REVIEW ARTICLE Human haemoglobin A new paradigm for oxygen binding involving two types of ab contacts Keiji Shikama 1,2 and Ariki Matsuoka 3 1 Biological Institute, Graduate School of Life Sciences, Tohoku University, Sendai, Japan, 2 PHP Laboratory for Molecular Biology, Sendai, Japan; 3 Department of Biology, Fukushima Medical University, Fukushima, Japan This review summarizes the most recent state of haemo- globin (Hb) research based on the literature and our own results. In particular, an attempt is made to form a unified picture for haemoglobin function by reconciling the cooperative oxygen binding with the stabilization of the bound dioxygen in aqueous solvent. The HbA molecule contains two types of ab contacts. One type is the a1b2or a2b1 contacts, called sliding contacts, and these are strongly associated with the cooperative binding of O 2 to the a 2 b 2 tetramer. The other type is the a1b1ora2b2 contacts, called packing contacts, but whose role in Hb function was not clear until quite recently. However, detailed pH-dependence studies of the autoxidation rate of HbO 2 have revealed that the a1b1anda2b2 interfaces are used for controlling the stability of the bound O 2 . When the a1b1ora2b2contactis formed, the b chain is subjected to a conformational con- straint which causes the distal (E7) histidine to be tilted slightly away from the bound dioxygen, preventing the proton-catalysed nucleophilic displacement of O 2 – from the FeO 2 by an entering water molecule. This is one of the most characteristic features of HbO 2 stability. Finally we discuss the role of the a1b1ora2b2 contacts by providing some examples of unstable haemoglobin mutants. These patho- logical mutations are found mostly on the b chain, especially in the a1b1 contact regions. In this way, HbA seems to differentiate two types of ab contacts for its functional properties. Keywords: ab contacts; distal (E7) histidine; HbA; heme oxidation; oxygen binding. Two types of ab contacts in HbA In haemoglobin (Hb) research, the central problem is understanding the mechanism for the cooperative oxygen binding to the a 2 b 2 tetramer. For human HbA, the a and b chains contain 141 and 146 amino acid residues, respect- ively, and a representative set of the successive oxygen- binding constants is given in terms of Torr )1 as follows: K 1 ¼ 0.0188, K 2 ¼ 0.0566, K 3 ¼ 0.407 and K 4 ¼ 4.28 in 0.1 M Bis/Tris buffer containing 0.1 M KCl at pH 7.4 and 25 °C [1]. In this reaction, major differences have been found between deoxyhaemoglobin and oxyhaemoglobin by comparing their X-ray crystal structures (e.g. [2–6]). These include a movement of the iron atom into the haem plane with a simultaneous change in the orientation 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 the one dimer relative to the other along the P axis by approximately 1 A ˚ . The latter two changes are accompanied by sequential breaking of the so-called salt bridges by C-terminal residues. Incidentally, the P is taken as an axis which is perpendicular to the dyads of both the liganded and unliganded Hb molecules. As illustrated in Fig. 1, there are two types of ab contacts in the Hb molecule. One is the a1b1(ora2b2) contact involving B, G, and H helices and the GH corner, and the other is the a1b2(ora2b1) contact involving mainly helices C and G and the FG corner [3,7]. When HbA goes from the deoxy to the oxy form, the a1b2anda2b1 contacts undergo the principal changes associated with cooperative oxygen binding, so that these are named the sliding contacts. As a result of the relative rotation of the a1b1anda2b2dimers, the gap between the b chains becomes too small to accommodate 2,3-diphosphoglyceric acid (DPG) that serves to reduce the oxygen affinity of HbA. At the a1b1anda2b2 interfaces, on the other hand, negligible changes are found insofar as the crystal structure has been examined. These are called the packing contacts accordingly, but their role in haemoglobin function was not clear for a very long time. To the packing contacts, we have recently assigned a key role for stabilizing the HbO 2 tetramer, as the formation of the a1b1ora2b2 contact greatly suppresses the haem oxidation, particularly of the b chain at acidic pH values [8,9]. Based on a nucleophilic displacement of O 2 – from the FeO 2 centre, kinetic analyses of HbO 2 oxidation were carried out with special focus on the proton-catalysed Correspondence to K. Shikama, PHP Laboratory for Molecular Biology, Nakayama-Yoshinari 1-16-8, Sendai 989-3203, Japan. E-mail: shikama@mail.cc.tohoku.ac.jp Abbreviations: Hb, haemoglobin; DPG, 2,3-diphosphoglyceric acid. Dedication:ThisreviewisdedicatedtoMaxF.Perutz (19 May 1914–6 February 2002), who laid the foundation for an entire field of haemoglobin research. According to a kind suggestion made by one of the referees, it should be added that Perutz once called haemoglobin a Ôhonorary enzymeÕ. Both haemoglobin and myoglobin are actually antienzymes, because they prevent the undesired electron transfer from Fe(II) to the bound O 2 as far as possible in aqueous solution. (Received 5 June 2003, revised 29 July 2003, accepted 13 August 2003) Eur. J. Biochem. 270, 4041–4051 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03791.x process performed by the distal (E7) histidine residue. Such examinations seem to be of primary importance, not only for a full understanding of the molecular mechanism of haemoglobin autoxidation, but also for planning new molecular designs for synthetic oxygen carriers that are highly resistant to haem oxidation under physiological conditions. Finally, we revisit haemoglobin function as seen from the two different types of ab contacts, and try to reconcile cooperative oxygen binding with stabilization of the bound dioxygen. With respect to this, we also give possible implications for the unstable haemoglobin mutants leading to the formation of Heinz bodies in red blood cells, resulting in haemolytic anaemia. Autoxidation reaction of HbO 2 and its constituent chains Biphasic nature of the autoxidation reaction The reversible and stable binding of molecular oxygen with the haem iron(II) is the basis of haemoglobin function. Even in air-saturated buffers, however, HbA is oxidized easily from the oxygenated form (HbO 2 ) to the ferric(III) met- form (metHb) with generation of the superoxide anion [10,11] as follows: HbO 2 ! k obs metHb þ O À 2 ð1Þ where k obs represents the first-order rate constant observed at a given pH value in terms of the constituent chains. This autoxidation reaction can be