Quaternary Structure Regulates Hemin Dissociation from Human Hemoglobin*

(Received for publication, February 11, 1997, and in revised form, May 2, 1997)

Mark S. Hargrove Dagger §, Timothy Whitaker Dagger , John S. Olson Dagger , Rita J. Vali and Antony J. Mathews

From the Dagger  Department of Biochemistry and Cell Biology and W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005-1892 and Somatogen Inc., Boulder, Colorado 80301-2857

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Rate constants for hemin dissociation from the alpha  and beta  subunits of native and recombinant human hemoglobins were measured as a function of protein concentration at pH 7.0, 37 °C, using H64Y/V68F apomyoglobin as a hemin acceptor reagent. Hemin dissociation rates were also measured for native isolated alpha  and beta  chains and for recombinant hemoglobin tetramers stabilized by alpha  subunit fusion. The rate constant for hemin dissociation from beta  subunits in native hemoglobin increases from 1.5 h-1 in tetramers at high protein concentration to 15 h-1 in dimers at low concentrations. The rate of hemin dissociation from alpha  subunits in native hemoglobin is significantly smaller (0.3-0.6 h-1) and shows little dependence on protein concentration. Recombinant hemoglobins containing a fused di-alpha subunit remain tetrameric under all concentrations and show rates of hemin loss similar to those observed for wild-type and native hemoglobin at high protein concentration. Rates of hemin dissociation from monomeric alpha  and beta  chains are much greater, 12 and 40 h-1, respectively, at pH 7, 37 °C. Aggregation of monomers to form alpha 1beta 1 dimers greatly stabilizes bound hemin in alpha  chains, decreasing its rate of hemin loss ~20-fold. In contrast, dimer formation has little stabilizing effect on hemin binding to beta  subunits. A significant reduction in the rate of hemin loss from beta  subunits does occur after formation of the alpha 1beta 2 interface in tetrameric hemoglobin. These results suggest that native human hemoglobin may have evolved to lose heme rapidly after red cell lysis, allowing the prosthetic group to be removed by serum albumin and apohemopexin.


INTRODUCTION

Human hemoglobin is protected against denaturation by encapsulation in red blood cells. The iron atoms are kept in the ferrous state by intracellular methemoglobin reductases, and Obardot 2 generated by spontaneous autooxidation is rapidly transformed into H2O and O2 by superoxide dismutase and catalase (1, 2). Heme loss is inhibited by maintenance of the reduced state, the high concentration of cytoplasmic hemoglobin, and the presence of a cell membrane which prevents dispersal and precipitation of any dissociated prosthetic group. When hemoglobin is released by a small amount of red cell lysis, it is diluted significantly in plasma. In the case of human hemoglobin, this dilution leads to formation of noncooperative, alpha 1beta 1 dimers which display epitopes that are recognized by circulating haptoglobin molecules (2, 3). Binding of dimers to haptoglobin facilitates rapid clearance from the blood stream (Fig. 1).


Fig. 1. Denaturation and clearance of hemoglobin in vivo (adapted from Bunn and Forget (2). The high concentration of hemoglobin inside red blood cells prevents formation of dimers, and reduction systems maintain the heme-iron in the Fe2+ state. Dilution of hemoglobin into the plasma after red cell lysis results in formation of dimers which facilitate autooxidation and hemin dissociation. Holo- and apohemoglobin dimers are removed by haptoglobin, and free hemin is rapidly taken up by serum albumin or apohemopexin.
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Hemoglobin dimers also autooxidize more rapidly and lose hemin more readily than hemoglobin tetramers (4-6). Rapid hemin loss from dilute, extracellular hemoglobin may be advantageous since the resulting free heme in plasma is readily taken up by serum albumin and apohemopexin and transported to the liver for recycling. Thus, we felt that it would be important to measure quantitatively how the state of aggregation affects the rate of hemin loss from the alpha  and beta  subunits of human hemoglobin.

Roughly 30 years ago, Bunn and Jandl (7) measured time courses for 59Fe-labeled hemin exchange between human adult and fetal hemoglobins and between adult hemoglobin and human serum albumin. Quantitative analysis suggested that the rate of hemin exchange with beta  subunits was 5-10-fold greater than with alpha  subunits. Benesch and Kwong (5) showed that partial hemin exchange between methemoglobin and human serum albumin can be followed spectrophotometrically at pH values >= 8. They measured rates of hemin dissociation from beta  subunits in a variety of native and mutant human hemoglobins. However, even at extremely high concentrations, human serum albumin is unable to extract significant amounts of hemin from alpha  subunits within intact hemoglobin. To overcome this problem, we developed a genetically engineered apoglobin for use as a colorimetric reagent to measure complete time courses of hemin dissociation from both myoglobins and hemoglobins (8).

