Heats of carbon monoxide binding by hemoglobin M Iwate.

The heat of reaction of CO gas with the alpha2Mmetbeta2 and alpha2Mbeta2 species of the alpha-chain mutant hemoglobin M Iwate has been studied in buffers with different heats of ionization of 25degrees and in the absence of organic phosphates. For the alpha2Mmetbeta2deoxy species we find a small Bohr effect (0.12 mol of H+/mol of CO) which is in correspondence with that found in equilibrium studies. The heat of reaction, when corrected for proton reaction with buffer, is -18.4 +/- 0.3 kcal/mol of CO at pH 7.4 At pH 9 the same value is observed within experimental error. This value compares closely with heats of reaction of CO with myoglobin and with van't Hoff determinations of the heat of oxygen binding to isolated hemoglobin alpha and beta chains after correction for the heat of replacement of O2 by CO. Furthermore, an analysis of the differential heat of ligand binding as a function of the extent of reaction indicated that, within experimental error, the heat of reaction with the first beta-chain heme in alpha2Mmetbeta2deoxy is the same as the second. Since the quaternary Tleads to R transition is blocked in this mutant hemoglobin, we compared it with Hb A to estimate the enthalpic component of the allosteric T leads to R transition in Hb A. The heats of reaction with CO(g) and Hb A are -15.7 +/- 0.5 and -20.9 +/- 0.5 kcal/mol at pH 7.4 and 9.0, respectively. In going from the T to the R state we find an enthalpy of transition of 9 +/- 2.5 kcal at pH 7.4 and -12 +/- 2.5 kcal at pH 9.0. From published free energies of transsition we conclude the T leads to R transition is enthalpically controlled at p/ 7.4 but entropically controlled at pH 9.0 A near normal Bohr effect is estimated from heats of reaction of CO with alpha2Mdeoxybeta2deoxy in various buffers. A large than normal heat of reaction (-21.6 +/- 0.5 kcal/mol of CO) is attributed to the abnormal alpha chains in Hb M Iwate.     

Electron-spin resonance of nitrosyl haemoglobins: normal alpha and beta chains and mutants Hb M Iwate and Hb Zürich.

At 77 K the electron spin resonance (ESR) spectra of the NO derivatives of the mutant haemoglobins Hb M Iwate and Hb Zurich as well as of the isolated chains of normal haemoglobin were studied. Two types of ESR spectra differing in the g-value and the hyperfine splitting at gzz were observed. The type II spectrum is characterized by a hyperfine structure at gzz = 2.005 with a splitting constant of deltaH = 23 G (14NO) or 32 G (15NO), respectively. In the type I spectrum the splitting constant of the hyperfine structure at gzz = 2.009 amounts to deltaH = 18 G (14NO) or 23 G (15NO), respectively. In some cases this hyperfine structure is coincident with another one at gxx = 2.064 with nearly identical splitting constant. In addition, the type I spectrum is characterized by an increased ESR absorption at gxx = 2.064. At neutral pH the NO derivatives of the isolated chains as well as of the mutant haemoglobins give rise to a type II spectrum. In correspondence with previous results gained with normal NO haemoglobin, the ESR spectra of the NO-alpha chains and NO-Hb Zurich show a transition to type I in the acid region. This transition is favoured by binding of 2,3-bisphosphoglycerate. On the other hand, the ESR spectra of the NO-beta chains and NO-Hb M Iwate are of the type II also at acid pH. The NO-beta chains show a transition of the ESR spectrum from type II to type I only at alkaline pH. These results indicate that in the tetrameric NO haemoglobin only the alpha chains are responsible for the transition of the ESR spectrum from type II to type I in the acid region. The two types of ESR spectra are interpreted in terms of two kinds of haem-NO complexes differing in the iron-NO and iron-imidazole distances. The type II spectrum is attributed to a complex with a relatively short iron-imidazole distance which is responsible for a weakened sigma-bond in trans position. The type I spectrum arises then from a complex with a larger iron-imidazole bond leading to an approach of the NO molecule to the iron. The influence of the protein conformation upon the iron-imidazole bond length is discussed with regard to the ESR spectra of the mutant NO haemoglobins and considering the influence of agents modifying the protein structure.     

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.     

Carbon 13 resonances of 13CO2 carbamino adducts of alpha and beta chains in human adult hemoglobin.

The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains.     

Photodissociation of CO and O2 from alpha and beta hemoglobin chains studied by using picosecond absorption spectroscopy.

The picosecond photodissociation of the CO and O2 forms of alpha and beta chains of hemoglobin were studied by following pi pi Soret absorption changes using a Nd3+ phosphate-glass laser, 531-nm pump pulse, 8 ps full width half maximum, and a pump-probe double-beam absorption apparatus. Three intermediates were observed within the first 50 ps after photon absorption. The most notable differences between the two monomers are the extent and rate of geminate recombination with the two ligands. We attribute this result to differences between the tertiary protein structure of the alpha and beta forms of Hb, both distal and proximal.     

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.     

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.     

Binding of human hemoglobin and its polypeptide chains with haptoglobin coupled to an agarose matrix.

The interactions of human haptoglobin covalently linked to agarose with human hemoglobin and with p-chloromercuribenzoic-acid-treated alpha and beta chains (alpha* and beta* chains) were studied by flow chromatography and equilibrium binding. The results indicate that in solid state, haptoglobin maintains the same binding characteristics as in solution, the order of binding affinities being: hemoglobin greater than alpha* chain greater than beta* chain. The study of the binding parameters of the alpha* chain shows an heterogeneity of binding sites on the haptoglobin and an average affinity constant Ka of 3.6 X 10(4)l/mol.     

