The Molecular Mechanism of Autoxidation for Human Oxyhemoglobin
TILTING OF THE DISTAL HISTIDINE CAUSES NONEQUIVALENT OXIDATION IN THE beta  CHAIN*

Mie Tsuruga, Ariki Matsuoka, Akira HachimoriDagger , Yoshiaki Sugawara§, and Keiji Shikama

From the Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Human oxyhemoglobin showed a biphasic autoxidation curve containing two rate constants, i.e. kf for the fast autoxidation due to the alpha  chains, and ks for the slow autoxidation of the beta  chains, respectively. Consequently, the autoxidation of the HbO2 tetramer produces two different curves from the pH dependence of kf and ks. The analysis of these curves revealed that the beta  chain of the HbO2 tetramer does not exhibit any proton-catalyzed autoxidation, unlike the alpha  chain, where a proton-catalyzed process involving the distal histidine residue can play a dominant role in the autoxidation rate. When the alpha  and beta  chains were separated from the HbO2 tetramer, however, each chain was oxidized much more rapidly than in the tetrameric parent. Moreover, the separated beta  chain was recovered completely to strong acid catalysis in its autoxidation rate. These new findings lead us to conclude that the formation of the alpha 1beta 1 contact produces in the beta  chain a conformational constraint whereby the distal histidine at position 63 is tilted away slightly from the bound dioxygen, preventing the proton-catalyzed displacement of Obardot 2 by a solvent water molecule. The beta  chains have thus acquired a delayed autoxidation in the HbO2 tetramer.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The reversible and stable binding of molecular oxygen to the heme iron(II) is the basis of hemoglobin function. Consequently, much attention has been directed to the elucidation of the molecular mechanism of cooperative oxygen binding to the hemoglobin tetramer (1). However, the oxygenated form of hemoglobin, as well as of myoglobin, is known to be oxidized easily to the ferric(III) met form, which cannot bind molecular oxygen and is therefore physiologically inactive, with generation of the superoxide anion (2-5).

To this autoxidation reaction, it has been widely accepted that hemoglobin is much more resistant as compared with myoglobin. Unlike myoglobin, Mansouri and Winterhalter (6) reported that oxyhemoglobin (HbO2) showed a biphasic autoxidation reaction with a fast and a slow component. They also demonstrated that the alpha chain was oxidized more rapidly than the beta  chain in hemoglobin tetramer. At the same time, however, there have been a number of reports that such a rate difference was not observed between the alpha  and beta  chains in the autoxidation reaction of HbA (7-9). Rather, Zhang et al. (9) showed that the rate of autoxidation was markedly enhanced when the HbO2 tetramer dissociates into alpha beta dimers. To clarify these discrepancies, we have recently examined systematically the effect of hemoglobin concentration on the autoxidation rate at several different values of pH, and found that human HbO2 exhibits a biphasic autoxidation curve only in the pH range from neutral to acidic (5). By dissociation of tetramers into alpha beta dimers, the rate of autoxidation for the fast component (due to the alpha  chain) was also found to increase markedly at the acidic pH, but the addition of 2,3-diphosphoglyceric acid offered no significant effect on the increment of the autoxidation rate (5).

In the present paper, we have made, for the first time, a full comparison between tetrameric HbO2 and the separated alpha  and beta  chains in terms of a detailed pH dependence for the autoxidation rate over the wide range of pH 5-11. On the basis of these kinetic results, our present goal is to solve the most important questions: whether each of the separated alpha  and beta  chains has its own different stability, and, if not, what the origin is of such a nonequivalence of the chains in the autoxidation reaction. This information is essential, not only for a full understanding of the molecular nature of hemoglobin autoxidation, but also for planning new molecular designs for synthetic oxygen carriers that are highly resistant against the heme oxidation in protic, aqueous solution under physiological conditions.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals-- Sephadex G-25 was a product of Amersham Pharmacia Biotech. CM-cellulose (CM-32) and DEAE-cellulose (DE-32) were purchased from Whatman. Sodium p-hydroxymercuribenzoate (p-MB)1 was an Aldrich product. Mes, Pipes, Mops, Hepes, Tris, Taps, and Caps for buffer systems, 2-mercaptoethanol and all other chemicals were of reagent grade from Wako Pure Chemicals, Osaka, Japan. Solutions were made with deionized and glass-distilled water.

Oxyhemoglobin Preparation-- Human hemoglobin A was prepared from freshly drawn blood (of 30 ml each time) by the method of Williams and Tsay (10). After the addition of 4 ml of CPD solution (CPD: 90 mM sodium citrate, 15 mM citric acid, 15 mM sodium biphosphate, and 130 mM glucose in 1% NaCl solution), the blood was centrifuged at 5000 rpm for 10 min. The packed red cells (~5 ml) were then washed three times with five volumes of 1% NaCl solution; 2 ml of the cells, the other portion being kept at -80 °C until further use, were hemolyzed with four volumes of cold distilled water. Stroma were removed by centrifugation at 18,000 rpm for 30 min. For the most part, all procedures were carried out at low temperature (4 °C). The hemolysate was then fractionated with ammonium sulfate between 20% and 80% saturation at pH 7.0. The hemoglobin precipitate was centrifuged down at 18,000 rpm for 30 min and dissolved in a minimum volume of 5 mM Hepes buffer (pH 7.9). The solution was then dialyzed against the same buffer and applied on a DEAE-cellulose column (2.5 × 9 cm) equilibrated with 5 mM Hepes buffer, pH 7.9. After passing a minor component (HbA2) through the column with 10 mM Hepes, the major band of HbA was eluted out completely with 20 mM Hepes at the same pH of 7.9. The HbO2 solution (approximately 25 ml) thus obtained was condensed by centrifugation in a Centriprep-10 tube (Amicon), and kept at low temperature (4 °C) until use. The concentration of hemoglobin (in heme) was determined, after conversion into cyanomet form, using the absorption coefficient of 10.4 mM-1 cm-1 at 540 nm. This value was obtained on the basis of the pyridine hemochromogen method (11).