The reagent is a myoglobin mutant in which the distal histidine (His-64) was replaced with tyrosine. The phenolate side chain coordinates to the iron atom giving the ferric form of the mutant holoprotein a "green" color and an absorbance spectrum very different from those of native metmyoglobins and methemoglobins which appear "brown." In addition, Val-68(E11) was replaced with Phe to enhance the stability of the apoprotein and to increase its affinity for hemin. When methemoglobin Ao is mixed with an excess of the H64Y/V68F apomyoglobin reagent, complete hemin exchange occurs and the observed time course is markedly biphasic (see Fig. 2). Using valence and mutant hybrid hemoglobins, we were able to confirm that the faster phase represents hemin loss from ferric beta  subunits and the slower phase hemin loss from alpha  subunits (8).


Fig. 2. Effects of inositol hexaphosphate on rates of hemin dissociation from hemoglobin. Hemin dissociation from 1 µM hemoglobin was measured in 0.2 M Bis-Tris, 0.45 M sucrose, pH 7.0, ± 0.2 mM inositol hexaphosphate. The rate of hemin dissociation from beta  subunits was 16 h-1 in the absence of inositol hexaphosphate and 3 h-1 in the presence of 0.2 mM inositol hexaphosphate. The decrease in k-H for beta  subunits is due to inositol hexaphosphate-induced aggregation to tetramers.
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Our rate constants for hemin loss, which were measured at low hemoglobin concentrations (1-10 µM), were significantly larger than those reported by Bunn and Jandl (7), which were measured at high protein concentration. We speculated that the differences were due to more rapid hemin dissociation from methemoglobin dimers that were present at the low concentrations used in our experiments. Benesch and Kwong (6) confirmed this idea directly by measuring the dependence of the rate of hemin dissociation from beta  subunits on hemoglobin concentration. However, since Benesch and Kwong (6) were using the human serum albumin assay, they were unable to examine the effects of dimer formation on hemin dissociation from alpha  subunits.

In this work, we have measured the rate constants for hemin loss from isolated alpha  and beta  subunits, alpha 1beta 1 dimers, native tetramers, and recombinant tetramers stabilized by alpha  gene fusion. The rate constants for hemin dissociation from native dimers and tetramers were obtained by analyzing the protein concentration dependence of the observed time courses. The results provide a quantitative description of the linkage between quaternary structure and hemin binding in the alpha  and beta  subunits. These data also explain why it is so difficult to prepare the aquomet forms of isolated chains. Finally, the high rates of hemin dissociation observed in dimers and monomers support the view that human hemoglobin has evolved to fall apart rapidly in dilute solution.


MATERIALS AND METHODS

Preparation of Proteins

A description of the properties of the H64Y/V68F apomyoglobin reagent and its use in measuring complete time courses of hemin loss from ferric myoglobins and hemoglobins are given in Hargrove et al. (8). Native human hemoglobin was prepared as described by Mathews et al. (9), and isolated alpha  and beta  subunits were purified by the method of Bucci (10). Recombinant wild-type human hemoglobin containing V1M replacements in alpha  and beta  subunits (rHb0.0)1 and genetically stabilized hemoglobins containing a glycine linker (rHb0.1, rHb1.1) between the C terminus of one alpha  subunit and the N terminus of another were purified as described by Looker et al. (11, 12). Some preparations of recombinant hemoglobin were purified by a modified procedure in which deoxygenated crude lysate was first heat treated at 65 °C for 30 min to precipitate Escherichia coli proteins. After cooling to 4 °C, polyethyleneimine was added, and the dense mass of E. coli proteins and nucleic acids was removed by centrifugation. Following centrifugation, hemoglobin was purified by sequential ion exchange chromatography using (1) Q-column, (2) S-column, and (3) Q-Sepharose Fast Flow resins (Pharmacia Biotech). These columns were equilibrated and eluted as described for columns Q2 and S1 in Looker et al. (11). In the present work, the Q-column chromatographic step was repeated after the S-column to remove additional methemoglobin. After the second Q-column procedure, ferrous hemoglobin containing fractions were pooled as above, concentrated to 50 mg/mL and stored as small aliquots in liquid nitrogen.

