Sulfhydryl groups in hemoglobin. A new molecular probe at the alpha1 beta 1 interface studied by Fourier transform infrared spectroscopy.

Infrared absorption bands due to sulfhydryl groups (VSH) of alpha-104(G11) and beta-112 (G14) cysteine residues of human carboxyhemoglobin (HbCO) have been observed near 2560 cm- minus 1 by use of Fourier transform infrared (FTIR) spectroscopy. The beta-93 cysteine SH groups absorb infrared radiation so weakly that they are not distinguished from background. Only single SH absorption bands due to the alpha-104 cysteines of pig and horse hemoglobin are observed. The SH absorption bands from human HbCO disappear in alkali, are broadened by detergent or guanidinium chloride, and show a complex titration curve, and an isotopic frequency shift (VSD/VSH equals to 0.7267) virtually identical with that reported for methanethiol. The integrated absorption coefficient (epsilonmM(area)) for 0.1 M ethanetiol increased with H-bond acceptor solvents in the order: CCl4 (0.07), water (0.21), acetone (0.43), and N,N-dimethyl-acetamide (1.35 mM- minus 1 cm- minus 2). Comparison of the integrated absorption coefficients for the alpha-104 cysteine SH (2.43), and the beta-112 SH (0.80), of human HbCO with those of ethanetiol solutions suggested specifically H-bonded structures with peptide carbonyl groups 4 (or 3) residues back in the G helices. This was found to agree with a molecular model of the alpha-chain G helix of horse HbO2 built to coordinates from M. F. Perutz.     

Interaction between bound cupric ion and spin-labeled cysteine beta-93 in human and horse hemoglobins

The location of the various copper binding sites for horse and human hemoglobin was probed using spin labels attached to the beta-93 cysteine residue. Dipole-dipole interactions between the spin label and bound copper produce a decrease in the amplitude of the spin label spectrum which was used to estimate the Cu(II) spin label distance. By comparing the results with horse and human hemoglobin at 298 and 77 K four different Cu(II) binding sites were identified. The low affinity horse hemoglobin site with the sulfhydryl blocked (site 1) was found to be located 10-13 A from the sulfhydryl spin label on the surface of the molecule. Only with a free sulfhydryl is the site (site 2) in the pocket between the F and H helices closer to the SH-group and the iron populated. It is site 2 which is responsible for the oxidation. In frozen solutions a Cu-nitroxide distance of about 17 A was determined with human hemoglobin. This distance is consistent with the previously postulated location of the "high affinity" human hemoglobin site near the amino terminus of the beta-chain. At 298 K a much shorter Cu-nitroxide distance of about 7 A was calculated for human hemoglobin. This shorter distance at higher temperature also correlated with a slightly smaller value of g11 and A11 for the Cu(II) ESR spectrum. It is postulated that in solution cross-linking between nitrogenous ligands in the region of the amino terminus of one beta-chain and the carboxyl terminus of the other beta-chain can explain this shorter distance. This cross-link could involve histidine beta-143, which is one of the ligands thought to be also involved in site 1. Binding to the "high-affinity" site in solution thus stabilizes the "low-affinity" site 2 relative to site 1 explaining the reported interaction between the "high-affinity" and "low-affinity" sites.     

Collective motion of iron and exogenous ligands in hemoglobin

M?ssbauer studies of deoxyhemoglobin (Hb), oxyhemoglobin (HbO2), and (carbonmonoxy)-hemoglobin (HbCO) have been performed providing information regarding the center shift (CS) up to 250 K and the recoil-free fraction (f) in the "solid state regime". The temperature dependence of the CS indicates that the effective mass of the iron in liganded hemoglobin includes the exogenous ligand, suggesting that vibrations of the iron are strongly coupled to those of the ligand. As a result of this coupled motion, the recoil-free fraction (f) provides a valuable probe for the shape of the potential energy surface in the ligand pocket. Thus HbO2 experiences a stronger restoring force than HbCO, which can be attributed to a steeper potential well for the oxygen into the ligand pocket.     

