable resistance against the acidic autoxidation in a manner of contacting with the a chain, no matter which valency state t he latter partner is in, the ferrous or the ferric. These new ®ndings have led us to conclude that the packing contact produces a conforma- tional constraint in the b chain whereby the distal (E7) histidine at position 63 is tilted slightly away from the bound dioxygen, so as to prevent the acid-catalyzed displacement of O 2 ± from the FeO 2 center by an entering water molecule. Thus, the remarkable stability of the HbO 2 tetramer can b e ascribed mainly to the delayed autoxidation of the b chains in acidic pH range. More speci®cally, the b chain h as acquired this stability by blocking out the proton catalysis performed by the distal histidine residue (Eqn 4). Similarly, Shaanan [31] reported the stereochemistry of the iron-dioxygen bond in human HbO 2 by single-crystal X-ray analysis. In the a chain, the distance between N e of His (E7) and the terminal oxygen atom (O-2) is found to be 2.7 A Ê , and the geometry favors a similar hydrogen bond as in oxymyoglobin [29]. In the b chain, however, N e of His (E7) is located further away from both O-2 and O-1 (3.4 and 3.2 A Ê , respectively), i ndicating that the hydrogen bond, even if formed, must be very weak. Recently, Lukin et al.[32] claimed t hat a hydrogen bond is formed between O 2 and t he distal histidine in both a and b chains of human HbO 2 ,as revealed by heteronuclear NMR spectra of the chain- selectively labeled samples. In 0.1 M phosphate buffer at pH 8.0 and 29 °C, the (H e2 ,N e2 ) cross-peaks o f the distal histidyl residues were clearly observed as doublets in the ( 1 H, 15 N) spectrum of HbO 2 ,at 1 H chemical shifts of 4.79 p.p.m. for b63His and 5.42 p.p.m. for a58His. These were taken as an indication that the H e2 proton is stabilized against solvent water exchange by a hydrogen bond between the distal His and the O 2 ligand in both a and b chains. At the same t ime, they reported that much wider separation of 1.17 p.p.m. appears on the H e1 resonances of the two distal histidine r esidues, showing that b63Hisisdifferentfrom a58His in either the orientation o r distance or both, with respect to the heme-bound dioxygen. Such m arked differ- ences between the two distal heme pockets may also b e responsible for our kinetic results of the a and b chains in the HbO 2 tetramer. I n this context, NMR spectra of the separa ted bO 2 chain must be most informative if available, because the autoxidation reaction of the b chain contains a very strong proton-catalysis in the isolated form but not in the HbO 2 tetramer. Ó FEBS 2002 The a1b1 contact in HbO 2 autoxidation (Eur. J. Biochem. 269) 209 As for the dimer, as well as the tetramer, effect on the oxidation rate, our explanations are as follows. At b asic pH, both isolated a and b chains are q uite susceptible to autoxidation. Each heme pocket seems to be suf®ciently open to allow e asier attack of the solvent hydroxyl ion on the FeO 2 center. As a result, there occurs a very rapid formation of hydroxide-metHb, the rate being dependent directly on the concentrations of OH ± ion. In a1b1 dimers, conformational constraints would greatly suppress accessi- bility of the displacing nucleophile to each heme pocket. However, OH ± ion is one of the strongest nucleophiles in vivo, so that practically no rate difference was observed between the a and b chains, resulting in the monophasic autoxidation rate over the basic pH range. To the acidic autoxidation, essentially the same explanation is valid. At acidic pH, the displacing nucleophile is an entering water molecule, but its concentration is always taken as 55.5 M in aqueous solution. Therefore, participation of the catalytic proton should be of p rimary importance to give a strong pH dependence on t he autoxidation rate. As the HbO 2 sample is diluted, the heme pocket of the a chain becomes freed from conformational c onstraints that would decrease accessibility of a water molecule and a catalytic proton as well. As a consequence, the rate of displacing O 2 ± from the FeO 2 approaches to that of the isolated a chain. In contrast to this, the heme pocket of t he b chain still obstructs easy access of a water molecule as well as a proton, so that the b chains can keep a constant