Measurement of Hemin Dissociation

The general procedures for measurement of hemin dissociation are described in Hargrove et al. (8). Unless otherwise indicated, experiments were performed at 37 °C in 0.15 M potassium or sodium phosphate buffer, pH 7.0 and 0.45 M sucrose. In all experiments, the concentration of apomyoglobin (H64Y/V68F) was at least twice the total Hb (heme) concentration. Under these conditions, the observed rate is equal to the first order rate of hemin dissociation from methemoglobin (8).

Below 12 µM Hb, dissociation time courses were monitored at 410 nm. Between 12 and 35 µM Hb, time courses were monitored at 600 nm. It was not possible to record continuous time courses above 35 µM Hb due to turbidity caused by precipitation of large amounts of apohemoglobin. Consequently, at high hemoglobin concentrations (200-600 µM heme) small aliquots were withdrawn at appropriate time points from a stock reaction mixture and centrifuged briefly (~30 s) at 14,000 rpm in a refrigerated bench top microcentrifuge. A measured volume of supernatant was then diluted into 10 mM sodium phosphate buffer, pH 8, and the visible spectrum recorded quickly. The changes in absorbance at 410 and 600 nm were used to monitor the formation of H64Y/V68F holomyoglobin and the concomitant disappearance of methemoglobin (see Fig. 3A).


Fig. 3. A, hemin dissociation from 1 and 600 µM native hemoglobin. The open circles represent data at high concentration and the small dots, data at 1 µM hemoglobin. B, hemin dissociation from 10 µM recombinant human hemoglobin (rHb(0.0)) and 5 µM genetically cross-linked human hemoglobin (rHb(0.1)). Each curve was fitted to a two exponential expression which was forced to have equal amplitudes. Genetic stabilization of hemoglobin tetramers clearly lowers the rate of hemin dissociation from beta  subunits.
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Analysis of Hemin Dissociation Time Courses

Absorbance traces at 410 and 600 nm were exported into data analysis software and analyzed as the sum of two independent exponential decay processes. In the latter stages of the reactions (>4 h), the absorbance traces were frequently distorted for samples at initial Hb concentrations greater than 20 µM due to precipitation of apohemoglobin. In these cases the most useful method of analysis was to truncate the absorbance trace to avoid regions showing absorbance increases due to apoprotein aggregation.


RESULTS

Results for Native Human Hemoglobin

Time courses for complete hemin loss from hemoglobin Ao are biphasic under all conditions. As shown in Fig. 2, addition of inositol hexaphosphate at low protein concentrations preferentially decreases the rate of the fast or beta  subunit phase of the reaction from ~16 h-1 to ~3 h-1. The rate constant for the slow phase was unaffected and remained at ~0.5 h-1. This result suggests that tetramer formation stabilizes hemin in beta  subunits since inositol hexaphosphate promotes tetramer formation by binding to the positively charge cleft between the beta  subunits of tetrameric hemoglobin (13, 14).

Time courses for hemin loss from native human hemoglobin at high and low protein concentrations are shown in Fig. 3A. As observed by Benesch and Kwong (6), the rate of hemin loss from beta  subunits (fast phase) is reduced almost 10-fold when hemoglobin concentration is increased from 1 to 600 µM. Time courses analogous to those in Fig. 3A were collected at 12 different protein concentrations, and the dependence of the fitted rate constants on heme concentration is shown in Fig. 4.