monitored by the spectral changes with time, after fresh HbO 2 was placed in 0.1 M buffer containing 1 m M EDTA at 35 °C. The spectra evolved to the final state of each run, which was identified as the usual ferric met-form, with a set of isosbestic points. Consequently, the process was fol- lowed by a plot of experimental data as _ ln([HbO 2 ] t / [HbO 2 ] 0 ) vs. time t, where the ratio of HbO 2 concentra- tion after time t to that at time t ¼ 0 can be obtained by the absorbance changes at 576 nm for the a-peak of human HbO 2 . Fig. 2 shows such examples of the first-order plot for the autoxidation reaction of human HbO 2 at two different pH values. At pH 6.2, HbA exhibited a biphasic curve that can be described by the first-order kinetics containing two rate constants as follows: ½HbO 2  t ½HbO 2  0 ¼ P  expðÀk f  tÞþð1 À PÞÂexpðÀk s  tÞ ð2Þ In this equation, a fast first-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 HbO 2 tetramer. P is the molar fraction of the rapidly reacting haems. This conclusion is based on the rapid chain separation experiment of partially (30%) oxidized HbO 2 on polyacrylamide gel [8,12]. By iterative least-squares procedures inserting various values for k f and k s into Eqn (2), the best fit to the experimental data was obtained as a function of time t.In these computations, the value of P was also allowed to vary across a large range from 0.40 to 0.60 [8,9]. In this way, the following parameters were established at pH 6.2: k f ¼ 0.82 (± 0.03) · 10 )1 h )1 , k s ¼ 0.13 (± 0.01) · 10 )1 h )1 ,and P ¼ 0.52 (± 0.04) in 0.1 M Mes buffer at 35 °C. At pH 9.2, on the other hand, the reaction was described completely by a single first-order rate constant of 0.99 (± 0.02) · 10 )2 h )1 (i.e. k f ¼ k s with P ¼ 0.50) in 0.1 M Caps buffer at 35 °C. We have also studied the effect of DPG on the autoxidation rate of HbA at 35 °C. DPG was added to stripped HbO 2 (0.13 m M )atmolarexcessesof5,14and24,butthis allosteric effector offered no significant effect on either k f or k s values at pH 6.5 and 8.5 [13]. Fig. 2. First-order plots for the autoxidation reaction of human HbO 2 in 0.1 M buffer at 35 °C. Each curve was obtained by a least-squares fitting to the experimental points, based on Eqn (2). At pH 6.2, HbA showed a biphasic autoxidation curve containing two rate constants, k f and k s , respectively. At pH 9.2, however, the reaction was mono- phasic. Redrawn from Yasuda et al.[9]. Fig. 1. Schematic diagram of HbA tetramer showing the two different types of ab contacts. HbA has a molecular dyad axis (which is per- pendicular to the plane of the figure) relating the a1b1 dimer to the a2b2dimer. 4042 K. Shikama and A. Matsuoka (Eur. J. Biochem. 270) Ó FEBS 2003 pH-Dependencies of the autoxidation rate If the values of k f and k s are plotted against the pH of the solution, we can obtain a pH profile for the stability of HbO 2 . Fig. 3 shows such profiles for both of the a and b chains in the HbO 2 tetramer over the range pH 5–11, under air-saturated conditions in 0.1 M buffer at 35 °C. In the acidic range of pH 7–5, the logarithmic values of k f increased very rapidly with increasing hydrogen ion con- centration. The values of k s also increased with increasing proton concentration but much less so than for k f .Rather, the k s values exhibited a rate saturation behaviour on the acidic extreme. In a plot of log(k obs ) vs. pH, its slope showed a value of n ¼ )1fork f , whereas a value close to n ¼ )0.6 was for k s . In the basic side higher than pH 8, on the other hand, practically no difference was observed between the k f and k s values, indicative of the oxidation curve being monophasic. Nevertheless, it is also true that both graphs depend strongly upon the pH of the solution, having a parabolic part with a minimum rate appearing at pH 8.5. At this point, the most important questions have arisen as to whether the constituent a and b chains each has its own different stability, and, if not, what the origin is of nonequivalence of the chains in haem oxidation. In this regard, it should be noted that such a chain heterogeneity of HbO 2 oxidation can be retained even in very diluted concentrations of haemoglobin [13]. When human HbO 2 is placed in dilution, the tetrameric species is known to dissociate into ab dimers along the a1b2ora2b1 interface, so that the dimers produced are of the a1b1ora2b2type [14,15]. Accordingly, these results strongly suggest that the formation of the a1b1ora2b2 contact must be responsible for the remarkable stability of the b chain against the acidic autoxidation. This was the next step to be clarified. Stability property of the separated a and b chains In separated a and b chain solutions, the protein is known to exist in an equilibrium of a ÀÀ* )ÀÀ a 2 and b ÀÀ* )ÀÀ b 4 respectively. Under our experimental conditions, the mono- meric form (87%) was predominant in the a chain, while the tetrameric form (99%) was predominant in the b chain. This estimation was made on the basis of the results of McDonald et al. [16]. As for the tetrameric form of the b chain, Borgstahl et al. [7] have reported the 1.8 A ˚ structure with carbonmonoxy-b 4 (COb 4 ) derivative, and compared subunit–subunit contacts between three types of interfaces (a1b1, a1b2, and a1a2) of HbO 2 and the corresponding COb 4 interfaces. As a result, they found that the b1b2 interface of the COb 4 tetramer is less stable and more loosely packed than its a1b1 counterpart in HbO 2 .In particular, there are significant packing differences at the end of the B helix between these homologous interfaces; the B helix–H helix contact region is spread apart by approxi- mately 1 A ˚ in COb 4 relative to oxyHb. Specifically, the b1b2 interface of the COb 4 tetramer does not include close contacts between residues Pro-125 (H3) and Val-33 (B15), Gln-127 (H5) and Val-34 (B16), and Ala-128 (H6) and Val- 34 (B16). The side chain disorder also makes the centre of the b1b2 interface packed less tightly in the COb 4 tetramer. Therefore, the b1b2 contact sites in the b 4 tetramer are indeed different from the corresponding a1b1 contact sites in the HbA tetramer. Anyway, we have revealed that over the wide range of pH 5–10, the separated a and b chains are both oxidized much more rapidly than in the parent HbO 2 tetramer. Fig. 4 represents such pH-dependencies of the observed rate constants, k a obs and k b obs , for autoxidation of the isolated a and b chains in 0.1 M buffer at 35 °C. It thus becomes evident that the b chain, when separated from the HbO 2 tetramer, does