resistance against the acidic autoxidation, even if the HbO 2 tetramer is diluted into ab dimers. Indeed, this is the most characteristic feature of hemoglobin autoxidation. In relevance to a clinical aspect, it should be noted that a quite large number of unstable hemoglobins have been reported so far [24,33]. M any of the mutants which occur at the a1b2 interface have altered oxygen af®nity, but bulk of evidence suggests that the a1b1 i nterface is much more important in maintaining normal hemoglobin stability than is the a1b2 interface. As a matter of fact, hemolytic anemia is known to result from substitutions affecting the a1b1 interface or the heme pocket. If such mutations occur, the heme iron will be more easily oxidized, and a sequence of events leads to the globin precipitation or Heinz body formation in r ed blood cells that causes hemolytic anemia. Typical examples of such variants are: E [b26(B8)Glu ® Lys], Volga [ b27(B9)Ala ® Asp], Genova [b28(B10)- Leu ® Pro], St Louis [b28(B10)L eu ® Gln], Tacoma [b30(B12)Arg ® Ser], Abraham Lincoln [b32( B14)Leu ® Pro], Castilla [b32 (B14 )Leu ® Arg], Philly [b35(C1)Tyr ® Phe], Rush [b101(G3)Glu ® Gln], Peterborough [b111(G13)Val ® Phe], Madrid [b115(G17)Ala ® Pro], Khartoum [b124(H2)Pro ® Arg],J.Guantanamo[b128- (H6)Ala ® Asp], Wien [b130(H8)Tyr ® Asp], Leslie [b131(H9)Gln ® deleted], Torino [a43(CD1)Phe ® Val], L.Ferrara [a47(CD5)Asp ® Gly], Setif [a94(G1)Asp ® Tyr], St. Lukes [a95(G2)Pro ® Arg]. Surprisingly, almost all of these pathological mutations are f ound on the b chain, especially in the a1b1 contact regions. It follows from our present study that in these v ariant hemoglobins the a1b1 c ontact becomes loose or disruptive, and that the autoxidation of the b chain must have been ac celerated, just like the separated one, with irreversible hemichrome formation. In conclusion, human hemoglobin seems to differentiate the two types of the ab contact quite properly for its functional properties. The a1b2ora2b1 contact is associ- ated with the cooperative oxygen binding, whereas the a1b1 or a2b2 contact is use d for controlling the stability of the bound O 2 . We can thus form, f or the ®rst time, a uni®ed picture of hemoglobin function by closely integrating the reversible and the s table binding of mo lecular oxygen by iron(II) in protic, aqueous solvent. ACKNOWLEDGEMENT This work was partly supported by grants-in-aid for Scienti®c Research (07640896 & 10440248) from the Ministry of Education, Culture and Science of Japan. REFERENCES 1. Wever, R., Oudega, B. & Van Ge lder, B.F. (1973) Generation of superoxide radicals during the autoxidation of mammalian oxyhemoglobin. Biochim. Biophys. Acta 302, 475±478. 2. G otoh, T. & Shikama, K. (1976) Generation of the superoxide radical during the autoxidation of oxymyoglobin. J. Biochem. (Tokyo) 80, 397±399. 3. Manso uri, A. & Winterhalter, K.H. (1973) Nonequ ivalence of chains in hemoglobin oxidation. Biochemistry 12, 4946±4949. 4. Zhang, L., Levy, A. & Rifkind, J.M. (1991) Autoxidation of hemoglobinenhancedbydissociationintodimers.J. Biol. Chem. 266, 24698±24701. 5. Tsuruga, M. & Shikama, K. (1997) Biphasic nature in the autoxidation reaction of human oxyhemoglobin. Biochim. Biophys. Acta 1337, 96±104. 6. Shikama, K. (1988) Stability properties of dioxygen-iron(II) porphyrins: an overview f rom simple complexes to myoglobin. Coordination Chem. Rev. 83, 73±91. 7. Shikama, K. (1998) The molecular mechanism of autoxidation for myoglobin and hemoglobin: a venerable puzzle. Chem. Rev. 98, 1357±1373. 8.Tsuruga,M.,Matsuoka,A.,Hachimori,A.,Sugawara,Y.& Shikama, K. (1998) The molecular mechanism o f autoxidation for human oxyhemoglobin: tilting o f the distal histidine causes nonequivalent oxidation in the b chain. J. Biol. Chem. 273, 8607±8615. 9. Williams, R.C. Jr & Tsay, K. (1973) A convenient chromato- graphic method for the preparation of hu man hemoglobin . Anal. Biochem. 54, 137±145. 10. D e Duve, C. (1948) A spectrophotometric method for t he simul- taneous determination of myoglobin and hemoglobin in extracts of human muscle. Acta Chem. Scand. 2, 264±289. 11. Geraci, G., Parkhurst, L.J. & Gibson, Q.H. (1969) Preparation and properties of a-andb-ch ains from hum an hemoglobin. J. Biol. Chem. 244, 4664±4667. 12. Turci, S.M. & McDonald, M.J. ( 1985) Isolation of normal and variant human hemoglobin subunits. J. Chromatogr. 