Fig. 4. Dependence of rate constants for heme dissociation on protein concentration. Hemin dissociation was measured at concentrations ranging from 1 to 600 µM in heme. Each time course was fitted to a two-exponential expression with equal amplitudes. The slow and fast phases were assigned to hemin dissociation from alpha  and beta  subunits, respectively. The protein concentration dependence of the rate constants for hemin dissociation from alpha  and beta  subunits was then fitted to Equations 1 and 2. Data for native alpha  and beta subunits are shown as open and closed circles, respectively; data for wild-type recombinant (rHb0.0) alpha  and beta  subunits are shown as open and closed squares; data for the alpha  and beta  subunits of genetically cross-linked hemoglobin (rHb0.1 and rHb1.1) are shown as open and closed triangles, respectively. In panel B, the dashed-dotted line represents at fit using Equations 1 and 2 and a fixed value of K4,2 = 1 µM. The dotted line assumes no protein concentration dependence of k-H for alpha  subunits in native hemoglobin rHb1.1 and rHb0.1. The dashed line represents the average value for alpha subunits in rHb0.0.
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Time courses for hemin dissociation from native hemoglobin were biphasic under all conditions. The rate constant for the faster or beta  phase decreased markedly with increasing protein concentration whereas the rate of the slow or alpha  phase showed little change (Fig. 4). Analysis of these data is simplified by the fact that the rate of formation and dissociation of hemoglobin tetramers is very rapid compared with that for hemin dissociation. The rate constants for tetramer dissociation and dimer aggregation are 1-10 s-1 and 1-5 × 105 M-1 s-1, respectively, for R-state forms of hemoglobin (3, 15, 16), whereas the rate constants for hemin dissociation are <= 0.005 s-1 (Table I). Thus, the observed rate of hemin loss from an alpha  or beta  subunit is a weighted sum of the rate constants that apply for the subunit in dimers and in tetramers.
k<SUB><UP>−H</UP></SUB>(<UP>obs</UP>)<UP> = </UP>k<SUP><UP>dimer</UP></SUP><SUB><UP>−H</UP></SUB><UP>&ggr;<SUB>dimer</SUB> + </UP>k<SUP><UP>tetramer</UP></SUP><SUB><UP>−H</UP></SUB>(<UP>1 − &ggr;</UP><SUB><UP>dimer</UP></SUB>) (Eq. 1)
where gamma dimer is the fraction of heme groups that are present in dimers. gamma dimer is given by (6, 17)
<UP>&ggr;<SUB>dimer</SUB> = </UP><FR><NU><UP>−</UP><FR><NU>K<SUB>4,2</SUB></NU><DE><UP>H</UP><SUB>0</SUB></DE></FR>+<RAD><RCD><FENCE><FR><NU>K<SUB>4,2</SUB></NU><DE><UP>H</UP><SUB>0</SUB></DE></FR></FENCE><SUP>2</SUP>+4<FR><NU>K<SUB>4,2</SUB></NU><DE><UP>H</UP><SUB>0</SUB></DE></FR></RCD></RAD></NU><DE>2</DE></FR> (Eq. 2)
where K4,2 is the equilibrium dissociation constant describing the dissociation of hemoglobin tetramers into dimers (K4,2 = [Hb2]2/[Hb4]) and H0 is the total concentration of heme groups (H0 = 2[Hb2] + 4[Hb4]).

Table I. Rate constants for hemin dissociation from human hemoglobin monomers, dimers, and tetramers at pH 7.0, 37 °C


Hemoglobin k-H monomer k-H dimer k-H tetramer

h-1
 alpha (native) 12 0.6 0.3
 alpha (0.0) 0.5 0.6
 alpha (0.1) 0.3
 alpha (1.1) 0.5
 beta (native) 40 15 1.5
 beta (0.0) 33 1.7
 beta (0.1) 1.5
 beta (1.1) 2.5

The solid line in Fig. 4A for beta  subunits within native hemoglobin represents a fit in which the values of k-Hdimer and K4,2 were varied, and k-Htetramer was fixed at 1.5 h-1, the value observed for beta  subunits in genetically stabilized rHb0.1. The fitted parameters are listed in Table I. The value obtained for K4,2 was 1.5 µM, which is very close to that determined for native hemoglobin by Edelstein et al. (17) using flash photolysis and ultracentrifugation techniques. Convergence was more difficult when all three parameters were varied. Similar values of k-Hdimer and k-Htetramer were obtained when K4,2 was fixed to 1 µM (17). Regardless of the exact analysis, the results in Fig. 4A show that k-H for beta  subunits decreases greater than 10-fold when alpha 1beta 1 dimers aggregate to form tetramers in agreement with the previous work of Benesch and Kwong (6).

In contrast, the rate of hemin dissociation from alpha  subunits shows little dependence on hemoglobin concentration. If K4,2 is fixed at 1.5 µM, the fitted values of k-H for alpha  subunits are 0.6 h-1 in dimers and 0.3 h-1 in tetramers. Fits of similar quality were obtained by assuming no dependence and an average value of 0.4 h-1. Regardless of the exact analysis, the results show that dissociation into dimers has little effect on hemin affinity in alpha  subunits.