not show any rate saturation behaviour at low pH. Rather, its rate increased very rapidly with increasing hydrogen ion concentration, exhibiting a value close to n ¼ )1 for the slope against the acidic pH. We can therefore conclude that the intrinsic oxidation rate is almost thesamewiththeseparateda and b chains, completely freed from the remarkable differences between them in the autoxidation reaction of the parent HbO 2 tetramer. Mechanism of the haem oxidation for HbO 2 FeO 2 bonding and its nucleophilic displacement of O 2 – It has been widely accepted that HbA is much more resistant to autoxidation than myoglobin. However, it is now evident that the constituent a and b chains, once separated from the parent HbO 2 , are oxidized more rapidly than most Fig. 3. Differential pH-dependencies of k f and k s for the autoxidation reaction of human HbO 2 in 0.1 M buffer at 35 °C. Apairofthe observed first-order rate constants, k f (s)andk s (d), was obtained by a least-squares fitting to each of the oxidation curves at different pH values. In the acidic range of pH 7–5, the logarithmic plots of k f give a slope of n ¼ )1againstthepH,butn¼ )0.6 for k s .Redrawnfrom Tsuruga et al.[8]. Ó FEBS 2003 A unified picture for Hb function (Eur. J. Biochem. 270) 4043 mammalian oxymyoglobins. Such enhancements in the oxidation rate have been frequently attributed to the increased concentration of the deoxygenated species in HbO 2 or MbO 2 solution, since the deoxy form is certainly the preferred target for many kinds of oxidants. This simple mechanism, however, cannot explain the above-mentioned results for the separated a and b chains, because it has been definitively established that both chains have a much higher oxygen affinity with fewer deoxygenated species than the parent HbO 2 tetramer. In 0.1 M phosphate buffer at pH 7.0 and 30 °C, indeed, Tyuma et al. [17] reported the P 50 values of 1.00 Torr for the a chains and 0.45 Torr for the b chains, whereas HbA showed P 50 ¼ 16.59 Torr in the absence of DPG. Certainly, dioxygen is a powerful oxidizing agent in a triplet ground state, 3 P À g , whose biradical electronic configuration is given by the following notation: O 2 ðr1sÞ 2 ðr à 1sÞ 2 ðr2sÞ 2 ðr à 2sÞ 2 ðr2p z Þ 2 ðp2p x Þ 2 ðp2p y Þ 2 ðp à 2p x Þ 1 ðp à 2p y Þ 1 ðr à 2p z Þ 0 ð3Þ Dioxygen therefore has a very strong tendency to take electrons from other substances and to make the com- plete electron-pairing in its unoccupied orbitals. This property leads to the sequential production of the so- called active oxygen species such as superoxide anion (O 2 – ), peroxide anion (O ¼ 2 Þ and hydroxyl radical (HO • ). For O 2 at 760 Torr 1 , pH 7 and 25 °C, its midpoint oxidation-reduction potential is + 0.81 V for the com- plete, four-equivalent reduction to water, showing a total free energy change of )74.7 kcalÆmol )1 ()312 kJÆmol )1 ). Nevertheless, the addition of the first electron to O 2 is an unfavourable, uphill process with a low redox potential of e°¢(O 2 /O 2 – ) ¼ )0.33 V [18]. All of the steps subsequent to water are downhill. In this sense, molecular oxygen is a rather poor one-electron acceptor, and this thermo- dynamic barrier to the first step seems to be the crucial ridge located between the stabilization and the activation of dioxygen bound to the haemoproteins [19]. Using a value of + 0.150 V for the oxidation–reduction potential of human Hb at pH 7 and 30 °C[20],wemay write the primary step for the autoxidation reaction of HbO 2 as follows: In this scheme, the reaction from left to right is associated with a change in redox potential (De°¢)of)0.48 V, which corresponds to a positive free energy change of + 11.0 kcalÆ mol )1 (+ 4 6 . 0 k J Æmol )1 ). Accordingly, a considerable energy barrier accompanies the reduction of O 2 to O 2 – by deoxy- Hb, so this one-electron transfer cannot occur spontane- ously. In many respects, the spontaneous dissociation of O 2 – from the FeO 2 centre is an energetically unfavourable process, so that there must be involved some specific mechanism that causes very rapid generation of O 2 – from HbO 2 , as formulated in Eqn (1), in aqueous solution. Recently, Shikama [21] has carefully evaluated various mechanisms proposed so far for the autoxidation reaction of myoglobin and haemoglobin, including the effects of pH, oxygen pressure, and subsequent side reactions with the H 2 O 2 produced by the spontaneous dismutation of O 2 – .As a result, he concluded that a displacement mechanism is needed to make it possible to yield O 2 – so readily from the FeO 2 centre. In essence, 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 haem pocket from the surrounding solvent. The iron is thus converted to the ferric met-form, and the water molecule or the hydroxyl ion remains bound to the Fe(III) at the sixth 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 processes [19,21–24]: Mb(II)(O 2 ÞþH 2 O ! k 0 Mb(III)(OH 2 ÞþO À 2 ð5Þ Mb(II)(O 2 ÞþH 2 O þ H þ À! k H Mb(III)(OH 2 ÞþHO 2 ð6Þ Mb(II)(O 2 ÞþOH À À! k OH Mb(III)(OH À ÞþO À 2 ð7Þ In these equations, k 0 is the rate constant for the basal displacement by H 2 O, k H is the rate constant for the Fig. 4. pH profiles for the autoxidation rate of the separated a and b chains in 0.1 M buffer at 35 °C. Both of the computed curves were obtained by a least-squares fitting to the experimental points over the whole range of pH studied, based on Eqn (8). Redrawn from Tsuruga et al.[8]. ð4Þ 4044 K. Shikama and A. Matsuoka (Eur. J. Biochem. 270) Ó FEBS 2003 proton-catalysed displacement by H 2 O, and k OH is the rate constant for the displacement by OH – . The extent of contribution of these elementary processes to the observed or 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 dependence on pH. The reductive displacement of the bound dioxygen as O 2 – byH 2 O can proceed without any protonation, but it has been clearly shown that the rate is enormously accelerated with the proton assistance by a factor of 10 6 per mole, as formulated by Eqn (6). In this proton catalysis, the distal histidine, which forms a hydrogen bond to the bound dioxygen [25], appears to facilitate the effective movement of a catalytic proton from the solvent to the bound, polarized dioxygen via its imidazole ring and by a proton-relay mechanism [21,24]. In this way, such a nucleophilic displacement mechanism has successfully been applied to detailed pH-dependence studies of the k f and k s values, both for the HbO 2 tetramer and the separated chains, over the wide range of pH 5–11 in 0.1 M buffer at 35 °C [8]. Numerical analyses of the pH-dependence curves In the autoxidation reaction, pH can affect the rate in many different ways. To work out definitely the kinetic and thermodynamic parameters contributing to each k obs vs. pH profile, we have proposed some mechanistic models for each case. The rate equations derived therefrom were tested for their fit to the experimental data with the aid of a computer. As a result, the pH-dependence curves for the autoxidation rate of the separated a and b chains have been analysed completely in terms of an Ôacid-catalysed two-state modelÕ [8]. In this kinetic formulation, it is assumed that a single, dissociable group, XH with pK 1 , is involved in the reaction. Consequently, there are two forms of the oxygenated chain, represented by A and B, at molar fractions of F and Y (¼ 1–F), respectively, which are in equilibrium with each other but which differ in dissociation state for the group XH. These forms can be oxidized to the ferric met-form by a nucleophilic displacement of O 2 – from the FeO 2 centre by an entering water molecule or hydroxyl ion. By using the rate constants defined in the preceding section, the observed first-order rate constant, k a obs or k b obs in Eqn (1), can be reduced to: k a obs ðor k b obs Þ¼f k A 0 ½H 2 Oþ k A H ½H 2 O½H þ gðUÞ þf k B 0 ½H 2 Oþ k B H ½H 2 O½H þ þ k B OH ½OH À gðWÞð8Þ where U ¼ ½H þ  ½H þ þK 1 and W ¼ð1 À UÞ¼ K 1 ½H þ þK 1 ð9Þ By iterative least-squares procedures inserting various values for K 1 , the adjustable parameter in Eqn (9), the best fit to more than 60 experimental points was obtained for each of k a obs and k b obs as a function of pH (see Fig. 4). In this way, the rate constants and the acid dissociation constant involved in the autoxidation reaction of the separated a and b chains were established in 0.1 M buffer at 35 °C, as summarized in Table 1. These results clearly indicate that both a and b chains are inherently quite susceptible to haem oxidation over the whole range of pH studied. For example, their k B 0 values are even higher (by 2.5–4.5-fold) than that of bovine MbO 2 (k B 0 ¼ 0.17 · 10 )3 h )1 Æ M )1 )in0.1 M buffer at 35 °C [26]. It becomes also evident that the proton-catalysed processes with the rate constants k A H and k B H promote most of the autoxidation reaction of each chain, above the basal processes in water with the rate constants k A 0 and k B 0 .In fact, the catalytic proton enhances the rate dramatically both in the separated a and b chains, by a factor of more than 10 6 per mole for state A and state B as well. In this proton catalysis, the distal histidine (the dissociable group XH with pK 1 ¼ 6.1), which is located at position 58 for the a chain and at position 63 for the b chain, appears to participate by a proton-relay mechanism the same as in mammalian oxymyoglobins [21,24]. Indeed, random and undirected access of a proton to the bound dioxygen cannot yield such an enzyme-like, catalytic effect on the acidic autoxidation of MbO 2 and HbO 2 as well. In the HbO 2 tetramer, on the other hand, a marked difference was found between the a and b chains in the oxidation rate. As seen in Fig. 3, the values of k f (due to the a chain) were suppressed considerably over the wide range of pH 7–11, but its pH-dependence was quite similar in shape to that of the separated a chain. By the same mechanism as described in Eqn (8) therefore, we can obtain the best fit to more than 75 experimental points of k f over thewholepHrangeasfollows: Table 1. Rate constants and acid dissociation constants obtained from the pH-dependence curves for the autoxidation rate of the separated a and b chains in 0.1 M buffer at 35 °C. Taken from Tsuruga et al. [8]. Ó FEBS 2003 A unified picture for Hb function (Eur. J. Biochem. 270) 4045 k f ¼f k A 0 ½H 2 Oþ k A H ½H 2 O½H þ gðUÞ þf k B 0 ½H 2 Oþ k B H ½H 2 O½H þ þ k B OH ½OH À gðWÞð10Þ Table 2 summarizes the rate constants and the acid dissociation constant involved in the autoxidation reac- tion of the a chain in the HbO 2 tetramer [8]. From these results, it is quite clear that the proton-catalysed processes with the rate constants k A H and k B H are mainly responsible for the acidic oxidation of human HbO 2 .In this proton catalysis, the distal histidine at position 58 should also participate as the dissociable group XH with pK 1 ¼ 6.2. In sharp contrast to the a chain, the autoxidation of the b chain in the HbO 2 tetramer exhibited a rate-saturation behaviour below pH 5. Unfortunately, at more acidic pH data points could not be obtained due to denaturation of the protein. By a simple Ôtwo-state modelÕ, however, we have reached the best fit to more than 80 values of k s over the whole range of pH studied, in a quite acceptable way as seen in Fig. 3. In this mechanism, we assumed that a single, dissociable group (XH with pK 1 )isalsoinvolvedinthe reaction, but the proton-catalysed processes (with the rate constants k A H and k B H ) were totally omitted from Eqn (10) as follows: k s ¼f k A 0 ½H 2 OgðUÞþf k B 0 ½H 2 Oþ k B OH ½OH À gðWÞ ð11Þ where the molar fractions of F and Y for the states A and B can be given by Eqn (9). According to the same fitting procedures, the rate constants and the acid dissociation constant involved in the autoxidation of the b chain in the HbO 2 tetramer were established in 0.1 M buffer at 35 °C, as summarized in Table 2 also. In these kinetic analyses, one of the most remarkable features is that in the HbO 2 tetramer, the b chain does not show any proton-catalysed process that has the term of k H [H 2 O][H + ] containing the distal histidine as its catalytic residue. Instead, the b chain shows the involvement of a dissociable group (XH) with pK 1 ¼ 5.1 in 0.1 M buffer at 35 °C. For this group the most probable candidate would also be the distal histidine at position 63. This residue however, if compared to the corresponding His58 (with pK 1 ¼ 6.2) of the a chain, seems to be less accessible to solvent protons, titrating at a lower pH by almost one pH unit. Moreover, this residue in the b chain would probably be located a little more apart from the bound O 2 so as to lose its catalytic effect on the acidic autoxi- dation. Key role of the a1b1 contact in stabilizing the HbO 2 tetramer Tilting of the distal histidine residue in the b chain As is evident from Fig. 3, the remarkable stability of human HbO 2 can be ascribed mostly to the delayed oxidation of the b chain in acidic pH range. It is also evident that the b chain has obtained this stability by blocking out the proton catalysis (Eqn 6) from