Rapid preparation of native alpha and beta chains of human hemoglobin.

1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is less than 0.1%. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.     

Enthalpy changes for inositol hexaphosphate binding to hemoglobins A and M Iwate.

Enthalpies of inositol hexaphosphate (IHP) binding to deoxy and carbonmonoxy (CO) HbA and HbM Iwate have been determined calorimetrically and compared as functions of pH. Values for deoxy HbA and for deoxy HbM Iwate are similar with CO HbM Iwate yielding slightly less heat of reaction. The results support the existence of both deoxy and CO HbM Iwate in T-like structures with only minor modifications occurring upon CO binding. For HbA observed heats of IHP binding have been corrected for heats of extraction of reacting protons from buffer. The resulting intrinsic IHP binding enthalpies show consistent values of ?7 to ?11 kcal/mol proton absorbed in binding. We suggest that a major driving force for organic phosphate binding is the exothermic protonation of histidine and/or a α-amino nitrogens induced by proximity of phosphate negative charges.     

Use of heme spin-labeling to probe heme environments of alpha and beta chains of hemoglobin.

A spin label attached to a propionic acid group of the heme has been used to probe the heme environment of the alpha and beta chains of hemoglobin in both the subunit and tetrameric forms. The electron paramagnetic resonance (EPR) studies of hemoglobin hybrids in which the spin label is attached to either the alpha- or beta-heme (alpha2SLbeta 2 or alpha2beta2SL) and spin-labeled isolated chains (alphaSL and betaSL) show that: 1) alpha- and beta-hemes have different environments in the tetrameric forms of oxy-, deoxy-, and methemoglobins as well as in isolated single chains; 2) when isolated subunits associate to form hemoglobin tetramers, the environment of the alpha-heme changes more drastically than that of the beta-heme; 3) upon deoxygenation of hemoglobin, the structure in the vicinity of the alpha-heme changes more drastically than that of the beta-heme; and 4) upon the addition of organic phosphates to methemoglobin, the change in the spin state of the heme irons mainly arises from beta-heme. The results demonstrate conclusively that the alpha and the beta subunits of hemoglobin are structurally nonequivalent as are their structural changes as the result of ligation. The relationship of EPR spectrum and structure of hemoglobin is discussed.     

Overproduction of alpha chains provides a proton-insensitive component to the bluefish hemoglobin system

Expression of alpha and beta chains and their post-translational assembly into alpha(2)beta(2) tetramers is fundamental to the formation and function of most vertebrate hemoglobins. There is a strong evolutionary bias that favors expression of equal amounts of the two types of chains, because cooperativity, pH sensitivity, and anionic control of function occurs only for the alpha(2)beta(2) tetramers. Remarkably, an over-production of alpha chains, as in the pathological condition known as beta thalassemia in humans, is adaptive rather than pathological in the bluefish hemoglobin system. The thalassemia of the bluefish is a novel means of providing for oxygen uptake and delivery when low pH conditions incapacitate the highly pH-sensitive Root effect hemoglobins of the fish. Although fish often have pH-insensitive along with highly pH-sensitive hemoglobins, having pH-insensitive alpha chain monomers in circulation is an unusual structural variation. The role of bluefish alpha chains in oxygen transport is enabled by their remarkably lower oxygen affinity relative to human alpha chains. This is the first reported case of a thalassemic condition that is maintained in a species as an adaptive advantage.     

Understanding mechanisms in a cooperative protein: the CO ligation intermediates of hemoglobin.

Hemoglobin is a regulatory component of the oxygen transport to the tissues, and for decades has been a prototype to develop new strategies for the study of the structure/function relationships in proteins. One of the most difficult, and so far, unattained objectives of hemoglobin research has been the study of the hemoglobin molecules in a state of partial ligation with oxygen, or intermediates, as a means of testing theories of cooperativity. A cryogenic technique has been developed for the isolation, identification and quantification of the reaction intermediates of hemoglobin and CO, which in many aspects is a close approximation to the physiological ligand. The technical features that are crucial for the evaluation of the significance of the experimental data obtained using this technique and various approaches to the analysis of the data are reported. The discussion points out the importance of accessing direct information on the nature and concentrations of the intermediates in solution to clarify mechanisms of cooperativity as opposed to the less informative studies of the bulk properties of the solution.     

NMR characterization of a diamagnetic model of unliganded alpha chains from human hemoglobin.

This paper reports the reconstitution and spectroscopic characterization of a complex between alpha globin from human adult hemoglobin and protoporphyrin IX-Zn(II). Optical and proton one-dimensional (1-D) NMR spectra indicate that the prosthetic group binds in a 1:1 stoichiometry to the apoglobin in a single conformation. Using 2-D proton NMR techniques we assigned resonances corresponding to the majority of porphyrin substituents and to several side chains of amino acids in contact with the porphyrin. Analysis of nuclear Overhauser enhancement interactions between identified protons indicated that the complex contains only one rotation isomer of the prosthetic group. The diamagnetic Zn(II) ion is coordinated to the proximal histidine (His87) and does not bind O2 or CO as a sixth ligand. The ring current effects on protons from the distal valine (Val62) are considerably higher than in the liganded form providing strong evidence for a more compact ligand binding pocket relative to the carbon monoxy state. Therefore, protoporphyrin-Zn(II)/alpha globin complex is a suitable diamagnetic model for unliganded alpha chains and will be used for structure determination by NMR and modeling methods.     

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.