Peculiar features of the aggregation effect of silver(I) ion on hemoglobin

Silver(I) ion has been shown to produce aggregation effect on bovine oxyhemoglobin (HbO(2)) in Tris buffer even when taken in amounts corresponding to only two or less silver ions per one HbO(2) tetramer. The extent of produced effect is comparable to those previously observed for Hg(II), Cd, Zn, and Ni in spite of significantly different electronic configurations of the ions in question. Aggregation effect of the silver is ascribed to an interaction of the reactive thiol group sulfur-bound silver atom with the carboxylate residues surrounding the reactive thiol group-bearing cysteine beta93 group of hemoglobin. Mercury ligands, in particular, Tris molecules and OH(-) anions markedly suppress the protein coagulation, thereby supporting the proposed protein aggregation mechanism.     

Electron spin resonance investigation of semiquinone radicals formed from the reaction of ubiquinone 0 with human oxyhemoglobin.

The redox properties and thiol reactivity of quinones play critical roles in their therapeutic and toxicological properties. The present study was undertaken to investigate the binding activity of ubiquinone 0 (UQ(0)) to human oxyhemoglobin (HbO(2)) using electron spin resonance (ESR). Addition of UQ(0) to HbO(2) resulted in the immediate detection of a five-line ESR spectrum characteristic of the semiquinone radical of UQ(0) (UQ(0)). With time the HbO(2) adduct with UQ(0), which was characterized by a broad immobilized ESR spectrum, was gradually formed. Matrix-assisted laser desorption/ionization time-of-flight mass spectra analysis showed that UQ(0) bound to the beta-chain of HbO(2). Superoxide dismutase dose-dependently suppressed the intensity of the broad spectrum and accelerated its formation. However, N-ethylmaleimide, a thiol-blocking agent, completely eliminated its formation. The nonspecific protease mixture pronase also prevented its formation and resulted in the gradual appearance of a 4-line spectrum from the 5-line spectrum of UQ(0). The structure of the species responsible for the 4-line spectrum was confirmed and identified by the reaction of UQ(0) with reduced glutathione. In human red blood cells, UQ(0) rapidly bound to glutathione but more slowly to HbO(2). These results suggest that UQ(0) reacted with both ferrous heme and the reactive beta-93 cysteinyl residue of HbO(2) to generate its corresponding semiquinone radical. Subsequently UQ(0) bound to the beta-93 cysteinyl residue of HbO(2) to form a covalent-binding adduct responsible for the broad spectrum.     

Heme-heme interactions in tetramers and dimers of hemoglobin subunits: DeVoe theory calculations

Detectable exciton couplets arising from heme-heme interactions in the hemoglobin (Hb) tetramers of HbO(2) and deoxyHb were predicted by DeVoe theory. This prediction was supported by the observation of an exciton couplet in the CD difference spectrum between the Hb tetramer and the alphabeta dimer of HbCO. In this paper, DeVoe theory is used to calculate the heme-heme interactions in the CO complex of the Hb tetramer (alpha(2)beta(2)) and dimer (alphabeta), the systems studied by Goldbeck et al. The couplet strength of the resulting theoretical CD difference spectrum agrees well with experiment, thus confirming that heme-heme interactions contribute significantly to the CD of HbCO. Given that the heme-heme distances in HbCO are 25 A and more, it is highly likely that heme-heme interactions also contribute significantly to the CD of other multi-heme proteins, e.g., cytochrome c(3), cytochrome oxidase, cytochrome bc(1), etc., where the hemes are in closer proximity.     

Mechanism of electron transfer to coordinated dioxygen of oxyhemoglobins to yield peroxide and methemoglobin. Protein control of electron donation by aquopentacyanoferrate(II)