the acidic oxidation. At this point, it should be emphasized that such a stability characteristic of the HbO 2 tetramercanberetainedeveninthelow concentrations of haemoglobin corresponding to appreci- able dissociation into a1b1ora2b2 dimers [13]. The mechanism whereby the b chain acquires the enhanced stability in the HbO 2 tetramer must therefore be associated with the formation of the a1b1ora2b2 contact. These recent findings have led us to conclude that the packing contact produces in the b chain a conformational constraint whereby the distal (E7) histidine at position 63 is tilted away from the bound dioxygen, so as to prevent the acid- catalysed displacement of O 2 – from the FeO 2 centre by an entering water molecule. Similarly, Shaanan [27] reported the stereochemistry of the iron-dioxygen bond in human HbO 2 bysingle-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 be2.7A ˚ , and the geometry favours a similar hydrogen bond as in the case of sperm whale MbO 2 [25]. In the b chain, however, N e (or N e2 relative to C e1 )ofHis(E7) is located further away from both O-2 and O-1 (3.4 and 3.2 A ˚ , respectively), indicating that the hydrogen bond, even if formed, must be very weak. Recently, Lukin et al. [28] claimed that a hydrogen bond is formed between O 2 and the distal histidine in both a and b chains of human HbO 2 , as revealed by heteronuclear NMR spectra of the chain-selectively labelled samples. In 0.1 M phosphate buffer at pH 8.0 and 29 °C, the (H e2 ,N e2 ) cross-peaks of 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 Table 2. Rate constants and acid dissociation constants obtained from the pH-dependence curves for the autoxidation rate of HbO 2 tetramer in 0.1 M buffer at 35 °C. Taken from Tsuruga et al. [8]. 4046 K. Shikama and A. Matsuoka (Eur. J. Biochem. 270) Ó FEBS 2003 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 time, they reported that much wider separation of 1.17 p.p.m. appears on the H e1 resonances of the two distal histidine residues, showing that b63His is different from a58His in either the orientation or distance or both, with respect to the haem-bound dioxygen. Such marked differences between the two distal haem pockets must also be responsible for our kinetic results of the a and b chains in the HbO 2 tetramer. Figure 5 illustrates in a very schematic way the structure of human HbO 2 , as seen in the a1b1(ora2b2) contact leading to the nonequivalence of the a and b chains. The four haem pockets are all exposed at the surface of the molecule, so that each FeO 2 centre is always subject to the nucleophilic attack of an entering water molecule or hydroxyl ion. In the a chain, the distal histidine at position 58 can stabilize the bound O 2 by hydrogen bond formation. Nevertheless, it is also true that this residue participates, via its imidazole ring and by a proton-relay mechanism, in facilitating the effective movement of a catalytic proton from the solvent to the bound, polarized dioxygen. This proton-assisted nucleophilic displacement of O 2 – from the FeO 2 centre by an entering water molecule, that is an S N -2 type process with proton assistance [21,24], can account for most of the autoxidation reaction at acidic pH side. In the b chain, on the other hand, the remarkable stability is produced by the formation of the a1b1and a2b2 contacts, which give rise to a conformational constraint whereby the distal histidine at position 63 is tilted away from the bound O 2 . As a result, the constituent b chains lose a proton-catalysed process and thus provide the HbO 2 tetramer with the enhanced stability against the acidic oxidation. To understand more quantitatively the effect of the a1b1 or a2b2 contact on the haem oxidation, the next step was to construct the iron valency hybrid tetramers containing either the a or b chains in the ferric met-form, and to test their stability as compared with the native HbO 2 tetramer as well as the separated a and b chains. Further evidence from the iron valency hybrid haemoglobins By mixing equivalent amounts of the separated a and b chains whose sulfhydryl groups were completely recovered, we can prepare the reconstructed HbO 2 and its valency hybrid tetramers such as (a 3+ ) 2 (bO 2 ) 2 and (aO 2 ) 2 (b 3+ ) 2 .To obtain the ferric met-form for each chain, the oxygenated species was oxidized by the addition of potassium ferri- cyanide. The mixed chain solution containing either the a or b chain in the ferric met-form was then applied to a CM-cellulose column to separate each hybrid tetramer from its unassociated chains [9]. When the iron valency hybrids are placed in air-saturated buffers, the oxygenated chains of each tetramer are oxi- dized easily to the ferric met-form. Fig. 6 represents such first-order plots to show wide differences in the oxidation rate of the b chain, when it exists as the separated (bO 2 ) 4 , valency hybrid (a 3+ ) 2 (bO 2 ) 2 , and reconstructed HbO 2 tetramers in 0.1 M Mes buffer at pH 6.2 and 35 °C. In this way, the resulting rate constants for the a and b chains are compared between the native, separated, reconstructed, and valency hybrid haemoglobins at several pH values [9]. At pH 6.2, for instance, native HbO 2 gives the rate constants of k f ¼ 0.82 · 10 )1 h )1 and k s ¼ 0.13 · 10 )1 h )1 in its biphasic curve. As listed in Table 3, almost the same oxidation rates were obtained for the reconstructed HbO 2 with a biphasic ratio of k f /k s ¼ 6.1. Among those, the most remarkable effect was found on the b chain. The separated b chain in itself undergoes quite rapid oxidation with a rate constant of k obs ¼ 0.10 h )1 ,butthisratewas dramatically suppressed up to k s ¼ 0.14 · 10 )1 h )1 (by sevenfold) in the reconstructed HbO 2 ,asisinnativeHbO 2 . More importantly, such a retarded k s value could be maintained totally in the valency hybrid (a 3+ ) 2 (bO 2 ) 2 tetramer. All of these features were essentially the same at other pH values. Certainly, the biphasic nature of the autoxi- dation rate of HbO 2 became much slower at pH 7.5, and even disappeared at pH 9.0. Nevertheless, the rate 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 haemoglobin, either it is native or recon- structed or even valency hybrid species. The similar situation was also found in the a chain, but its effect on the stability of human HbO 2 wasmuchlesscrucialthan the b chain. It thus becomes evident that the b chain has acquired a remarkable resistance against the acidic oxidation in a manner of contacting with the a chain, no matter which valency the latter partner is in, the ferrous or the ferric state. From these recent findings, we conclude that the packing contact produces a conformational constraint in the Fig. 5. Schematic representation of human oxyhaemoglobin as seen in the a1b1 contact to produce tilting of the distal histidine in the b chain. In HbO 2 , the four haem pockets are all exposed at the surface of the molecule. By the formation of the a1b1contact,theb chain is subject to a structural constraint whereby the distal histidine at position 63 is tilted slightly away from the bound O 2 . Ó FEBS 2003 A unified picture for Hb function (Eur. J. Biochem. 