Isolation of Mercuribenzoated alpha  and beta  Chains-- All separations were carried out with HbO2 at low temperature (0-4 °C) by a two-column method. The procedure was essentially the same as described by Geraci et al. (12) and by Turci and McDonald (13). p-MB (100 mg) was dissolved in 2 ml of 0.1 M NaOH and neutralized with 1 M CH3COOH. This was reacted with 10 ml of HbO2 solution (4-5 mM in heme) in 30 mM phosphate buffer, pH 6.0, and in the presence of 0.1 M NaCl. The solution was left overnight, and the flocculent precipitate was removed by centrifugation. To obtain alpha p-MB chains, the mercurated HbO2 solution was adjusted to pH 8.2 by filtration through a Sephadex G-25 column (2.5 × 40 cm) equilibrated with 15 mM Hepes buffer (pH 8.2). The resultant solution was then passed through a DEAE-cellulose column (3 × 12 cm) equilibrated with the same buffer. Under these chromatographic conditions, the alpha p-MB chains were readily eluted out, whereas the beta  chains and unsplit HbO2 were retained on the top of the column. To obtain beta p-MB chains, on the other hand, the mercurated HbO2 solution was adjusted to pH 6.7 by filtration through Sephadex G-25 in 10 mM phosphate buffer, pH 6.7. The resultant solution was then applied to a CM-cellulose column (3 × 12 cm) equilibrated with the same buffer. The unreacted HbO2 and the mercurated alpha  chains were retained on the top of the column at this pH, and only the beta p-MB chains were eluted out completely.

Removal of Mercuribenzoate from alpha  and beta  Chains-- p-MB was removed from the alpha  chains by incubating with 15 mM 2-mercaptoethanol in 10 mM Pipes buffer (pH 6.5) for 10 min at 0 °C. The mixture was then applied to a CM-cellulose column (2.5 × 6 cm), which had been equilibrated with 10 mM Pipes buffer, pH 6.5. The column was washed first with the same buffer containing 15 mM mercaptoethanol for 30 min at a flow rate of 70 ml/h. The mercaptoethanol was then removed by washing with the buffer alone for another 1 h, and the regenerated alpha  chains were finally eluted in the oxy form with 30 mM Hepes buffer at pH 8.2. For beta p-MB, the chains were incubated with 20 mM 2-mercaptoethanol in 5 mM phosphate buffer (pH 8.8) for 10 min at 0 °C. The solution was then placed on a DEAE-cellulose column (2.5 × 6 cm) equilibrated with 5 mM phosphate buffer, pH 8.8. The column was washed with the same buffer containing 20 mM mercaptoethanol over a period of 3 h at a flow rate of 100 ml/h. The mercaptoethanol was then removed by washing with 10 mM Hepes buffer (pH 8.2) for another 3 h, and the regenerated beta  chains were finally obtained by changing the buffer concentration to 50 mM at the same pH. The alpha  and beta chains thus separated were kept stably in the oxy form in liquid nitrogen until use. The concentration of each chain was determined on the basis of the pyridine hemochromogen method (11).

Determination of SH Groups-- According to the method of Boyer (14), sulfhydryl groups were titrated spectrophotometrically at 250 nm with p-hydroxymercuribenzoate at pH 7.0. In the separation procedure employed above, the regenerated alpha  and beta  chains gave uniformly 1.0 (1.05 ± 0.08) and 2.0 (2.01 ± 0.08) free SH groups, respectively (15).

Autoxidation Rate Measurements-- The autoxidation rate of HbA was measured in 0.1 M buffer over a wide range of pH at 35 °C according to our standard procedure. A 0.5-ml solution containing 0.2 M appropriate buffer was placed in a small tube and incubated in a water bath maintained at 35 (± 0.1) °C. The reaction was started by adding the same volume of fresh HbO2 solution (180-300 µM). For spectrophotometry, the reaction mixture was then quickly transferred to a quartz cell (of 1-mm path length) held at 35 (± 0.1) °C. The cell was sealed with a piece of Sealon film (Fuji) to prevent evaporation, and changes in the absorption spectrum from 450 to 700 nm were recorded on the same chart at measured intervals of time. For the final state of each run, the hemoglobin was completely converted to its ferric met form by the addition of potassium ferricyanide. For separated alpha  and beta  chains, the rate measurements were carried out in a 1-cm cell with 10 µM solution (in heme), and in the presence of 20% (v/v) glycerol. The addition of such a protein stabilizer was not essential, but was effective in preventing precipitation during the course of the autoxidation reaction over a long period of time at 35 °C.

The buffers used were Mes, maleate, Pipes, Mops, phosphate, Hepes, Tris, Taps, bicarbonate, and Caps. The pH of the reaction mixture was carefully checked, before and after the run, with a Hitachi-Horiba pH meter (model F-13).

Spectrophotometric Measurements-- Absorption spectra were recorded in a Hitachi two-wavelength double-beam spectrophotometer (model 557 or U-3210) equipped with a thermostatically controlled cell holder. Temperature was controlled by a water bath (Advantec, thermocool LCH-190) maintained to within ±0.1 °C.

EPR Measurements-- Electron paramagnetic resonance (EPR) spectra were recorded in a Varian EPR spectrometer (model 112) operating at 9.0-9.2 GHz. Experiments were carried out with ferric protein solution (~600 µM in heme) in 10 mM maleate buffer (pH 6.2), and in the presence of 50% (v/v) glycerol over a magnetic field of 0-500 mT at 8.0 K. An Oxford flow cryostat (ESR-900) was used for liquid helium temperature measurements.