Recombinant Hemoglobins Stabilized by alpha  Subunit Fusion

Fig. 3B shows time courses for hemin dissociation from recombinant, wild-type human hemoglobin (rHb0.0) and a recombinant human hemoglobin stabilized against dimer formation by fusion of two alpha  subunit genes into a single gene (rHb0.1). In both genes the codons for the N-terminal valines in both subunits were replaced with methionine codons to initiate translation in E. coli. Since the initiator methionine is retained, each subunit has effectively a V1M mutation (11). In rHb0.1, a glycine residue connects the C terminus of one alpha  chain with the N terminus of a second alpha  chain. This subunit fusion prevents dissociation of the (alpha -alpha )beta 2 tetramer into dimers (11).

Although both proteins were at low concentrations (5-10 µM), the rate of hemin dissociation from beta  subunits in the di-alpha containing hemoglobin was 10-fold slower than that from beta  subunits in the wild-type control (Fig. 3B). The absolute value (~1.5 h-1) is roughly equal to that observed for beta  subunits within native human hemoglobin at very high heme concentrations (600 µM trace in Fig. 3A). As shown in Fig. 4, the rate of hemin loss from beta subunits in genetically stabilized hemoglobin (rHb0.1) shows no dependence on protein concentration and serves to fix the value of k-H for these subunits in hemoglobin tetramers. In contrast, the rate of hemin loss from beta  subunits in the wild-type control (rHb0.0) shows a dependence on protein concentration which is similar to that observed for native HbA0 (Fig. 4A). The fitted values of beta  k-Hdimer and K4,2 for (rHb0.0) were 33 h-1 and 2.6 µM, respectively, when k-Htetramer was fixed at 1.5 h-1. Thus, the V1M mutations in the recombinant protein appear to increase the rate of hemin loss from beta  subunits in dimers approximately 2-fold. As in the case of native hemoglobin, the rate of hemin loss from alpha  subunits appears to be independent of protein concentration and is unaffected by genetic fusion (Fig. 4, Table I).

We examined rates of hemin dissociation from rHb1.1 which, in addition to the V1M mutations and fused alpha  subunits, contains the Presbyterian mutation (beta  N108K). The latter substitution was added to enhance the O2 transport properties of the recombinant protein (11). As with rHb0.1, no dependence on protein concentration was observed, and the only difference was a small increase in k-H for beta  subunits which is presumably due to the N108K mutation in this subunit.

Hemin Dissociation from alpha  and beta  Monomers

Time courses for hemin dissociation from native isolated alpha  and beta  chains were measured in a stopped flow apparatus because of their high rates of hemin loss and the difficulty of sample preparation. A slight excess of potassium ferricyanide was added to a syringe containing 20 µM of the oxygenated forms of either alpha  or beta  chains to generate the corresponding ferric forms. Immediately after formation of the ferric subunit, the contents of this syringe were reacted with 40 µM H64Y/V68F apomyoglobin. Time courses for hemin dissociation were measured at 600 nm to avoid background absorbance by excess ferricyanide (lambda max = 400 nm). Slow absorbance increases were observed at the end of each reaction due to precipitation of the newly generated apoglobin chains. Hemin dissociation rate constants were estimated from the initial portions of the time courses by fitting to one or two exponential expressions with an offset (Fig. 5). Hemin dissociation from alpha  chains appears to be monophasic with a rate constant equal to ~12 h-1 at 37 °C, pH 7. Time courses for hemin dissociation from isolated beta  subunits were biphasic, with observed rate constants equal to ~40 h-1 and ~2 h-1 for the fast and slow phases. McGovern et al. (18) estimated that the equilibrium constant for dissociation of beta  tetramers into monomers is 1.25 × 10-12 M3 at pH 7. This value of K4,1 predicts a significant amount of beta  tetramers at the concentrations used in our experiments. Thus, the simplest interpretation of the isolated beta  chain time course is that hemin dissociation from monomeric beta  chains is very rapid (k-H approx  40 h-1), whereas hemin loss from beta  tetramers is slow (k-H approx  2 h-1) and comparable to that from beta  subunits within tetrameric hemoglobin.