Reactions of human oxyhemoglobin A with iron(II) compounds have been investigated. Human oxyhemoglobin (HbO2) reacts with aquopentacyanoferrate(II), Fe(II)(CN)5H2O3-, to yield hydrogen peroxide, aquomethemoglobin and Fe(III)(CN)5H2O2-. The reaction follows a second order rate law, first order in the pentacyanide and in HbO2. Since reaction rates are lower in the presence of catalase, the H2O2 produced must promote metHb formation in reactions independent of pentacyanide. Changes in concentrations of effectors (e.g. H+, inositol hexaphosphate, Cl-, and Zn2+), alkylation of beta-93 cysteine with N-ethylmaleimide, and substitution at distal histidine (as in Hb Zurich with beta-63 His----Arg) in each case can markedly affect pentacyanide reaction rates demonstrating a fine control of rates by protein structure. Hexacyanoferrate(II) (ferrocyanide) reacts with HbO2 to produce cyano-metHb as well as aquo-metHb but the reaction with the hexacyanide is much slower than with the aquopentacyanide. Iron(II) EDTA converts HbO2 to deoxy-Hb with no evidence for formation of metHb as an intermediate. These findings support a mechanism in which the pentacyanide anion reacts directly with coordinated dioxygen. One-electron transfers to O2 from both pentacyanide iron(II) and heme iron(II) result in the formation of a mu-peroxo intermediate, HbFe(III)-O-O-Fe(III) (CN)5(3-). Hydrolysis of this intermediate yields metHb . H2O, H2O2, and FeIII(CN)5H2O2-. The reaction of HbO2 with Fe(CN)6(4-) must follow an outer sphere electron transfer mechanism. However, the very slow rate that is seen with Fe(CN)6(4-) could arise entirely from the pentacyanide produced from loss of one cyanide ligand from the hexacyanide. Fe(II)EDTA reacts rapidly with free O2 in solution but can not interact directly with the heme-bound O2 of HbAO2. The dynamic character of the O2 binding sites apparently permits access of the Fe2+ of the pentacyanide to coordinated dioxygen but the protein structure is not sufficiently flexible to allow the larger Fe2+EDTA molecule to react with bound O2. It is necessary for maintenance of the oxygen transport function of the red cell for reductants such as the methemoglobin reductase system, glutathione, and ascorbate to be able to reduce metHb to deoxy-Hb. It is also important for these reductants to be unable to donate an electron to HbO2 to yield H2O2 and metHb. Thus, a mechanistic requirement for the delivery of one-electron directly to the dioxygen ligand, if peroxide is to be produced, enables the protein to protect the oxygenated species from those electron donors normally present in the cell by denying these reductants steric access to coordinated O2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Laser-induced time-resolved photoacoustic calorimetry study on photo-dissociation of human and bovine oxyhemoglobin

The dynamics of the enthalpy and volume changes related to the photo-dissociation of oxygen from human and bovine oxyhemoglobin are investigated by nanosecond time-resolved photoacoustic calorimetry (PAC). The values of enthalpy and volume change associated with the above process are deltaH = 37.8 +/- 3 kcal/mol, deltaV = 5.0 +/- 1 ml/mol for human HbO(2); and deltaH = 35.7 +/- 3.5 kcal/mol, deltaV = 4.8 +/- 1 ml/mol for bovine HbO(2), respectively. A possible explanation for the similar values between both human and bovine oxyhemoglobin is proposed. In addition, the PAC results for human HbO(2) and HbCO are compared and discussed.     

Effector-induced structural fluctuation regulates the ligand affinity of an allosteric protein: binding of inositol hexaphosphate has distinct dynamic consequences for the T and R states of hemoglobin

The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 degrees C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the micros-ms time scale, with the exception of several amino acid residues, that is, beta109Val and beta132Lys in deoxy-Hb A, and alpha40Lys in HbCO A. The mobility of alpha40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between alpha40Lys and beta146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of beta109Val, which is located in the intradimer (alpha 1beta 1 or alpha 2beta 2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify alpha40Lys in HbCO A, but does not significantly affect the flexibility of beta109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (alpha 1beta 2 or alpha 2beta 1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal beta92His in the beta-heme pocket, as well as some other residues located in the flexible joint (betaC helix-alphaFG corner) and switch (alphaC helix-betaFG corner) regions that play an important role in the dimer-dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the beta-chain. In contrast, no significant perturbation of the dynamic features on the ms-micros time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the beta-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate ( R ex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.     