Curve Fittings-- Biphasic curves for the autoxidation reaction were analyzed as described previously (5). The curve fittings for a plot of log(kobs) versus pH were made by an iterative least-squares method on a personal computer (NEC PC-9821 V12) with graphic display, according to our previous specifications (16, 17).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Biphasic Nature in the Autoxidation Reaction of HbO2-- In air-saturated buffers, the oxygenated form of HbA is oxidized easily to its ferric met form (metHb) with generation of the superoxide anion (2, 18) as shown by Reaction 1. 
<UP>HbO</UP><SUB>2</SUB> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>obs</UP></SUB></UL></LIM> <UP>metHb</UP>+<UP>O&cjs1138;<SUB>2</SUB></UP>
<UP><SC>Reaction</SC> 1</UP>
In this reaction, kobs represents the first-order rate constant observed at a given pH in terms of each subunit. Fig. 1 shows such an example for the spectral changes with time when fresh HbO2 (10 µM) was oxidized in 0.1 M Mes buffer (pH 6.5) at 35 °C and in the presence of 1 mM EDTA. The spectra evolved to the final state, which was identified as acidic (or aquo) metHb, with a set of isosbestic points at 524 and 591 nm. This process of autoxidation was therefore followed by a plot of experimental data as -ln([HbO2]t/[HbO2]0) versus time t, where the ratio of HbO2 concentration after time t to that at time t = 0 can be monitored by the absorbance changes at 576 nm (the alpha -peak of human HbO2).


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Fig. 1.   Spectral changes with time for the autoxidation reaction of human HbO2 in 0.1 M Mes buffer at pH 6.5 and 35 °C. Scans were made at 270-min intervals after 0.5 ml of the fresh HbO2 solution (20 µM) was added to the same volume of 0.2 M Mes buffer in the presence of 1 mM EDTA. The final spectrum was that of the acidic met form.

Fig. 2 shows first-order plots for the autoxidation reaction of HbO2 tetramer at two different values of pH. At pH 6.5, HbA showed a biphasic autoxidation curve that can be described completely by first-order kinetics containing two rate constants as follows.
<FR><NU>[<UP>HbO</UP><SUB>2</SUB>]<SUB>t</SUB></NU><DE>[<UP>HbO</UP><SUB>2</SUB>]<SUB>0</SUB></DE></FR>=P·<UP>exp</UP>(<UP>−</UP>k<SUB>f</SUB>·t)+(1−P) · <UP>exp</UP>(<UP>−</UP>k<SUB>s</SUB>·t) (Eq. 1)
In this equation, kf and ks represent first-order rate constants for the fast and slow autoxidation processes, respectively, and P is the molar fraction of the rapidly reacting hemes. By iterative least-squares procedures inserting various values for kf and ks, the best fit to the experimental data was obtained as a function of time. In these computations, as described previously (5), the initial value for each of the rate constants was taken from the corresponding slope of a biphasic curve, and was refined by the step sizes of 0.01 h-1 to 0.001 h-1 to find the best values of kf and ks. The value of P was also allowed to vary a large range (from 0.40 to 0.60) in all cases. In this way, the following parameters in Equation 1 were established: kf = 0.078 (± 0.007) h-1, ks = 0.011 (± 0.001) h-1, and P = 0.48 (± 0.04) in 0.1 M Mes buffer at pH 6.5 and 35 °C. In the autoxidation reaction at pH 8.0, however, the process could be described completely by a single first-order rate constant of 0.008 (± 0.001) h-1 (i.e. kf triple-bond  ks, P = 0.50), and the final state of the run was identified as hydroxide-metHb.


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Fig. 2.   First-order plots for the autoxidation of human HbO2 in 0.1 M buffer at 35 °C. At pH 6.5, HbA showed a biphasic autoxidation curve that can be described by a first-order kinetics containing two rate constants, kf for the initial fast oxidation and ks for the second slow oxidation, respectively. At pH 8.0, however, the autoxidation of HbO2 was monophasic. The rate measurements were carried out in the presence of 1 mM EDTA with 10 µM HbO2 in 0.1 M Mes buffer at pH 6.5, and with 15 µM HbO2 in 0.1 M Mops buffer at pH 8.0.

As for the biphasic nature of HbA, we have confirmed that the alpha  chain is oxidized more rapidly than the beta  chain, for instance, by a factor of not less than 3 at pH 6.0 and 35 °C. This examination was made by the rapid chain separation of partially (30%) oxidized HbO2 on a 7.5% polyacrylamide gel (19). Our result is in good accord with that of Mansouri and Winterhalter (6). Therefore, we can conclude unequivocally that the rate constant of kf is due to the autoxidation of the alpha  chain, while the value of ks is for the beta  chain of the HbO2 tetramer. When HbO2 is placed in dilution, on the other hand, the tetrameric species is known to dissociate into alpha beta dimers (20). To keep the tetramer concentration as high as possible, the following experiments were therefore carried out with 90-150 µM HbO2 using a quartz cell of 1-mm path length. In such concentrated solutions, the equilibrium fraction of tetrameric species was estimated to be more than 90% (19). Under these conditions, a pair of the observed first-order rate constants involved in Equation 1 was determined by a least-squares fitting to each of the reaction curves obtained at more than 75 different values of pH.

If the values of kf and ks are plotted against the pH of the solution, we can obtain a pH profile for the stability of HbO2. Fig. 3 shows such a profile for both of the alpha  and beta  chains in the HbO2 tetramer over the wide range of pH 5-11, under air-saturated conditions in 0.1 M buffer at 35 °C. This graph clearly demonstrates the biphasic nature emerged in the autoxidation reaction of HbO2. In the acidic range of pH 7 to 5, the logarithmic values of ks increased rapidly with increasing hydrogen ion concentration, but much less so than for kf. The latter has a value of n = -1 for the slope against the pH, while the former shows a value close to n = -0.6. In the range higher than pH 8.0, on the other hand, practically no difference was found between the values of kf and ks, indicative of the reaction 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. 


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Fig. 3.   Differential pH dependences of kf and ks for the autoxidation reaction of human HbO2 in 0.1 M buffer at 35 °C. The rate measurements were carried out with fresh HbO2 solution (90-150 µM) in the presence of 1 mM EDTA. A pair of the observed first-order rate constants, kf (open circle ) and ks (bullet ), was obtained by a least-squares fitting to each of the reaction curves at different values of pH. In the acidic range of pH 7 to 5, the logarithmic values of ks increased rapidly with increasing hydrogen ion concentration, but much less so than for kf, the latter having a value close to n = -1 for the slope against the pH of the solution.

