Proton nuclear magnetic resonance and biochemical studies of oxygenation of human adult hemoglobin in deuterium oxide.

The proton nuclear magnetic resonance spectrum of human adult deoxyhemoglobin in D2O in the region from 6 to 20 ppm downfield from the proton resonance of residual water shows a number of hyperfine shifted proton resonances that are due to groups on or near the alpha and beta hemes. The sensitivity of these resonances to the ligation of the heme groups and the assignment of these resonances to the alpha and beta chains provide an opportunity to investigate the cooperative oxygenation of an intact hemoglobin molecule in solution. By use of the nuclear magnetic resonance correlation spectroscopy technique, at least two resonances, one at approximately 18 ppm downfield from HDO due to the beta chain and the other at approximately 12 ppm due to the alpha chain, can be used to study the binding of oxygen to the alpha and beta chains of hemoglobin. The present results using approximately 12% hemoglobin concentration in 0.1 M Bistris buffer at pD 7 and 27 degrees C with and without organic phosphate show that there is no significant line broadening on oxygenation (from 0 to 50% saturation) to affect the determination of the intensities or areas of these resonances. It is found that the ratio of the intensity of the alpha-heme resonance at 12 ppm to that of the beta-heme resonance at 18 ppm is constant on oxygenation in the absence of organic phosphate but decreases in the presence of 2,3-diphosphoglycerate or inositol hexaphosphate, with the effect of the latter being the stronger. On oxygenation, the intensities of the alpha-heme resonance at 12 ppm and of the beta-heme resonance at 18 ppm decreases more than the total number of deoxy chains available as measured by the degree of O2 saturation of hemoglobin. This shows the sensitivity of these resonances to structural changes which are believed to occur in the unligated subunits upon the ligation of their neighbors in an intact tetrameric hemoglobin molecule. A comparison of the nuclear magnetic resonance data with the populations of the partially saturated hemoglobin tetramers (i.e., hemoglobin with one, two, or three oxygen molecules bound) leads to the conclusion that in the presence of organic phosphate the hemoglobin molecule with one oxygen bound maintains the beta-heme resonance at 18 ppm but not the alpha-heme resonance at 12 ppm. These resluts suggest that some cooperativity must exist in the deoxy quaternary structure of the hemoglobin molecule during the oxygenation process. Hence, these results are not consistent with the requirements of two-state concerted models for the oxygenation of hemoglobin. In addition, we have investigated the effect of D2O on the oxygenation of hemoglobin by measuring the oxygen dissociation curves of normal adult hemoglobin as a function of pH in D2O andH2O media. We have found that (1) the pH dependence of the oxygen equilibrium of hemoglobin (the Bohr effect) in higher pH in comparison to that in H2O medium and (2) the Hill coefficients are essentially the same in D2O and H2O media over the pH range from 6.0 to 8.2...     

Spin label studies on conformational changes of aphohemoglobin due to heme binding.

Human apohemoglobin (globin) was spin-labeled at the beta-93 sulfhydryl groups with 2,2,5,5-tetramethyl-3-aminopyrrolidine-I-oxyl. Spin-labeled globin exhibited an EPR spectra that is less immobilized than that of spin-labeled hemoglobin, indicating the conformational difference in the vicinity of the label between hemoglobin and globin. Spectrophotometric titration of spin-labeled globin with protohemin showed that 1 mol of globin (on the tetramer basis) combines with 4 mol of hemin, producing a holomethemoglobin spectrophotometrically indistinguishable from native methemoglobin. The EPR spectrum was also changed strikingly upon the addition of protohemin. This change, however, was not proportional to the amount of hemin added, but marked changes occurred after 3 to 4 mol of hemin were mixed with 1 mol of spin-labeled globin. The EPR spectrum of spin-labeled hemoglobin thus prepared was identical with that prepared by direct spin labeling to methemoglobin. These results suggest the preferential binding of hemin to alpha-globin chains in the course of heme binding by globin. This assumption was further confirmed by preparing spin-labeled semihemoglobin in which only one kind of chain contained hemin (alpha h betaO SL and alpha O beta h SL). The EPR spectrum of the alpha h beta O SL molecule showed a slightly immobilized EPR spectrum, similar to that of spin-labeled globin mixed with 50% of the stoichiometric amount of hemin. On the other hand, the alpha O beta h SL molecule showed a distinctly different EPR signal from that of globin half-saturated with hemin, and showed an intermediate spectrum between those of beta h SL and alpha h beta h SL. These results indicate that heme binding to globin chains brings about a major conformational change in the protein moiety and that chain-chain association plays a secondary role. We conclude that hemin binds preferentially to alpha-globin chains and that the conformation of globin changes rapidly to that of methemoglobin after all four hemes are attached to globin heme pockets.     