270) 4047 b chain, so that the proton-catalysed process performed by the distal histidine residue disappears from its acidic autoxidation. Furthermore, spectral examinations have disclosed that the formation of the a1b1ora2b2 contact also protects the b chain from its haemichrome conversion. As a matter of fact, the oxidation product of the isolated b chain was not for the usual ferric met-form but for its admixture with haemichrome. In this way, the noticeable stability of human HbO 2 depends largely upon the very unique property of the b chain on the a1b1ora2b2 interface. Concluding remarks: a unified picture for Hb function In HbA, the four haem pockets are all exposed at the surface of the molecule. From the X-ray crystal structures (e.g. [2–6]), however, it becomes apparent that the ligands ) including O 2 andCOtotheferrousformand H 2 O, OH – ,N 3 – and CN – to the ferric form ) cannot gain access to the closed haem pockets of haemoglobin as in the case of myoglobin. Karplus and McCammon [29] expressed this situation by the following passage in a satirical way. If the structure of sperm whale myoglobin was so rigid that the rotations of side chains were impossible, an oxygen molecule might take many billions of years to enter or leave the haem pocket across high energy kinetic barriers: the time would be much longer than a whale’s lifetime. Consequently, the thermal fluctu- ations of side chain amino acid residues are essential for the penetration of ligands from the surrounding solvent through the globin matrix to the haem pocket [29–32]. In this respect, much attention has been paid to the possible roles of the distal (E7) histidine residue in myoglobin and haemoglobin functions. It has been suggested that it acts as a gate [29] or a swinging door [33,34] for ligand entry into the haem pocket, and that it stabilizes the bound dioxygen by hydrogen-bond formation [25], as well as it stabilizes the axial water molecule of the ferric, high-spin species [35–37]. Furthermore, the distal histidine via its imidazole ring participates in a proton-relay mechanism as a catalytic residue for the acidic oxidation of MbO 2 and HbO 2 [8,21,24]. Fig. 6. First-order plots to compare the autoxidation rate of the b chain between three different haemoglobin derivatives in 0.1 M maleate buffer at pH 6.2 and 35 °C. Each curve was obtained by a least-squares fitting to the experimental points, based on Eqn (2). The oxidation of the separated b chains could be described by a single rate constant of k obs ¼ 0.10 h )1 in the presence of 20% (v/v) glycerol. This inherent rate was dramatically suppressed not only in the reconstructed HbO 2 but also in the valency hybrid (a 3+ ) 2 (bO 2 ) 2 as well. Redrawn from Yasuda et al.[9]. Table 3. Comparison of the autoxidation rate constants between the whole, separated, reconstructed, and hybrid haemoglobins in 0.1 M buffer at pH 6.2 and 35 °C. Taken from Yasuda et al. [9]. 4048 K. Shikama and A. Matsuoka (Eur. J. Biochem. 270) Ó FEBS 2003 To make clear the functional role of the distal histidine residue in the autoxidation reaction, Brantley et al.[38] were the first to use systematically the site-directed mutagenesis of sperm whale myoglobin. They showed that mutations of the distal His at position 64, such as those of H64G, H64V, H64L and H64Q, caused dramatic increases in the autoxidation rate. At pH 7.0, for instance, the H64V mutant MbO 2 was oxidized 400 times more rapidly than the wild-type (H64H) MbO 2 .Usingthese mutant myoglobins, we have also carried out detailed pH-dependence studies of the autoxidation rate over the wide range of pH 5–12 in 0.1 M buffer at 25 °C[39].The resulting pH-profiles were then compared with those of the corresponding myoglobins occurring in nature. As a result, if the distal (E7) histidine was replaced by other amino acid residues, all such mutant oxymyoglobins were found to contain no proton-catalysis in the autoxidation reaction. Their pH profiles could be formulated by the kinetic equations lacking in the rate constants k A H and k B H accordingly. Along with these lines of evidence, we have recently proposed that the distal histidine can play a dual role in the nucleophilic displacement of O 2 – from MbO 2 or HbO 2 [39]. One is in a proton-relay mechanism via the imidazole ring at acidic pH. Insofar as we have examined, such a proton- catalysed process could never be observed in the autoxi- dation reaction of myoglobins lacking the usual distal histidine residue, no matter what the protein is, the naturally occurring or the distal His mutant [39]. As a matter of fact, even if the distal residue is a histidine, it cannot manifest any proton-catalysis when the residue is tilted away from the precise E7 position. This is just the case we have described here for the b chainintheHbO 2 tetramer. The other role of the distal histidine would be in the maximum protection of the FeO 2 centre against a water molecule or a hydroxyl ion that can enter the haem pocket from the surrounding solvent [38]. This is relevant to the considerable stability of MbO 2 and HbO 2 in the neutral pH range. In this way, the distal histidine provides the delicate balance of catalytic and steric factors necessary for controlling the reversible oxygen binding to myoglobin and haemoglobin in aqueous solution. It is now clear that the constituent a and b chains, once separated from the HbO 2 , are oxidized much more easily than in the parent tetramer over the whole range of pH 5–10. Moreover, their rates come to be almost equal to each other and exhibit a very strong acid catalysis. This inherently high oxidation rate of each chain can be suppressed dramatically by the formation of a1b1(or a2b2) contact. In particular, the b chain provides a further effect on the stability of HbO 2 by preventing the proton- catalysed oxidation at acidic pH. In order to explain such unique properties of human HbO 2 , a nucleophilic displace- ment