A Kinetic Formulation for the Autoxidation Reaction of the HbO2 Tetramer-- In the autoxidation reaction, pH can affect the rate in many different 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 Obardot 2 from MbO2, but is due to a nucleophilic displacement of Obardot 2 from MbO2 by a water molecule or a hydroxyl ion that can enter the heme pocket from the surrounding solvent (3, 21-23). 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 to form aqua- or hydroxide-metMb, respectively. Even the complicated pH profile for the autoxidation rate can thereby be explained primarily in terms of the following three types of displacement process (3, 17, 21, 24).
<UP>Mb</UP>(<UP>II</UP>)(<UP>O</UP><SUB>2</SUB>)+<UP>H</UP><SUB>2</SUB><UP>O</UP> <LIM><OP><ARROW>⇀</ARROW></OP><UL>k<SUB>0</SUB></UL></LIM> <UP>Mb</UP>(<UP>III</UP>)(<UP>OH</UP><SUB>2</SUB>)+<UP>O&cjs1138;</UP><SUB><SUB><UP>2</UP></SUB></SUB>
<UP>Mb</UP>(<UP>II</UP>)(<UP>O</UP><SUB>2</SUB>)+<UP>H</UP><SUB>2</SUB><UP>O</UP>+<UP>H<SUP>+</SUP></UP> <LIM><OP><ARROW>⇀</ARROW></OP><UL>k<SUB><UP>H</UP></SUB></UL></LIM> <UP>Mb</UP>(<UP>III</UP>)(<UP>OH</UP><SUB>2</SUB>)+<UP>HO</UP><SUB>2</SUB>
<UP>Mb</UP>(<UP>II</UP>)(<UP>O</UP><SUB>2</SUB>)+<UP>OH<SUP>−</SUP></UP> <LIM><OP><ARROW>⇀</ARROW></OP><UL>k<SUB><UP>OH</UP></SUB></UL></LIM> <UP>Mb</UP>(<UP>III</UP>)(<UP>OH<SUP>−</SUP></UP>)+<UP>O&cjs1138;</UP><SUB><SUB><UP>2</UP></SUB></SUB>
<UP><SC>Reactions</SC> 2–4</UP>
In these reactions, k0 is the rate constant for the spontaneous displacement by H2O, kH is the rate constant for the proton-catalyzed displacement by H2O, and kOH is the rate constant for the displacement by OH-. The extent of contribution of these elementary processes to the observed autoxidation rate, kobs in Reaction 1, can vary with the concentrations of H+ or OH- ion. Consequently, the stability of MbO2 shows a very strong pH dependence having a parabolic shape. The reductive displacement of the bound dioxygen as Obardot 2 by H2O 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 more than 106/mol, as formulated by Reaction 3. 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 dioxygen via its imidazole ring by a proton-relay mechanism (3). In fact, such a proton-catalyzed process can never be observed in the autoxidation reaction of myoglobins lacking the distal histidine residue, such as Aplysia Mb where the His(E7) is replaced by Val (17, 24).

On the basis of this view, we have investigated two different types of pH dependences emerged from the autoxidation of the alpha  and beta  chains in the HbO2 tetramer in 0.1 M buffer at 35 °C. To know definitely the kinetic and thermodynamic parameters contributing to each kobs versus 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.

For the alpha  chain, its pH dependence curve has thus been described in terms of an "acid-catalyzed two-state model". In this model, it is assumed that a single, dissociable group, AH with pK1, is involved in the reaction. Consequently, there are two forms of the oxygenated alpha  chain, represented by A and B, at molar fractions of Phi  and Psi , respectively, which are in equilibrium with each other but which differ in dissociation state for the group AH. These forms can be oxidized to ferric met form by displacement of Obardot 2 from the FeO2 center by an entering water molecule or hydroxyl ion. Using the rate constants defined above, therefore, the reaction scheme may be written as shown below.
<AR><R><C><UP>HbO</UP><SUB>2</SUB> (<UP>AH</UP>)</C><C><LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB>1</SUB></UL></LIM></C><C><UP>HbO</UP><SUB>2</SUB> (<UP>A<SUP>−</SUP></UP>)</C></R><R><C>k<SUP><UP>A</UP></SUP><SUB>0</SUB>⇃ k<SUP><UP>A</UP></SUP><SUB><UP>H</UP></SUB>⇃</C><C></C><C>k<SUP><UP>B</UP></SUP><SUB>0</SUB>⇃ k<SUP><UP>B</UP></SUP><SUB><UP>H</UP></SUB>⇃ k<SUP><UP>B</UP></SUP><SUB><UP>OH</UP></SUB>⇃</C></R><R><C><UP>metHb</UP></C><C></C><C><UP>metHb</UP></C></R></AR>
<UP><SC>Scheme</SC> 1</UP>
For the mechanism delineated in Scheme 1, the observed rate constant, kobsf (triple-bond  kf) in h-1, for the autoxidation of the alpha  chain can be reduced to the following equation,
k<SUP>f</SUP><SUB><UP>obs</UP></SUB> (≡ k<SUB>f</SUB>)={k<SUP><UP>A</UP></SUP><SUB>0</SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>]+k<SUP><UP>A</UP></SUP><SUB><UP>H</UP></SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>][<UP>H<SUP>+</SUP></UP>]}(&PHgr;)+{k<SUP><UP>B</UP></SUP><SUB>0</SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>]+k<SUP><UP>B</UP></SUP><SUB><UP>H</UP></SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>][<UP>H<SUP>+</SUP></UP>]+k<SUP><UP>B</UP></SUP><SUB><UP>OH</UP></SUB>[<UP>OH<SUP>−</SUP></UP>]}(&PSgr;), (Eq. 2)
where
&PHgr;=<FR><NU>[<UP>H<SUP>+</SUP></UP>]</NU><DE>[<UP>H<SUP>+</SUP></UP>]+K<SUB>1</SUB></DE></FR> (Eq. 3)
and
&PSgr;=(1−&PHgr;)=<FR><NU>K<SUB>1</SUB></NU><DE>[<UP>H<SUP>+</SUP></UP>]+K<SUB>1</SUB></DE></FR>. (Eq. 4)
By iterative least-squares procedures inserting various values for K1, the adjustable parameter in Equations 3 and 4, the best fit to more than 75 values of kf was obtained over the whole range of pH 5-11, as demonstrated in Fig. 4. As a reference, Fig. 5 represents the sum of the squared residuals as a function of pK1 values inserted in this computation. In this way, the rate constants and the acid dissociation constant involved in the autoxidation reaction of the alpha  chain were established in 0.1 M buffer at 35 °C, and are summarized in Table I.