Carboxy- and oxyhemoglobin in pregnant ewe and fetus after inhalation of marijuana, marijuana placebo and tobacco cigarette smoke

We measured carboxyhemoglobin (HbCO) and oxyhemoglobin (HbO2) percent saturations and blood gases in four near-term pregnant ewes and their fetuses, during and for 6 hours after 9-12 minutes of smoke inhalation from one high-potency marijuana cigarette (M), a marijuana placebo cigarette (P), and a reference tobacco cigarette (T). Maternal HbCO reached maximum levels at or soon after the exposure (M, 2.8%; P, 3.5%; T, 6.3% above baseline) and fell to baseline values by 6 hours. Fetal HbCO rose slowly reaching a plateau at 3 hours (M, 0.7%; P, 1.1%; T, 2.0% above baseline) which was maintained for at least three additional hours. Reductions in maternal and fetal HbO2 after exposure to marijuana placebo and reference tobacco cigarettes reflected these rises in HbCO. After exposure to marijuana cigarettes, however, fetal HbO2 dropped precipitously by 17% of baseline and showed a prolonged rate of return to presmoking HbO2 levels. Although P exposure caused a greater change in HbCO in the fetus than did M, it had a less-profound effect on fetal oxygenation.     

Spectroscopic markers of the T<-->R quaternary transition in human hemoglobin

In this work, we use a sol-gel protocol to trap and compare the R and T quaternary states of both the deoxygenated (deoxyHb) and carbonmonoxide (HbCO) derivatives of human hemoglobin. The near infrared optical absorption band III and the infrared CO stretching band are used to detect the effect of quaternary structure on the spectral properties of deoxyHb and HbCO; comparison with myoglobin allows for an assessment of tertiary and quaternary contributions to the measured band shifts. The R<-->T transition is shown to cause a blue shift of the band III by approximately 35 cm(-1) for deoxyHb and a red shift of the CO stretching band by only approximately 0.3 cm(-1) for HbCO. This clearly shows that quaternary structure changes are transmitted to the heme pocket and that effects on deoxyHb are much larger than on HbCO, at least as far as the band energies are concerned. Experiments performed in the ample temperature interval of 300-10K show that the above quaternary structure effects are "static" and do not influence the dynamic properties of the heme pocket, at least as probed by the temperature dependence of band III and of the CO stretching band. The availability of quaternary structure sensitive spectroscopic markers and the quantitative measurement of the quaternary structure contribution to band shifts will be of considerable help in the analysis of flash-photolysis experiments on hemoglobin. Moreover, it will enable one to characterize the dynamic properties of functionally relevant hemoglobin intermediates and to study the kinetics of both the T-->R and R-->T quaternary transitions through time-resolved spectroscopy.     

Apparent specific volume of human hemoglobin: effect of ligand state and contribution of heme

The apparent specific volumes of human deoxy-, oxy-, met-, and CN-met hemoglobin (Hb) were measured with a vibrating tube densitometer. The values were calculated from the difference in density between protein solutions and solvents with which they were in dialysis equilibrium. The results obtained were very similar to the value for horse HbCO often used for sedimentation studies of Hbs. The apparent specific volumes of oxy- and CN-metHb are approximately 0.0020 cm(3)/g higher than those of deoxy- and metHb. This small reproducible difference could be due either to a small conformational difference between the liganded and unliganded species or to different interactions with components of the solvent. On the basis of these results, a simple method for the determination of the contribution of the heme to the apparent specific volume is proposed. The contribution can be estimated from the difference between the measured volume of each molecular species and that calculated from the amino acid composition.     

Solution 1H NMR study of the active site molecular structure and magnetic properties of the cyanomet complex of the isolated, tetrameric beta-chain from human adult hemoglobin