mechanism has successfully been applied to detailed pH-dependence studies of the autoxidation rate. As for the dimer and tetramer effects on haem oxidation, probable explanations are as follows. At basic pH, the separated a and b chains are both quite susceptible to autoxidation. Each haem pocket seems to be consid- erably open to allow easier attack of the solvent hydroxyl ion on the FeO 2 centre. As a result, there occurs a very rapid formation of hydroxide-met species, its rate being dependent directly upon the concentration of OH – ion. When the a1b1(ora2b2) contact is formed, accessibility of OH – ion to the haem pocket would be greatly reduced by conformational constraints. As OH – ion is one of the strongest nucleophiles in vivo, practically no rate difference could be observed between the a and b chains on the basic pH side, so that the autoxidation curve would become monophasic regardless of the ab dimer and the HbO 2 tetramer. On the acidic side from neutral pH, the displacing nucleophile is an entering water molecule and its concen- tration is always taken as 55.5 M in aqueous solution. Participation of the catalytic proton via the distal histidine residue should therefore be a most decisive factor in accelerating the displacing rate of O 2 – from FeO 2 with H 2 O. This is just the case with the separated a and b chains, both exhibiting a very strong acid catalysis in their oxidation rate. Once the a1b1(ora2b2) contact is established, the b chain is subjected to a conformational constraint whereby the distal histidine at position 63 is tilted away from the bound dioxygen so as to be free from the proton-catalysed displacement. In this way, the b chain can acquire a remarkable resistance against the acidic autoxidation, and this is one of the most characteristic features of the HbO 2 stability. In relevance to a clinical aspect, it is interesting to note that a quite large number of unstable haemoglobins have been reported so far in the medical literature [3,4,40]. Many of the mutants which occur at the a1b2 interface have altered oxygen affinity, but bulk of evidence suggests that the a1b1 interface is much more important in maintaining normal haemoglobin stability than is the a1b2 interface. In fact, haemolytic anaemia is known to result from substitu- tions affecting the a1b1 interface or the haem pocket. If such mutations occur, the haem iron will be more easily oxidized, and a sequence of events leads to the globin precipitation or Heinz body formation in red blood cells. Typical examples of such variants are: Tacoma [b30(B12)Arg fi Ser], Abraham Lincoln [b32(B14)Leu fi Pro], Castilla [b32 (B14)Leu fi Arg], Philly [b35(C1)Tyr fi Phe], Peterbor- ough [b111(G13)Val fi Phe], Madrid [b115(G17)Ala fi Pro], Khartoum [b124(H2)Pro fi Arg],J.Guantanamo [b128(H6)Ala fi Asp], Leslie [b131(H9)Gln fi deleted] and so on. Surprisingly, most of the pathological mutations are found on the b chain, especially in the a1b1 contact regions. In these unstable haemoglobins, the a1b1 contact would become loose or disruptive due to many different causes including: the insertion of proline (Abraham Lincoln, Madrid), the substitution with a too-small amino acid side chain (Tacoma) or a too-large side chain (Peterborough), the introduction of a charged or very polar group (Castilla, Khartoum, J. Guantanamo), and the deletion of amino acid residue (Leslie). The transport and storage of molecular oxygen by haemoglobin and by myoglobin are essential to life. The iron(II)-dioxygen bond in these haem proteins plays a vital role in their physiology. It is in the ferrous form that haemoglobin or myoglobin can bind molecular oxygen reversibly and carry out its physiological function. From known changes in valency of the haem iron, one can write the functional cycle of the haemoglobin molecule as follows: Ó FEBS 2003 A unified picture for Hb function (Eur. J. Biochem. 270) 4049 During reversible oxygen binding, the oxygenated form of haemoglobin, as well as of myoglobin, is oxidized easily to the ferric met-form with generation of the superoxide anion. The met-haemoglobin or met-myoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In red blood cells and muscle tissues, however, an NADH-cytochrome b 5 oxidoreductase is present which can reduce metHb or metMb to the ferrous deoxy-species again and thus prevent the continued accumulation of the ferric met-form in situ. The enzyme is called methaemoglobin reductase [41] and metmyoglobin reductase [42], respec- tively, and is known to have a FAD group that can accept electrons from NADH. As a matter of fact, a strong and cyclic reduction of the iron(III) species by these enzymes is a basis for the continuity of haemoglobin and myoglobin functions in vivo, since the autoxidation reaction is inevitable in nature for all oxygen-binding haem proteins [21,23,24], as well as for all synthetic dioxygen carriers [43,44]. In fact, it is a matter of our experience that the metMb content in myoglobin extracts from various muscle tissues is com- monly about 40%, while the metHb content of freshly drawn blood is usually maintained within 1–2% but by a very strong reductive environment. In conclusion, human haemoglobin seems to differentiate two types of ab contacts quite properly for its functional properties. The a1b2ora2b1 contact is associated with the cooperative oxygen binding, whereas the a1b1ora2b2 contact is used for controlling the stability of the bound O 2 . We can thus form a unified picture for haemoglobin function by closely integrating the cooperative and the stable binding of molecular oxygen with iron(II) in aqueous solvent. Acknowledgements The materials of our previous publications were used with permission from Publishers including: American Society for Biochemistry and Molecular Biology, Inc. (J. Biol. Chem.) and Blackwell Publishing Ltd. (Eur. J. Biochem.). References 1. Imai, K. (1994) Adair fitting to oxygen equilibrium curves of hemoglobin. Methods Enzymol. 232, 559–576. 2. Baldwin, J. & Chothia, C. (1979) Haemoglobin: the structural changes related to ligand binding and its allosteric mechanism. J. Mol. Biol. 129, 175–220. 3. Dickerson, R.E. & Geis, I. (1983) Hemoglobin: Structure, Func- tion, Evolution and Pathology. Benjamin Cummings Publishing Co.Inc.MenloPark,CA,USA. 4. 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. 5. Perutz, M. (1990) Mechanisms of Cooperativity and Allosteric Regu- lation in Proteins. Cambridge University Press, Cambridge, UK. 6. Perutz, M.F., Wilkinson, A.J., Paoli, M. & Dodson, G.G. (1998) The stereochemical mechanism of the cooperative effects in hemoglobin revisited. Annu. Rev. Biophys. Biomol. Struct. 