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Fig. 4.   A plot of log(kf) versus pH for the autoxidation of the alpha  chain of HbO2 tetramer in 0.1 M buffer at 35 °C. The computed curve (------) was obtained by a least-squares fitting to the experimental data (open circle ) over the whole range of pH 5-11, based on Equation 2, derived from an acid-catalyzed two-state model (see "Results"). In this procedure, three kinetic parameters for state B were first established so as to best cover a parabolic part of the pH profile, since its component (- - -) was manifested by the term kB = k0B[H2O] + kHB[H2O][H+] + kOHB[OH-] in the alkaline range, where the molar fraction of Psi  approaches unity in Equation 2. HbO2 concentration, 90-150 µM.


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Fig. 5.   A graphic representation of sum of the squared residuals (SSR) as a function of pK1 values inserted into Equation 2. After the values of k0B, kHB, and kOHB had been fixed in Equation 2, iterative least-squares procedures inserting various values for K1, the adjustable parameter in Equations 3 and 4, were carried out so as to obtain the best fit to more than 75 values of kf over the whole range of pH 5-11. At the minimum of the sum of the squared residuals (SSR), the conjugate values of k0A and kHA, as well as the value of pK1, are established. The resulting kinetic constants and acid dissociation constant are summarized in Table I.

                              
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Table I
Rate constants and acid dissociation constants obtained from the pH dependence curves for the autoxidation rate of HbO2 tetramer in 0.1 M buffer at 35 °C

From these results, it becomes evident that the proton-catalyzed processes with the rate constants kHA and kHB promote the autoxidation of the alpha  chain above the spontaneous processes in water with the rate constants k0A and k0B. In fact, the catalytic proton enhances the rate enormously, by a factor of 5.6 × 106 mol-1 for state A and by a factor of 3 × 107 mol-1 for state B. In this proton catalysis, the distal histidine at position 58 (the dissociable group AH with pK1 = 6.2) appears to be involved by the same mechanism as proposed previously in mammalian oxymyoglobins (3, 17, 23, 24).

In sharp contrast to the alpha  chain, the rate of autoxidation of the beta  chain in the HbO2 tetramer exhibited a saturation behavior below pH 5. Unfortunately, in more acidic pH range, data points could not be obtained due to the denaturation of the protein. Nevertheless, we could finally establish the best fit to more than 80 values of ks by a "two-state model" in a quite acceptable way, as shown in Fig. 6. In this mechanism, we assume that a single, dissociable group (AH with pK1) is also involved in the reaction. Employing the same notations defined above, the autoxidation reaction of the beta  chain may therefore be written as shown by Scheme 2. 
<AR><R><C><UP>HbO</UP><SUB>2</SUB> (<UP>AH</UP>)</C><C><LIM><OP><ARROW>⇌</ARROW></OP><UL>K<SUB>1</SUB></UL></LIM></C><C><UP>HbO</UP><SUB>2</SUB> (<UP>A<SUP>−</SUP></UP>)</C></R><R><C>k<SUP><UP>A</UP></SUP><SUB>0</SUB>⇃</C><C></C><C>k<SUP><UP>B</UP></SUP><SUB>0</SUB>⇃ k<SUP><UP>B</UP></SUP><SUB><UP>OH</UP></SUB>⇃</C></R><R><C><UP>metHb</UP></C><C></C><C><UP>metHb</UP></C></R></AR>
<UP><SC>Scheme</SC> 2</UP>
For this reaction, the observed rate constant, kobss (triple-bond  ks) in h-1, is given by Equation 5,
    k<SUP>s</SUP><SUB><UP>obs</UP></SUB> (≡ k<SUB>s</SUB>)={k<SUP><UP>A</UP></SUP><SUB>0</SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>]}(&PHgr;)+{k<SUP><UP>B</UP></SUP><SUB>0</SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>]+k<SUP><UP>B</UP></SUP><SUB><UP>OH</UP></SUB>[<UP>OH<SUP>−</SUP></UP>]}(&PSgr;), (Eq. 5)
where
&PHgr;=<FR><NU>[<UP>H<SUP>+</SUP></UP>]</NU><DE>[<UP>H<SUP>+</SUP></UP>]+K<SUB>1</SUB></DE></FR> (Eq. 6)
and
&PSgr;=(1−&PHgr;)=<FR><NU>K<SUB>1</SUB></NU><DE>[<UP>H<SUP>+</SUP></UP>]+K<SUB>1</SUB></DE></FR>. (Eq. 7)
By the same fitting procedures as for the alpha  chain, the rate constants and the acid dissociation constant involved in the autoxidation reaction of the beta  chain were established over the whole range of pH 5-11 in 0.1 M buffer at 35 °C, and are also summarized in Table I.


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Fig. 6.   A plot of log(ks) versus pH for the autoxidation of the beta  chain of HbO2 tetramer in 0.1 M buffer at 35 °C. The computed curve (------) was obtained by a least-squares fitting to the experimental data (bullet ) over the whole range of pH studied, based on Equation 5, derived from a two-state model (see "Results"). The resulting kinetic constants and acid dissociation constant are listed in Table I. HbO2 concentration, 90-150 µM.

In these kinetic formulations, one of the most remarkable features is that the beta  chain does not show any proton-catalyzed process having the term of kH[H2O][H+], such as the one that can play a dominant role in the autoxidation reaction of the alpha  chain, involving the distal histidine as its catalytic residue. Instead, the beta  chain contains a dissociable group (AH) with pK1 = 5.1 in 0.1 M buffer at 35 °C. Although it is hazardous to identify a dissociable group only by its pKa value, we suggest that the most probable candidate for the group AH in Scheme 2 is the distal histidine residue at position 63. In the beta  chain, however, this residue seems to be less accessible to protons, titrating at a lower pH by a factor of almost one pH unit compared with the value of pK1 = 6.2 for the alpha  chain. At the same time, this residue would probably be located apart from the bound O2, since a lack of hydrogen bonding with the terminal oxygen atom could reduce the autoxidation rate of the beta  chain. As mentioned in Reaction 3, the proton transfer from the distal histidine to the bound, polarized dioxygen can facilitate displacement of Obardot 2 as the hydroperoxyl radical HO2. Therefore, our next step was to examine whether each of the separated alpha  and beta  chains has its own different susceptibility to aqueous autoxidation.