Fig. 5. Hemin dissociation from isolated alpha  and beta  subunits and intact native methemoglobin at pH 7, 37 °C. The concentrations of all three proteins was ~10 µM after mixing in a stopped slow apparatus as described in the text. The hemin dissociation rates for the beta  and alpha  subunits in native hemoglobin were 7 and 0.5 h-1, respectively. Hemin dissociation from isolated alpha  chains showed a single phase with k-H = 12 h-1. Hemin dissociation from isolated beta  chains showed a fast phase (k-H = 40 h-1), presumably associated with beta  monomers, and a slower phase (k-H approx  2 h-1) associated with beta  tetramers.
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DISCUSSION

The quaternary structure of methemoglobin has a profound effect on the rate of hemin dissociation (Table I). The monomeric forms of the isolated alpha  and beta  chains lose hemin 30-40 times more rapidly than the corresponding subunits in a tetramer. Hargrove et al. (19) have shown that the association rate constant for the binding of monomeric heme to apoglobins is always ~1 × 108 M-1 s-1 regardless of the exact protein structure. Thus, the equilibrium constant for hemin dissociation can be computed as K-H = k-H/(1 × 108 M-1 s-1) where k-H is converted from units of h-1 to s-1. Equilibrium dissociation constants for hemin binding to monomeric, dimeric, and tetrameric native hemoglobin are listed in Table II and compared with the value for sperm whale myoglobin under the same conditions.

Table II. Comparison of the equilibrium dissociation constants for hemin binding to the alpha  and beta  subunits of native human hemoglobin and sperm whale myoglobin at pH 7.0, 37 °C

The K-H values in picomolar were calculated using the dissociation rate constants in Table I and assuming that the association rate constant is ~1 × 108 M-1s-1 for all three apoproteins, regardless of quaternary state (24).
Protein K-H monomer K-H dimer K-H tetramer

pM
Sperm whale myoglobin 0.03
 alpha (native) 33 1.7 0.8
 beta (native) 110  42 4.2

The results in Tables I and II provide a quantitative explanation for why the aquomet forms of isolated alpha  and beta  chains are so unstable. The half-times for hemin dissociation are 1-3 min at 37 °C, and although the equilibrium constants are still ~10-10 M, rebinding has to compete with irreversible hemin aggregation and precipitation. In addition, the apoprotein forms of the isolated subunits are very unstable at 37 °C and precipitate almost immediately after heme removal.

The instability of apoglobin monomers and their low affinity for hemin may explain why it is difficult to express the alpha  and beta  subunits of human hemoglobin separately as soluble holoproteins in E. coli. In contrast, co-expression of the subunits yields high levels of soluble hemoglobin (11, 12, 20). Dimer and tetramer formation are required to stabilize the apoproteins and to increase hemin affinity. In contrast apomyoglobin is much more stable and has an affinity for hemin which is 1,000-3,000-fold greater than that of the isolated subunits of hemoglobin. This result accounts for the ease of expression of sperm whale holomyoglobin in bacteria (21-23).

Formation of alpha 1beta 1 dimers from monomers causes a 30-fold increase in the affinity of alpha  subunits for hemin, whereas only a 2-fold increase is observed for beta  subunits. The structural cause of this selectivity is not clear since the alpha 1beta 1 interface involves mostly hydrophobic contacts between the B, G, and H helices of the two subunits, regions which are far removed from the heme pocket in both proteins. Presumably, formation of these contacts stabilizes the overall, tertiary structure of alpha  but not beta  subunits. Association of dimers into tetramers is driven primarily by formation of the alpha 1beta 2 interface which involves more polar contacts between the C and N termini and the C-helices and FG corners of both subunits. The C-helix is near the heme group and the FG corner serves to position the proximal His(F8) for direct coordination to the iron atom. These interactions are required for strong hemin binding to beta  subunits. A more detailed structural interpretation will require systematic mutagenesis studies analogous to those carried out for sperm whale myoglobin (24).


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.
§   Recipient of graduate fellowships from the National Institutes of Health Training Grant GM-08280.
   Supported by United States Public Health Service Grants GM-35649 and HL-47020, Grant C-612 from the Robert A. Welch Foundation, and the W. M. Keck Foundation. To whom correspondence should be addressed: Dept. of Biochemistry and Cell Biology, Rice University, W. M. Keck Center for Computational Biology, Houston, TX 77005-1892. Tel.: 713-527-4015; Fax: 713-285-5154; E-mail: bio{at}rice.edu.
1   The abbreviations used are: rHb, recombinant hemoglobin; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.

ACKNOWLEDGEMENTS

We thank Dr. Douglas Lemon and Dr. Carol Cech at Somatogen, Inc. for reading the manuscript critically and making several helpful suggestions.


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