Kinetic resolution of ligand binding to the alpha and beta chains within human hemoglobin.

Nitric oxide has been used as a chain-specific, spin label of unliganded heme groups present in kinetic mixtures of human hemoglobin and n-butyl isocyanide. In these experiments, deoxyhemoglobin was reacted with n-butyl isocyanide for a controlled time and then mixed rapidly with a high concentration of nitric oxide to fill residual, unoccupied heme sites. The final mixture was frozen immediately after formation to prevent any displacement of bound isonitrile. The EPR spectrum of the frozen sample was resolved into alpha and beta nitric oxide components; these reflect the relative proportions of alpha- and beta-heme sites which were unoccupied by n-butyl isocyanide. Individual time courses for the alpha and beta subunits were obtained by varying the time between the formation of the isonitrile/hemoglobin mixture and its reaction with nitric oxide. At pH 7.0 only the beta chain time course exhibits an initial rapid phase; the alpha chain time course is monophasic, exhibiting almost, exponential behavior. This result shows unequivocally that the beta-hemes within deoxyhemoglobin react much more rapidly with n-butyl isocyanide than the alpha hemes.     

Studies on cobalt myoglobins and hemoglobins. Preparation of isolated chains containing cobaltous protoporphyrin IX and characterization of their equilibrium and kinetic properties of oxygenation and EPR spectra.

Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.     

Esterification of the propionate groups promotes alpha/beta hemoglobin chain homogeneity of CN-hemin binding

This study examines the post-translational role of peripheral propionate groups in the incorporation of the Fe-protoporphryin IX heme into nascent alpha- and beta-globin chains. Human apohemoglobin (a heme-free alpha/beta dimer) in 0.05 M potassium phosphate buffer, pH 7, at 20 degrees C was titrated with either CN-protohemin (native heme with two peripheral propionate groups), or CN-dimethylester hemin (a modified heme with two methyl ester groups in place of the propionate groups). Soret spectrophotometric CN-hemin titrations confirmed that a spectral shift resulted upon binding of protohemin, but no spectral shift occurred upon binding the dimethylester derivative. Recent studies have correlated a Soret spectral shift with the preferential heme binding to the alpha subunit of apohemoglobin. The absence of a Soret wavelength shift (in conjunction with molecular modeling) presented here suggested that the modification of heme propionate groups prevented the formation of an alpha-heme/beta-globin intermediate, a requisite step in the normal assembly of functional hemoglobin.     

NMR study of hybrid hemoglobins containing unnatural heme: effect of heme modification on their tertiary and quaternary structures