343, 168± 174. 13. B oyer, P.D. ( 1954) Spectrophotometric study of the reaction of protein sulfhydryl groups w ith organic mercurials. J. Am. Chem. Soc. 76, 4331±4337. 14. Rosemeyer, M.A. & Huehns, E.R. (1967) On the mechanism of the dissociation of haemoglobin. J. Mol. Biol. 25, 253±273. 15. Edelstein, S.J., Rehmar, M.J., Olson, J.S. & Gibson, Q.H. (1970) Functional aspects of the subunit associatio n-dissociat ion equi- libria of hemoglobin. J. Biol. Chem. 245, 4372±4381. 16. M cDonald, M.J., Turci, S.M., Mrabet, N .T., Himelstein, B.P. & Bunn, H.F. ( 1987) The kinetics of assembly of normal and variant human oxyhemoglobins. J. Biol. Chem. 262, 5951±5956. 210 J. p. Yasuda et al. (Eur. J. Biochem. 269) Ó FEBS 2002 17. Rifkind, J.M., Abugo, O., Levy, A. & Heim, J. (1994) Detection, formation and relevan ce of he michromes and hemochromes. Methods Enzymol. 231, 449±480. 18. Levy, A., Kuppusamy, P. & Rifkind, J.M. (1990) Multiple heme pocket subconformation s of m ethemoglo bin associated with distal histidine interactions. Biochemistry 29, 9311±9316. 19. Borgstahl, G.E.O., Rogers, P.H. & Arnone, A. (1994) The 1.8 A Ê structure of carbonmonoxy-b 4 hemoglobin. J. Mol. Biol. 236, 817±830. 20. Imai, K. ( 1994) Adair ®tting to oxyge n equilibrium curves of hemoglobin. Methods Enzymol. 232, 559±576. 21. Perutz, M. (1990) Mec hanisms of Cooperativity and Allosteric Regulation in Proteins. Cambridge University Press, Cambridge, UK. 22. Fermi, G. & Perutz, M.F. (1981) Haemoglobin and myoglobin. In Atlas of Molecular Structure in Biology ,Vol.2(Phillips,D.C. & Richards, F.M., eds), Clarendon Press, Oxford, UK. 23. Perutz, M.F., Wilkinson, A.J., Paoli, M. & Dodson, G.G. (1998) The stereochemical mechanism of the cooperative eects in hemoglobin revisited. Annu. Rev. Biophys. Biomol. Struct. 27, 1±34. 24. Dickerson, R.E. & Geis, I. (1983) Hemoglobin: Structure, Func- tion, Evolution and Pathology. The Benjamin/Cummings Publish- ing Co, Inc. Menlo Park, CA, USA. 25. Bald win, J. & Chothia, C. (1979) Haemoglobin: the s tructural changes related to ligand binding and its allosteric mechanism. J. Mol. Biol. 129, 175±220. 26. Satoh, Y. & S hikama, K. (1981) Autoxidation of oxymyoglobin: a nucleophilic displacement mechanism. J. Biol. Chem. 256, 10272±10275. 27. Shikama, K. (1984) A controversy on the mechanism of autoxi- dation of oxym yoglo bin and oxyhaemoglobin: oxidation, d isso- ciation, or displacement? Biochem. J. 223, 279±280. 28. Shikama, K. (1990) Autoxidation of oxymyoglobin: a meeting point of the stabilization and the activation of molecular oxygen. Biol. Rev. (Cambridge) 65, 517±527. 29. Phillips, S.E.V. & Schoenborn, B.P. (1981) Neutron diraction reveals oxygen-histidine hydrogen bond in oxymyoglobin. Nature (London) 292, 81±82. 30. Suzuki, T., Watanabe, Y H., Nagasawa, M., Matsuoka, A. & Shikama, K. (2000) Dual nature of the distal histidin e residue in the autoxidation reaction o f myoglobin and h emoglobin: com- parison of the H64 mutants. Eur. J. Biochem. 267, 6166±6174. 31. Shaanan, B. (1982) The iron-oxygen bond in human oxyhaemo- globin. Nature (London) 296, 683±684. 32. Lukin, J.A., Simplaceanu, V., Zou, M ., Ho, N.T. & Ho, C. (2000) NMR reveals hydrogen bonds between oxygen and distal histi- dines in oxyhemoglobin. Proc. Natl Acad. Sci. USA 97, 10354±10358. 33. Winslow, R.M. & Anderson, W.F. (1978) The hemoglobinopa- thies. In The M etabolic Basis of Inherited Disease (Stanbury, J.B., Wyngaarden, J.B. & Fredrickson, D.S., eds), 4th edn. Part 10, Chapter 62, pp. 1465±1507. McGraw-Hill, Inc., New York, USA. Ó FEBS 2002 The a1b1 contact in HbO 2 autoxidation (Eur. J. Biochem. 269) 211 . The a1 b1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen Further evidence from the iron valency hybrids Jun pei Yasuda 1 , Takayuki Ichikawa 1 , Mie Tsuruga 1 ,. 4). Similarly, Shaanan [31] reported the stereochemistry of the iron -dioxygen bond in human HbO 2 by single-crystal X-ray analysis. In the a chain, the distance between N e of His (E7) and the terminal. than its a1 b1 counterpart in HbO 2 . At all rates, the present spectral examinations clearly indicate that the formation of the a1 b1ora2b2 contact suppresses remarkably the a cidic a utoxidation

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