27, 1–34. 7. Borgstahl, G.E.O., Rogers, P.H. & Arnone, A. (1994) The 1.8 A ˚ structure of carbonmonoxy-b 4 hemoglobin. J. Mol. Biol. 236, 817–830. 8. Tsuruga, M., Matsuoka, A., Hachimori, A., Sugawara, Y. & Shikama, K. (1998) The molecular mechanism of autoxidation for human oxyhemoglobin: Tilting of the distal histidine causes nonequivalent oxidation in the b chain. J. Biol. Chem. 273, 8607–8615. 9. Yasuda, J., Ichikawa, T., Tsuruga, M., Matsuoka, A., Sugawara, Y. & Shikama, K. (2002) The a1b1 contact of human hemoglobin plays a key role in stabilizing the bound dioxygen. Further evidence from the iron valency hybrids. Eur. J. Biochem. 269, 202–211. 10. Brunori, M., Falcioni, G., Fioretti, E., Giardina, B. & Rotilio, G. (1975) Formation of superoxide in the autoxidation of the isolated a and b chains of human hemoglobin and its involvement in hemichrome precipitation. Eur. J. Biochem. 53, 99–104. 11. Gotoh, T. & Shikama, K. (1976) Generation of the superoxide radical during autoxidation of oxymyoglobin. J. Biochem. (Tokyo) 80, 397–399. 12. Mansouri, A. & Winterhalter, K.H. (1973) Nonequivalence of chains in hemoglobin oxidation. Biochemistry 12, 4946–4949. 13. Tsuruga, M. & Shikama, K. (1997) Biphasic nature in the autox- idation reaction of human oxyhemoglobin. Biochim. Biophys. Acta 1337, 96–104. 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 association-dissociation equili- bria of hemoglobin. J. Biol. Chem. 245, 4372–4381. 16. McDonald, 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. 17. Tyuma, I., Benesch, R.E. & Benesch, R. (1966) The preparation and properties of the isolated a and b subunits of hemoglobin A. Biochemistry 5, 2957–2962. 18. Sawada, Y., Iyanagi, T. & Yamazaki, I. (1975) Relation between redox potentials and rate constants in reactions coupled with the system oxygen-superoxide. Biochemistry 14, 3761–3764. 19. Shikama, K. (1990) Autoxidation of oxymyoglobin: a meeting point of the stabilization and the activation of molecular oxygen. Biol. Rev. (Cambridge) 65, 517–527. 20. Antonini, E., Wyman, J., Brunori, M., Taylor, J.F., Rossi-Fanelli, A. & Caputo, A. (1964) Studies on the oxidation-reduction potentials of heme proteins. I. Human hemoglobin. J. Biol. Chem. 239, 907–912. 21. Shikama, K. (1998) The molecular mechanism of autoxidation for myoglobin and hemoglobin. A venerable puzzle. Chem. Rev. 98, 1357–1373. 22. Satoh, Y. & Shikama, K. (1981) Autoxidation of oxymyoglobin. A nucleophilic displacement mechanism. J. Biol. Chem. 56, 10272– 10275. 23. Shikama, K. (1985) Nature of the FeO 2 bonding in myoglobin. An overview from physical to clinical biochemistry. Experientia 41, 701–706. 24. Shikama, K. (1988) Stability properties of dioxygen-iron (II) porphyrins: an overview from simple complexes to myoglobin. Coordination Chem. Rev. 83, 73–91. ð12Þ 4050 K. Shikama and A. Matsuoka (Eur. J. Biochem. 270) Ó FEBS 2003 [...]... autooxidation of myoglobin J Biol Chem 268, 6995–7010 Suzuki, T., Watanabe, Y.-H., Nagasawa, M., Matsuoka, A & Shikama, K (2000) Dual nature of the distal histidine residue in the autoxidation reaction of myoglobin and hemoglobin Comparison of the H64 mutants Eur J Biochem 267, 6166–6174 Winslow, R.M & Anderson, W.F (1978) The hemoglobinopathies In The Metabolic Basis of Inherited Disease 4th edn (Stanbury,... between oxygen and distal histidines in oxyhemoglobin Proc Natl Acad Sci USA 97, 10354– 10358 29 Karplus, M & McCammon, J .A (1986) The dynamics of proteins Sci Am 254, 30–39 30 Karplus, M & McCammon, J .A (1983) Dynamics of proteins: elements and function Annu Rev Biochem 52, 263–300 31 Tian, W.D., Sage, J.T & Champion, P.M (1993) Investigations of ligand association and dissociation rates in the ÔopenÕ and... diffraction reveals oxygen- histidine hydrogen bond in oxymyoglobin Nature (London) 292, 81–82 26 Sugawara, Y & Shikama, K (1980) Autoxidation of native oxymyoglobin Thermodynamic analysis of the pH profile Eur J Biochem 110, 241–246 27 Shaanan, B (1982) The iron -oxygen bond in human oxyhaemoglobin Nature (London) 296, 683–684 28 Lukin, J .A. , Simplaceanu, V., Zou, M., Ho, N.T & Ho, C (2000) NMR reveals... Mapping the pathways for O2 entry into and exit from myoglobin J Biol Chem 276, 5177–5188 35 Matsuoka, A. , Kobayashi, N & Shikama, K (1992) The Soret magnetic circular dichroism of ferric high-spin myoglobins A A unified picture for Hb function (Eur J Biochem 270) 4051 36 37 38 39 40 41 42 43 44 probe for the distal histidine residue Eur J Biochem 210, 337– 341 Shikama, K & Matsuoka, A (1994) Aplysia... the ÔopenÕ and ÔclosedÕ states of myoglobin J Mol Biol 233, 155–166 32 Ramadas, N & Rifkind, J.M (1999) Molecular dynamics of human methemoglobin: the transmission of conformational information between subunits in an ab dimer Biophys J 76, 1796–1811 33 Johnson, K .A. , Olson, J.S & Phillips, G.N Jr (1989) Structure of myoglobin-ethylisocyanide: Histidine as a swinging door for ligand entry J Mol Biol 207,... J.B., Wyngaarden, J.B & Fredrickson, D.S., eds) pp 1465–1507 McGraw-Hill, Inc., New York, NY, USA Yubisui, T., Miyata, T., Iwanaga, S., Tamura, M & Takeshita, M (1986) Complete amino acid sequence of NADH-cytochrome b5 reductase purified from human erythrocytes J Biochem (Tokyo) 99, 407–422 Livingston, D.J., McLachlan, S.J., La Mar, G.N & Brown, W.D (1985) Myoglobin: Cytochrome b5 interactions and the... unusual properties: another prototype in myoglobin and haemoglobin biochemistry Biol Rev (Cambridge) 69, 233–251 Quillin, M.L., Arduini, R.M., Olson, J.S & Phillips, G.N Jr (1993) High-resolution crystal structures of distal histidine mutants of sperm whale myoglobin J Mol Biol 234, 140–155 Brantley, R.E Jr, Smerdon, S.J., Wilkinson, A. J., Singleton, E.W & Olson, J.S (1993) The mechanism of autooxidation... Mar, G.N & Brown, W.D (1985) Myoglobin: Cytochrome b5 interactions and the kinetic mechanism of metmyoglobin reductase J Biol Chem 260, 15699–15707 Momenteau, M & Reed, C .A (1994) Synthetic heme dioxygen complexes Chem Rev 94, 659–698 Busch, D.H & Alcock, N.W (1994) Iron and cobalt ÔlacunarÕ complexes as dioxygen carriers Chem Rev 94, 585–623 . REVIEW ARTICLE Human haemoglobin A new paradigm for oxygen binding involving two types of ab contacts Keiji Shikama 1,2 and Ariki Matsuoka 3 1 Biological Institute,. mono- phasic. Redrawn from Yasuda et al.[9]. Fig. 1. Schematic diagram of HbA tetramer showing the two different types of ab contacts. HbA has a molecular dyad