The effect of heme modification on the tertiary and quaternary structures of hemoglobins was examined by utilizing the NMR spectra of the reconstituted [mesohemoglobin (mesoHb), deuterohemoglobin (deuteroHb)] and hybrid heme (meso-proto, deutero-proto) hemoglobins (Hbs). The heme peripheral modification resulted in the preferential downfield shift of the proximal histidine N1H signal for the beta subunit, indicating nonequivalence of the structural change induced by the heme modification in the alpha and beta subunits of Hb. In the reconstituted and hybrid heme Hbs, the exchangeable proton resonances due to the intra- and intersubunit hydrogen bonds, which have been used as the oxy and deoxy quaternary structural probes, were shifted by 0.2-0.3 ppm from that of native Hb upon the beta-heme substitution. This suggests that, in the fully deoxygenated form, the quaternary structure of the reconstituted Hbs is in an "imperfect" T state in which the hydrogen bonds located at the subunit interface are slightly distorted by the conformational change of the beta subunit. Moreover, the two heme orientations are found in the alpha subunit of deuteroHb, but not in the beta subunit of deuteroHb, and in both the alpha and beta subunits of mesoHb. The tertiary and quaternary structural changes in the Hb molecule induced by the heme peripheral modification were also discussed in relation to their functional properties.     

Binding modes of L35 to alpha- and beta-semihemoglobins: structural insights into the inequivalence of alpha- and beta-subunits of hemoglobin

It has been thought for several years that the greatly lowered oxygen affinity, high cooperativity, and heterotropic modulation displayed by tetrameric human hemoglobin (Hb) was an exclusive result of the assembly of high affinity alpha(1)beta(1) dimers into alpha(2)beta(2) tetramers. However, in recent times, it has been shown that alpha- and beta-semihemoglobins, namely alpha(heme)beta(apo) and alpha(apo)beta(heme), which are dimers of Hb characterized by a high affinity for oxygen and lack of cooperativity do respond to effectors such as 2-[4-(3,5-dichlorophenylureido) phenoxy]-2-methylpropionic acid (L35), a bezafibrate (BZF) related compound, by decreasing the ligand affinity to a considerable extent (between 60- and 130-fold). In order to shed some light on the structural basis of this phenomenon, we have developed a binding mode of L35 to semihemoglobins through docking analysis using the program GRID. Molecular modelling studies did identify sites on semihemoglobins where favourable interactions with L35 can occur. We found that the effector binds differently to the two semihemoglobins exhibiting high affinity only for the alpha chain heme pocket. The proposed binding models are consistent with the experimental findings and may be rationalized in terms of different hydrophobic and hydrophilic characteristics between alpha- and beta-heme pockets of Hb.     

A study of conformational changes in two beta-93 modified hemoglobin A's using a triphosphate spin label.

The binding of oxygen and 1-oxyl-2,2,6,6-tetramethylpiperidine 4-triphosphate (spin-labeled triphosphate) to normal adult human hemoglobin (HbA) covalently labeled at the beta-93 sulfhydryl groups with N-(2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (I) was studied. HbA-I was used as a model for HbA labeled at the beta-93 SH groups with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide (II) since the binding of SLTP to HbA-II could not be measured conveniently, in the presence of the paramagnetic resonance signal of II. Both HbA-I and HbA-II can be treated as variant hemoglobins with abnormal beta chains. The oxygen and SLTP binding data from HbA-I and oxygen binding data from HbA-II are consistent with a concerted transition model for cooperativity which assumes nonequivalence between alpha and beta subunits (GCT model). The distribution of environments "seen" by conformation sensitive probes such as II and trifluoracetone (19F NMR probe) attached to the beta-93 sulfhydryl groups of HbA can also be accounted for by the GCT model. It is proposed that the beta-93 probes sense the dramatic change in beta subunit structure resulting from the quaternary structure change (T leads to R) upon heme saturation as well as tertiary structure changes at the alpha1-beta2 contact region resulting from ligand binding to the beta-heme group. Structural changes caused by ligation of the alpha-hemes are not discussed.     

Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).     

One- and two-dimensional NMR investigations of the heme pocket in free alpha(CO) chains from human hemoglobin

Two-dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated alpha(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J-correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOSEY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu86, Leu-91, Val-93, Leu-101 and Leu-136. Starting from the known crystallographic structure of the alpha subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring-current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the alpha(CO) subunits in crystals of HbA(CO) and of the free alpha(CO) chains. The one-dimensional NMR spectra were used to monitor the pH-induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p-chloromercuribenzoate is bound to Cys-104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.     