The solution molecular structure and the electronic and magnetic properties of the heme pocket of the cyanomet complex of the isolated beta-chain of human adult hemoglobin, HbA, have been investigated by homonuclear 2D (1)H NMR in order to assess the extent of assignments allowed by (1)H NMR of a homo-tetrameric 65-kDa protein, to guide the future assignments of the heterotetrameric complex of HbA, and to compare the structure of the beta-chain to the crystallographically characterized complexes that contains the beta-chain. The target residues are those that exhibit significant (>|0.2| ppm) dipolar shifts, as predicted by a "preliminary" set of magnetic axes determined from a small set of easily assigned active site residues. All 104 target residues ( approximately 70% of total) were assigned by taking advantage of the temperature dependence predicted by the "preliminary" magnetic axes for the polypeptide backbone; they include all residues proposed to play a significant role in modulating the ligand affinity in the tetramer HbA. Left unassigned are the A-helix, the end of the G-helix and the beginning of the H-helix where dipolar shifts are less than |0.2| ppm. These comprehensive assignments allow the determination of a robust set of orientation and anisotropies of the paramagnetic susceptibility tensor that leads to quantitative interpretation of the dipolar shifts of the beta-chain in terms of the crystal coordinates of the beta-subunit in ligated HbA which, in turn, confirms a largely conserved molecular structure of the isolated beta-chain relative to that in the intact R-state HbA. The major magnetic axis, which is correlated with the tilt of the Fe-CN unit, is tilted approximately 10 degrees from the heme normal so that the Fe-CN unit is tilted toward the beta-meso-H in a fashion remarkably similar to the Fe-CO tilt in the beta-subunit of HbCO. It is concluded that a set of "preliminary" magnetic axes and the use of variable temperature 2D NMR spectra are crucial to effective assignments in the tetrameric cyanomet beta-chain and that this approach should be similarly effective in HbA.     

Subpicosecond resonance Raman spectroscopy of carbonmonoxy- and oxyhemoglobin.

In this paper we present the resonance Raman spectrum of the carbonmonoxy- (HbCO) and oxyhemoglobin (HbO2) photointermediates on a 800-900 fs timescale. In the case of HbCO, the frequencies of the so-called core-size markers (1500-1650 cm-1) are characteristic of a deoxylike photoproduct in a high spin state (S = 2) with a partially domed heme. The spectrum of the HbO2 photointermediate, on the other hand, is different, and may be characteristic of an excited-state species. These results are discussed in terms of a reaction scheme previously presented by Petrich, J. W., C. Poyart, and J. L. Martin (1988. Biochemistry. 27:4049-4060) and compared with those obtained in the literature on a 30-40 ps timescale. In both molecules a distinct downshift of the v4 mode was observed with respect to the equilibrium value, which is indicative of an elevated temperature of the heme after photodissociation.     

Interaction of sodium dodecyl sulfate with human native and cross-linked hemoglobins: a transient kinetic study

The interaction of sodium dodecyl sulfate (SDS) at a concentration range (0-515 microM) below the critical micelle concentration (CMC approximately 0.83 mM) with human native and cross-linked oxyhemoglobin (oxyHb) and methemoglobin (metHb) has been investigated by optical spectroscopy and stopped-flow transient kinetic measurements. It is observed that the interaction of SDS with human native and cross-linked oxyHb shows the disappearance of the bands of oxyHb at 541 and 576 nm and the appearance at 537 nm. The resultant spectra are characteristic of low spin (Fe(3+)) hemichrome. Similarly SDS has been found to convert human native and cross-linked high spin (Fe(3+)) metHb to low spin (Fe(3+)) hemichrome. The interaction of SDS with oxyHb suggests a conformational change of the protein in the heme pocket, which may induce the binding of distal histidine to iron leading to the formation of superoxide radical. The formation of hemichrome from metHb is found to be concentration-dependent with SDS. The stopped flow transient kinetic measurements of the interaction of SDS with metHb show that at least four molecules of SDS interact with one molecule of metHb. The interaction of SDS with human cross-linked oxy and met hemoglobin shows results similar to those for human native oxy and met hemoglobin indicating that the covalent modification does not alter the interaction of SDS with cross-linked hemoglobin.     