Stability Properties of the Separated alpha  and beta  Chains-- In separated chain solutions, the protein is known to exist in an equilibrium of alpha  right-left-harpoons  alpha 2 or beta  right-left-harpoons  beta 4, respectively. Under our experimental conditions, the monomeric form (87%) was predominant in the alpha  chains, while the tetrameric form (99%) was predominant in the beta  chains. This estimation was made on the basis of the results by McDonald et al. (26). Compared with the HbO2 tetramer, the separated alpha  and beta  chains were both oxidized much more rapidly over the whole range of pH 5-10, according to a simple, first-order kinetics based on Reaction 1. 

Fig. 7 shows such pH dependences of the observed rate constants, kobsalpha and kobsbeta , for the autoxidation reaction of the separated alpha  and beta  subunits in 0.1 M buffer at 35 °C. Surprisingly, it became thus evident that the separated beta  chain by itself does not show any saturation behavior in its rate at low pH. Rather, the rate increased rapidly with increasing hydrogen ion concentration, exhibiting a value close to n = -1 for the slope against the acidic pH. Consequently, each pH profile could be almost superimposed on the other, except for the rate minimum region where beta  chains became rather susceptible to autoxidation. We have therefore established the best fit to more than 60 experimental points, for each of kobsalpha and kobsbeta , as a function of pH by the same mechanism as delineated in Scheme 1. 
k<SUP>&agr;</SUP><SUB><UP>obs</UP></SUB> (<UP>or</UP> k<SUP>&bgr;</SUP><SUB><UP>obs</UP></SUB>)={k<SUP><UP>A</UP></SUP><SUB>0</SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>]+k<SUP><UP>A</UP></SUP><SUB><UP>H</UP></SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>][<UP>H<SUP>+</SUP></UP>]}(&PHgr;)+{k<SUP><UP>B</UP></SUP><SUB>0</SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>]+k<SUP><UP>B</UP></SUP><SUB><UP>H</UP></SUB>[<UP>H</UP><SUB>2</SUB><UP>O</UP>][<UP>H<SUP>+</SUP></UP>]+k<SUP><UP>B</UP></SUP><SUB><UP>OH</UP></SUB>[<UP>OH<SUP>−</SUP></UP>]}(&PSgr;) (Eq. 8)
Table II summarizes the kinetic and thermodynamic parameters involved in the autoxidation reaction of separated alpha  and beta  chains.


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Fig. 7.   pH profiles for the autoxidation rate of separated alpha  and beta  chains in 0.1 M buffer at 35 °C. The logarithmic values of the observed first-order rate constants, kobsalpha (open circle ) and kobsbeta (bullet ), for the autoxidation of separated alpha  and beta  chains are plotted against the pH of the solution. For each chain, the rate measurements were carried out with 10 µM solution (in heme) in the presence of 1 mM EDTA. 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 Equation 8. The resulting kinetic constants and acid dissociation constant are summarized for each chain in Table II.

                              
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Table II
Rate constants and acid dissociation constants obtained from the pH dependence curves for the autoxidation rate of separated alpha  and beta  chains in 0.1 M buffer at 35 °C

These results show that both alpha  and beta  chains are inherently quite susceptible to autoxidation over the whole range of pH studied. For example, the values of k0B are even higher (by a 2.5-4.5-fold) than that of bovine MbO2 (k0B = 0.17 × 10-3 h-1 M-1) in 0.1 M buffer at 35 °C (27). According to the view described already, our present results suggest that both of the heme pockets in alpha  and beta  chains are sufficiently open to allow easier attack of the solvent water molecule on the FeO2 center, with a consequent very rapid formation of the ferric species. Moreover, the beta  chain tetramer can manifest proton-catalyzed processes with the rate constants kHA and kHB, if it is placed free from the alpha  chain. In this proton catalysis, the distal histidine at position 63 (the dissociable group AH with pK1 = 6.1) appears to participate by recovering a hydrogen bond with the bound dioxygen.

Another important feature of the separated beta  chain was seen in the hemichrome formation. As already shown in Fig. 1, when the proteins exist in HbO2 tetramer, both of the alpha  and beta  chains were oxidized to its ferric met form, accompanying typical spectral changes due to the acid-alkaline transition with pKa = 7.9. However, things were quite different with the separated beta  chain, for its oxidation product carried with it a hemichromogen spectrum over the wide range of pH 5-10. Fig. 8 shows such an example for the spectral changes with time when the separated beta  chain (10 µM) was oxidized in 0.1 M maleate buffer, pH 6.2, at 35 °C. The final spectrum was not that of the acidic (or aquo) met form, but for its admixture of the hemichrome with a peak at 530 nm and a shoulder near 560 nm. In the framework of the accepted mechanisms both of the autoxidation reaction (3, 24) and the hemichrome formation (28), our present results imply that a nucleophilic displacement of Obardot 2 from the beta  chain by an entering water molecule or hydroxyl ion is the rate-limiting step, and that the subsequent conversion of the met form into a hemichrome must proceed very quickly. Therefore, the autoxidation of separated beta  chains can be described by the following scheme at a neutral pH.
<AR><R><C>&bgr;(<UP>II</UP>)(<UP>O</UP><SUB>2</SUB>)+<UP>H</UP><SUB>2</SUB><UP>O</UP></C><C> <LIM><OP><ARROW>⇀</ARROW></OP><UL>k<SUB>0</SUB></UL></LIM></C><C>&bgr;(<UP>III</UP>)(<UP>OH</UP><SUB>2</SUB>)+<UP>O&cjs1138;</UP><SUB><UP>2</UP></SUB></C></R><R><C></C><C></C><C><UP>⇃</UP><IT>k</IT><SUB><UP>X</UP></SUB></C></R><R><C></C><C></C><C>&bgr;(<UP>III</UP>)(<UP>X</UP>)</C></R></AR>
<UP><SC>Scheme</SC> 3</UP>
X represents a heme ligand endogenous to the protein, with the kinetic relationship of kX >>  k0. In separated beta  chains, the distal histidine residue at position 63 is the most probable candidate for X, and the observed first-order rate constant, kobsbeta , corresponds to the rate-limiting step for the reaction leading to the formation of a hemichrome.