Substitution of the heme binding module in hemoglobin alpha- and beta-subunits. Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin

In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin alpha-subunit converted the heme proximal structure of myoglobin into the alpha-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol. 283, 311-327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the betaalpha(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin beta-subunit was replaced by that of hemoglobin alpha-subunit. Based on the gel chromatography, the betaalpha(HBM)-subunit was preferentially associated with the alpha-subunit to form a heterotetramer, alpha(2)[betaalpha(HBM)(2)], just as is native beta-subunit. Deoxy-alpha(2)[betaalpha(HBM)(2)] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N(delta)H proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet alpha(2)[betaalpha(HBM)(2)] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the betaalpha(HBM)-subunit in tetrameric alpha(2)[betaalpha(HBM)(2)] was similar to that of the beta-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.     

Conformational studies on the beta subunits of human hemoglobin and their arginyl-COOH peptides.

The beta subunits of hemoglobin upon alkylation of the cysteinyl residues with iodoacetamide showed a sedimentation velocity with an S20w, near 1.8 as for monomeric subunits. They reacted with alpha chains to give a tetrameric hemoglobin with a sedimentation constant near 4.4. Their CD spectrum was indistinguishable from that of untreated beta chains below 270 nm, otherwise they showed some deviation that became pronounced in the Soret region, where the optical activity of the alkylated subunits was definitely lower than that of the native subunits. Upon removal of the heme the apo-beta subunits showed a decreased optical activity in the far-uv region of the spectrum indicating a substantial loss of helical content. Their sedimentation behavior was consistent with the presence of large aggregates, which dissociates into monomers upon reconstitution with cyanoheme. The apo-beta subunits could be renatured from 6 M guanidine hydrochloride. They showed a stoichiometric reaction with heme in the molar ratio 1:1. Upon reconstitution with the heme their optical activity became similar to that of the native beta chains in the far-uv region of the spectrum, but remained lower in the near-uv and Soret regions. After acylation of the lysyl residues with citraconic anhydride the apo-beta subunits were digested with trypsin and the arginyl-COOH peptides beta(1-30), beta(31-40), beta(41-104), and beta(105-146) were separated by gel chromatography. With the exception of the peptide beta/105-146), which was insoluble at neutral pH, the sedimentation behavior of the other peptides showed the presence of small polymers. The sedimentation behavior of the peptide beta(31-40) was not tested. The percentage of alpha helix, beta conformation, and of random coil (or unordered structure) of the various proteins and peptides was measured fitting their CD spectra in the far-uv region with the parameter published by Y.H. Chen et al. ((1974), Biochemistry 13, 3350) and by N. Greenfield and G.D. Fasman ((1969), Biochemistry 8, 4108). In this way the helical content of the native and reconstituted alkylated beta subunits appeared to be near 76%, a value very near to that present in the same subunits in the hemoglobin crystal. The helical content of the apo-beta subunits in 0.04 M borate buffer at pH 9.6 decreased to a value near 45%. The helical content of the isolated peptides in electrolyte solutions was in any case near 10% indicating an almost complete loss of the structure that they have in the hemoglobin crystal. Cyanoheme reacted with the peptide beta(41-104), however, the reaction was not stoichiometric indicating a low affinity of the heme for the peptide. With the exception of the peptide beta(31-104), all of the other peptides recovered some of their helical structure when dissolved in 50% methanol. Notably also the apo-beta subunits did so suggesting that the loss of structure upon the removal of the heme could be in part due to the exposure of the heme pocket to water.     

Spectral, conformational and chemical properties of opossum methemoglobin

Opossum methemoglobin differs from methemoglobin A in spectral, spin state, conformational and chemical properties. The primary structural alterations in opossum hemoglobin, including the critical substitution at alpha 58 (E7) His leads to Gln result in the following properties. (a) Major contribution of the spectral transitions due to inositol hexakisphosphate binding arises from the alpha chains. (b) The aquomet to hydroxymet (high-spin to low-spin) transition as a function of pH is slightly retarded resulting in considerable high spin at alkaline pH. (c) The tertiary conformation (t) around the beta hemes, upon transition to a T quaternary state, differs from the known hemoglobin t tertiary structure. (d) Both alpha and beta hemes are susceptible to rapid reduction by ascorbic acid (the reduction rate being tenfold faster than that of methemoglobin A). These properties suggest that the heme environments in both the alpha and beta subunits of opossum hemoglobin are different from those of human hemoglobin A.     