Parameters of oxygen-binding function of human hemoglobin modified by carbon oxide and UV-light

The parameters of oxygen-binding function of human hemoglobin, modified by carbon oxide and UV-radiation: the pressure of half-saturation with the ligand (P50), Hill's constant (n), and arterial-venous difference of HbO2 concentration in the sample were studied. The presence of carboxyform in blood in boundaries of admissible values (lower than 10 per cents) did not noticeably influence to the oxygenation parameters. Functional properties of hemoproteid were substantially modified in case of HbCO concentration increasing from 30 up to 80 percent. It has been discovered, that the leading mechanism of protection from hemic hypoxia in case of poisoning with CO is decreasing of degree of cooperative interactions and relative affinity of hemoglobin for ligands. The stimulating influence of UV-light to the functional properties of modified with carbon oxide human hemoglobin observed in case carboxyform hemoprotein concentration in solution was lower than 10 percent. The disturbance of oxygen-binding ability of hemoglobin at the influence of higher concentrations of Hb-CO was inconvertible and was not correct with UV-radiation.     

WAXS studies of the structural diversity of hemoglobin in solution

Specific ligation states of hemoglobin are, when crystallized, capable of taking on multiple quaternary structures. The relationship between these structures, captured in crystal lattices, and hemoglobin structure in solution remains uncertain. Wide-angle X-ray solution scattering (WAXS) is a sensitive probe of protein structure in solution that can distinguish among similar structures and has the potential to contribute to these issues. We used WAXS to assess the relationships among the structures of human and bovine hemoglobins in different liganded forms in solution. WAXS data readily distinguished among the various forms of hemoglobins. WAXS patterns confirm some of the relationships among hemoglobin structures that have been defined through crystallography and NMR and extend others. For instance, methemoglobin A in solution is, as expected, nearly indistinguishable from HbCO A. Interestingly, for bovine hemoglobin, the differences between deoxy-Hb, methemoglobin and HbCO are smaller than the corresponding differences in human hemoglobin. WAXS data were also used to assess the spatial extent of structural fluctuations of various hemoglobins in solution. Dynamics has been implicated in allosteric control of hemoglobin, and increased dynamics has been associated with lowered oxygen affinity. Consistent with that notion, WAXS patterns indicate that deoxy-Hb A exhibits substantially larger structural fluctuations than HbCO A. Comparisons between the observed WAXS patterns and those predicted on the basis of atomic coordinate sets suggest that the structures of Hb in different liganded forms exhibit clear differences from known crystal structures.     

Low temperature photodissociation studies of ferrous hemoglobin and myoglobin complexes by M?ssbauer spectroscopy.

57Fe-enriched complexes of hemoglobin and myoglobin with CO and O2 were photodissociated at 4.2 degrees K, and the resulting spectra were compared with those of the deoxy forms. Differences in both quadrupole splitting and isomer shift were noted for each protein, the photoproducts having smaller isomer shift and larger quadrupole splitting than the deoxy forms. The photoproducts of HbCO and HbO2 had narrow absorption lines, indicating a well-defined iron environment. The corresponding myoglobin species had broader absorption lines, as did both deoxy forms. The weak absorption lines of photodissociated NO complexes appeared to be wide, possibly indicating magnetic interaction with the unpaired electron of the nearby NO.     

Proton nuclear magnetic resonance studies of hemoglobin Malm?: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malm? (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.     

Conformational substates and dynamic properties of carbonmonoxy hemoglobin

Heme pocket dynamics of human carbonmonoxy hemoglobin (HbCO) is studied by Fourier transform infrared spectroscopy. The CO stretching band at various temperatures in the interval 300-10 K is analyzed in terms of three taxonomic A substates; however, in HbCO the band attributed to the A(1) taxonomic substate accounts for approximately 90% of the total intensity in the pH range 8.8-4.5. Two different regimes as a function of temperature are observed: below 160 K, the peak frequency and the bandwidth of the A(1) band have constant values whereas, above this temperature, a linear temperature dependence is observed, suggesting the occurrence of transitions between statistical substates within the A(1) taxonomic substate in this protein. The relationship between the heme pocket dynamics (as monitored by the thermal behavior of the CO stretching band), the overall dynamic properties of the protein matrix (as monitored by the thermal behavior of Amide II and Amide I' bands) and the glass transition of the solvent (as monitored by the thermal behavior of the bending band of water) is also investigated. From this analysis, we derive the picture of a very soft heme pocket of hemoglobin characterized by rather large anharmonic terms and strongly coupled to the dynamic properties of the solvent.