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Fig. 8.   Spectral changes with time for the autoxidation of separated beta  chains in 0.1 M maleate buffer at pH 6.2 and 35 °C. Scans were made at 40-min intervals after 1.0 ml of the oxygenated beta  chains (20 µM) was added to the same volume of 0.2 M maleate buffer at pH 6.2 in the presence of 1 mM EDTA and 20%(v/v) glycerol. The final spectrum was not for the acidic met form, but an admixture with hemichrome. Over the wide range of pH 5-10, such a hemichrome formation was not observed in separated alpha  chains, as well as in HbO2 tetramer (see Fig. 1).

In respect to the hemichrome formation, an EPR analysis of the ferric derivatives may provide us with more detailed information. Fig. 9 shows the 8 K EPR spectrum for the oxidation product of separated beta  chains in 10 mM maleate buffer at pH 6.2. 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 beta  chain exhibited a low spin spectrum with g1 = 2.77, g2 = 2.27, and g3 = 1.68, which differentiates this species from that of the hydroxide-type complex. According to Rifkind et al. (28), such low spin complexes characterized by the highest g value in the range of 2.83-2.75 and the lowest g value in the range of 1.69-1.63 have been designated as complex B, indicating crystal field parameters of the reversible hemichrome. They also suggest that the bis-histidine complex B may still have water retained in the heme pocket, and therefore in solution it is in rapid equilibrium with the high spin aquo complex (29). In the oxidized beta  chain, in fact, the molar fraction of the hemichrome (complex B) was estimated to be 85% at pH 6.2. These EPR interpretations agree very well with our present results concerning the lability of the distal histidine residue in the beta  chain.


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Fig. 9.   The 8 K EPR spectra for the oxidation product of separated beta  chains in 10 mM maleate buffer at pH 6.2. In addition to a typical high spin aqua species with g values of 5.86 and 1.99, the oxidized beta  chain exhibited a low spin spectrum with g values of 2.77, 2.27, and 1.68, which can be attributed to a hemichrome complex. These EPR signals are completely coincident with the results from optical absorption in Fig. 8.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

It has been widely accepted that hemoglobin tetramer is considerably more resistant to autoxidation than myoglobin. However, it becomes evident that the separated alpha  and beta  chains are oxidized more easily than most other mammalian oxymyoglobins. Enhancements in the oxidation rate of MbO2 or HbO2 have been frequently attributed to the increased concentration of the deoxygenated species, since each deoxy form is certainly the preferred target for many kinds of oxidant. This mechanism cannot explain the present results for the enhanced oxidation of the separated chains, because it has been definitively established that both of the chains have a much higher oxygen affinity with fewer deoxygenated species compared with the parent hemoglobin. In 0.1 M phosphate buffer at pH 7.02 and 30 °C, for instance, Tyuma et al. (30) reported the values of P50 = 1.00 mmHg for alpha  chains and 0.45 mmHg for beta chains, while HbA showed a value of P50 = 16.59 mmHg in the absence of 2,3-diphosphoglyceric acid.

For a very long time, the autoxidation reaction of HbO2 has been investigated at a single pH or within a limited range of pH. However, the present study has disclosed that the rate of autoxidation depends strongly upon the pH of the solution in a very complicated manner, and that the pH dependence curves can vary significantly among the separated alpha  and beta  chains and their tetrameric parent over the wide range of pH 5-11. On the basis of the molecular mechanism proposed for the myoglobin autoxidation (3, 24), we have made, for the first time, a full kinetic formulation for the autoxidation reaction of human HbO2 in terms of a nucleophilic displacement of Obardot 2 from the FeO2 center, and revealed that the beta  chain plays a crucial role in hemoglobin autoxidation. In this SN-2 mechanism, we did not consider whether geminate recombination of Obardot 2 plays a role in the reaction; so this point remains open to future study. However, it should be remarked that after photodissociation, the geminate rebinding of CO, NO, or O2 ligand to myoglobin or hemoglobin was observed on the pico- to nanosecond time scales at room temperature (31-33). It was also reported that in myoglobin the ligand association and dissociation rates could be controlled by a small fraction of the "open and closed" conformations observable for the distal heme pocket on a microsecond time scale (34). Compared with these processes, the autoxidation of HbO2 and MbO2 takes place only on the hour to day time scales.

At any rate, it becomes evident that the separated beta  chain, although it exists predominantly as a tetramer, is inherently quite susceptible to autoxidation. The FeO2 bond is always exposed to the nucleophilic attack of an entering water molecule with and without proton catalysis, and to the attack of an entering hydroxide anion. These nucleophiles can thus cause a strong pH dependence having a parabolic part. The proton-catalyzed displacement of Obardot 2, in which the distal histidine at position 63 appears to participate by a proton-relay mechanism, can also account for most of the enhanced rate occurring in the acidic pH range. Such properties are essentially the same as in the separated alpha  chain, except for the formation of hemichrome as an oxidized product of beta  chains. The conversion of the ferric met form into a hemichrome must involve changes of the protein conformation so that a nitrogenous residue (probably of the distal histidine at position 63) is coordinated as the sixth ligand of the ferric heme iron. The spontaneous formation of such a hemichrome was at variance with the alpha  chain, as well as with the HbO2 tetramer, during the course of the autoxidation reaction over the wide range of pH 5-11. Therefore, it appears that the distal heme pocket of the alpha  chains, as well as the beta chains in the HbA tetramer, is more rigid than that of the beta  chain tetramer.