Proton NMR study of methemoglobin and its isolated chains. Effect of the subunit association on the structure of the subunits.

In order to investigate the effect of the alpha beta subunit contacts on the subunit structure of human adult methemoglobin, the hyperfine shifted proton NMR spectra of several high spin complexes (water, cyanate, thiocyanate, formate, fluoride, and nitrite) and low spin complexes (imisazole, azide, and cyanide) of hemoglobin and its isolated subunits were characterized at 220 MHz and 22 degrees C. The spectra of ferric low spin derivatives of the isolated subunits were approximately superimposable on the corresponding hemoglobin spectra. On the other hand, the high spin spectra of the isolated subunits were greatly different from each other. The spectral anomaly in the ferric high spin complexes of the isolated beta subunit were interpreted to indicate other structural change than the hemichrome formation in the beta heme pocket. Difference in the subunit association effect between the high and low spin complexes of the isolated beta subunit was interpreted on the basis of a conformational change of the apoprotein dependent on the spin state of the beta heme iron.     

Monooxygenase activity of human hemoglobin: role of quaternary structure in the preponderant activity of the beta subunits within the tetramer

Catalysis of para hydroxylation of aniline was measured for human ferrihemoglobin and various derivatives in a reconstituted system consisting of the appropriate hemoprotein (at 4 microM heme), reduced nicotinamide adenine dinucleotide phosphate (NADPH), cytochrome P-450 reductase, and aniline under atmospheric O2. The isolated subunits of hemoglobin (alpha 3+ and beta 3+4) were prepared by treatment with p-(hydroxymercuri)benzoate. Semihemoglobin (alpha heme2 beta 02) was prepared from ferrihemoglobin and apohemoglobin. Converse valency hybrids alpha 3+2(beta 2+-CO)2 and (alpha 2+-CO)2 beta 3+2 were prepared from appropriately ligated alpha and beta subunits. After chromatography, the hemoglobin derivatives were characterized by visible and 1H NMR spectroscopy and electrophoresis. At the same concentration of aniline, the alpha and beta subunits were much less active than the normal tetramer. alpha-Semihemoglobin and the alpha 3+2(beta 2+-CO)2 hybrid also displayed lower hydroxylase activity. The (alpha 2+-CO)2 beta 3+2 hybrid was about as active as normal alpha 3+2 beta 3+2. This result suggests that the activity of tetrameric hemoglobin primarily involves the beta subunits. Also transfer of the beta subunits from the beta 4 molecular environment to the alpha 2 beta 2 state enhances their monooxygenase activity approximately 15-fold. The hemoglobin derivatives were differently susceptible to substrate inhibition, the beta 4 species being most sensitive. Estimates of Vmax from the linear portions of the corresponding Lineweaver-Burk plots showed agreement within a factor of 2.5 for all of the hemoglobin derivatives, suggesting that the intrinsic O2-activating capacities of the derivatives are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance raman studies of heme structural differences in subunits of deoxy hemoglobin

Low frequency resonance Raman (RR) spectra are reported for deoxy hemoglobin (Hb), its isolated subunits, its analogue bearing methine-deuterated hemes in all four subunits (Hb-d(4)), and the hybrids bearing the deuterated heme in only one type of subunit, which are [alpha(d4)beta(h4)](2) and [alpha(h4)beta(d4)](2). Analyzed collectively, the spectra reveal subunit-specific modes that conclusively document subtle differences in structure for the heme prosthetic groups in the two types of subunits within the intact tetramer. Not surprisingly, the most significant spectral differences are observed in the gamma(7) mode that has a major contribution from out of plane bending of the methine carbons, a distortion that is believed to relieve strain in the high-spin heme prosthetic groups. The results provide convincing evidence for the utility of selectively labeled hemoglobin hybrids in unraveling the separate subunit contributions to the RR spectra of Hb and its various derivatives and for thereby detecting slight structural differences in the subunits.     

Carnivora: primary structure of the hemoglobin from the spotted hyena (Crocuta crocuta, Hyenidae)

The primary structure of the alpha- and beta-chains of hemoglobin from spotted hyena (Crocuta crocuta, Hyenidae) is presented. The structure-function relationship is discussed. The separation of the chains directly from hemoglobin was performed by RP-HPLC. After tryptic digestion of the chains, the peptides were isolated by RP-HPLC. Amino-acid sequences were determined by Edman degradation in liquid- and gas-phase sequencers. The alignment of the tryptic peptides was made by homology with human and other Carnivora hemoglobins. The hemoglobin from spotted hyena (Crocuta crocuta) exhibits in its alpha- and beta-chains 22 and 20 exchanges, respectively, compared to human hemoglobin. In the alpha-chains, two alpha 1 beta 1-contacts are exchanged. In the beta-chains five exchanges involve one alpha 1 beta 1-contact, one alpha 1 beta 2-contact, one heme contact, and two 2,3-DPG-binding sites.     

Sequential assignment of the proton NMR spectrum of isolated alpha(CO) chains from human adult hemoglobin.

In this paper we report proton two-dimensional NMR experiments on isolated alpha chains from human hemoglobin A (HbA) in the monocarboxylated state. Several J-correlated and NOE spectra in water or deuterium water and phosphate buffer (100 mM) at 310 K and pH 5.6 were acquired and analysed for the sequential assignment of the proton resonances. In addition, we used the topological data obtained from the crystal structure of alpha subunits in the monocarboxylated HbA tetramer. The assigned resonances correspond to 70% of the amino acid residues. The present results provide information on the tertiary structure of isolated alpha chains in solution, particularly in the heme region. This structure may be compared with that of the a subunits in the tetrameric HbA(CO) in crystal by comparison of observed chemical shifts and those calculated from the X-ray atomic coordinates. Overall, the global folding of the two forms are highly similar. However, this analysis points out several local conformational differences in the heme pocket and the neighboring of the unique Trp residue. Possible explanations of these differences are discussed.     

Ligand-induced conformational changes in spin label-modified human hemoglobins and chains and their carboxypeptidase A-digested derivatives.

The reactive sulfhydryls of human adult and fetal hemoglobin and the single sulfhydryl of isolated gamma chains have been spin labeled with N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) iodoacetamide. Similar electron paramagnetic spectral differences between oxy- and deoxy-modified hemoglobins were observed for both these hemoglobins and for the isolated chains, indicating that ligand-induced conformational changes occur in isolated hemoglobin subunits as well as intact hemoglobin tetramers. Ligand induced changes in the reactivity of p-hydroxymercuribenzoate with the sulfhydryl groups of both intact hemoglobins and isolated subunits, observed by McDonald and Noble (1974) J. Biol. Chem. 249, 3161-3165), led them to draw a similar conclusion. Following carboxypeptidase A digestion of these modified hemoglobins and gamma chains, a procedure which specifically removes the two C-terminal residues of the beta or gamma chains, spectral differences between the liganded and unliganded spin-labeled derivatives still persisted. However, the magnitude of this difference was not only more reduced in the case of the hemoglobins than in that of the subunits but the spectra of both the oxy and deoxy derivatives of the hemoglobins were characteristic of the oxy derivative of a cooperative tetrameric hemoglobin. These findings support the premise that the COOH-terminal end of the beta or gamma chain contributes, although possibly to different extents, to the spectral differences exhibited by both the spin-labeled hemoglobins and chains.     

1H-NMR heme resonance assignments by selective deuteration in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl alpha-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole pi bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.