When HbO2 is placed in dilution, the tetrameric species is known to dissociate into alpha beta dimers along alpha 1beta 2 interface, so that the dimers formed are of the alpha 1beta 1 type (35, 36). In a previous paper, we reported that the alpha beta dimers can be oxidized to the ferric met form without any spectral evidence for the formation of hemichromes. In the alpha beta dimers, we also found that the slow component (ks) due to the beta  chain is quite resistant to the acidic autoxidation (5). These results imply that the intrinsic tendency of the beta  chain to form a hemichrome, as well as to produce an acid-catalyzed autoxidation, must have been suppressed by the formation of the alpha 1beta 1 (or alpha 2beta 2) contact.

In this connection, Borgstahl et al. reported the 1.8 Å structure of carbonmonoxy-beta 4 (CObeta 4) tetramer of human hemoglobin, and compared subunit-subunit contacts between three types of interfaces (alpha 1beta 1, alpha 1beta 2, and alpha 1alpha 2) of oxyHb and the corresponding CObeta 4 interfaces (37). In their examinations, interfaces were defined using a 3.5-Å cutoff and at least one interaction is <3.5 Å in each case. As a result, they found that, in contrast to the stable beta 1beta 4 interface, the beta 1beta 2 interface of the CObeta 4 tetramer is less stable and more loosely packed than its alpha 1beta 1 counterpart in oxyHb. 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 ~1 Å in CObeta 4 relative to oxyHb. Specifically, the CObeta 4 beta 1beta 2 interface 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 center of the CObeta 4 beta 1beta 2 interface packed less tightly. Therefore, the contact sites in the beta 4 tetramer are indeed different from the alpha 1beta 1 contact sites in the HbA tetramer. This supports explicitly our conclusion described above. As is evident from Fig. 3, the remarkable stability of HbO2 tetramer can be ascribed mainly to the delayed autoxidation of the beta  chains at acidic pH range. It is also clear that the beta  chain has acquired this stability against autoxidation by blocking out the proton catalysis (Reaction 3) from the aqueous autoxidation, in a manner that the distal histidine cannot act as its catalytic residue.

In this regard, Shaanan (38) investigated the stereochemistry of the iron-dioxygen bond in human HbO2 tetramer by single-crystal x-ray analysis. In the alpha  chain, the distance between Nepsilon of His(E7) and the terminal oxygen atom (O-2) is found to be 2.7 Å, and the geometry favors a similar hydrogen bond as in oxymyoglobin (25). In the beta  chain, however, Nepsilon of His(E7) is located further both from O-2 and O-1 (3.4 and 3.2 Å, respectively), indicating that the hydrogen bond, if formed, is much weaker. Our kinetic results seem to be in good accord with the crystal structural evidence for the FeO2 bonding in HbO2 tetramer.

Fig. 10 illustrates in a very schematic way the structure of HbO2 tetramer, as seen in the alpha beta contact leading to the nonequivalence of the chains. The four heme pockets are all exposed at the surface of the molecule. The distal histidine can stabilize the bound dioxygen by hydrogen bond formation. Nevertheless, it is also true that each FeO2 bonding is always subject to the nucleophilic attack of an entering water molecule including its conjugate ions OH- and H+. In this process, the distal histidine is proposed to participate, via its imidazole ring and a proton-relay mechanism, in facilitating the effective movement of a catalytic proton from the solvent to the bound, polarized dioxygen. This proton-catalyzed nucleophilic displacement of Obardot 2 from the FeO2 center by the entering water molecule, a SN-2 type process with proton assistance, can account for most of the autoxidation reaction occurring under physiological conditions. These features are essentially the same as those in the autoxidation of myoglobin (3, 23, 24).


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Fig. 10.   Schematic representation of human oxyhemoglobin as seen in the alpha 1beta 1 contact to produce tilting of the distal histidine in the beta  chain. In HbO2 tetramer, the heme pockets are all exposed at the surface of the molecule. By the formation of the alpha 1beta 1 contact, the beta  chain is subject to a structural constraint whereby the distal histidine at position 63 is tilted away slightly from the bound O2. As a result, the beta  chain loses a proton-catalyzed process, and HbO2 tetramer can acquire the enhanced stability against the autoxidation reaction.

In the hemoglobin molecule, however, there are two types of the alpha beta contacts. One is the alpha 1beta 1 (or alpha 2beta 2) contact involving B, G, and H helices and the GH corner, and other is the alpha 1beta 2 (or alpha 2beta 1) contact involving mainly helices C and G and the FG corner (37, 39). The mechanism whereby alpha  and beta  chains acquire the enhanced stability against the aqueous autoxidation must be associated with the formation of the alpha 1beta 1 (or alpha 2beta 2) contact. These packing contacts are also expected to produce in the beta  chains a conformational constraint, whereby the distal histidine at position 63 is tilted away slightly from the bound O2 so as to prevent the acid-catalyzed autoxidation from the beta  chains in the HbO2 tetramer. In this way, the hemoglobin molecule seems to differentiate two types of the alpha beta contacts quite properly for its own function. The alpha 1beta 2 and alpha 2beta 1 contacts are associated with the cooperative oxygen binding, whereas the alpha 1beta 1 and alpha 2beta 2 contacts are used for controlling the stability of the bound O2. In fact, the former sliding contacts are known to undergo the principal change when the hemoglobin molecule goes from its deoxy to oxy configuration during the course of the ligand binding (1, 39). To the latter packing contacts, for the first time, we have assigned a new role in the stabilization of the HbO2 tetramer against autoxidation.

    ACKNOWLEDGEMENT

We thank Dr. Y. Ohba (Institute for Chemical Reaction Science, Tohoku University) for EPR measurements.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Inst. of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Nagano 386-8567, Japan.

§ Present address: Dept. of Health Science, Hiroshima Prefectural Women's University, Hiroshima 734-8558, Japan.

To whom all correspondence should be addressed. Fax: 81-22-263-9206.

1 The abbreviations used are: p-MB, sodium p-hydroxymercuribenzoate; EPR, electron paramagnetic resonance; Mes, 2-(N-morpholino)ethanesulfonic acid; Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid); Mops, 3-(N-morpholino)propanesulfonic acid; Taps, 3-[tris(hydroxymethyl)methyl]aminopropanesulfonic acid; Caps, 3-(cyclohexylamino)propanesulfonic acid.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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