Proton nuclear magnetic resonance study of the ferrous derivatives of the dimeric and tetrameric hemoglobin from the mollusc Scapharca inaequivalvis

Proton NMR spectra have been measured for the two hemoglobins from the mollusc Scapharca inaequivalvis: HbI, a homodimer, and HbII, a heterotetramer. These hemoglobins are endowed with a unique subunit assembly, since the heme carrying E and F helices are involved in the major intersubunit contact. In the far-downfield region of hyperfine-shifted resonances the spectra of HbI and HbII in the deoxy state show respectively one (66.7 ppm) and two (67.8 and 63.6 ppm) exchangeable signals of the proximal histidine N delta H groups, the resonance position being indicative of a significant strain in the iron-imidazole interaction. In the hydrogen-bonded proton region, inter- and intrasubunit hydrogen-bonded proton signals have been detected for both hemoglobins. Deoxy-HbI shows two unique downfield resonances at 11.83 and 11.51 ppm which disappear in the oxygenated state, suggesting that the corresponding hydrogen bonds are involved in the stabilization of the tertiary and/or quaternary structure of the deoxy form. HbII shows even smaller changes in this region upon changes in ligation state. These results therefore provide further proof that, at variance with the vertebrate hemoglobin tetramer, the unique subunit assembly of these proteins is stabilized mainly by hydrophobic interactions.     

Structure and ligand selection of hemoglobin II from Lucina pectinata

Lucina pectinata ctenidia harbor three heme proteins: sulfide-reactive hemoglobin I (HbI(Lp)) and the oxygen transporting hemoglobins II and III (HbII(Lp) and HbIII(Lp)) that remain unaffected by the presence of H(2)S. The mechanisms used by these three proteins for their function, including ligand control, remain unknown. The crystal structure of oxygen-bound HbII(Lp) shows a dimeric oxyHbII(Lp) where oxygen is tightly anchored to the heme through hydrogen bonds with Tyr(30)(B10) and Gln(65)(E7). The heme group is buried farther within HbII(Lp) than in HbI(Lp). The proximal His(97)(F8) is hydrogen bonded to a water molecule, which interacts electrostatically with a propionate group, resulting in a Fe-His vibration at 211 cm(-1). The combined effects of the HbII(Lp) small heme pocket, the hydrogen bonding network, the His(97) trans-effect, and the orientation of the oxygen molecule confer stability to the oxy-HbII(Lp) complex. Oxidation of HbI(Lp) Phe(B10) --> Tyr and HbII(Lp) only occurs when the pH is decreased from pH 7.5 to 5.0. Structural and resonance Raman spectroscopy studies suggest that HbII(Lp) oxygen binding and transport to the host bacteria may be regulated by the dynamic displacements of the Gln(65)(E7) and Tyr(30)(B10) pair toward the heme to protect it from changes in the heme oxidation state from Fe(II) to Fe(III).     

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: The oxidation reaction

The oxidation by ferricyanide of the dimeric (HbI) and tetrameric (HbII) hemoglobins from the bivalve mollusc Scapharca inaequivalvis has been studied in static and kinetic experiments. Both hemoglobins give rise to hemichromes as stable oxidation products.Oxidation of deoxyHbI yields a hemichrome by a simple bimolecular process. No intermediate Met form can be detected during the reaction even in rapid mixing experiments. The HbI hemichrome undergoes a reversible pH-dependent dissociation into monomers. A simple model has been proposed to account for the linkage between proton binding and subunit dissociation.In the case of tetrameric HbII, oxidation yields an intermediate Met form. Thus, the kinetics of the oxidation reaction are always biphasic; the fast reaction is a bimolecular process and yields the Met derivative. The slow reaction is a monomolecular process and corresponds to the conversion of the Met form into the hemichrome: its rate is independent of the state of ligation of the ferrous protein and decreases with increase of pH. The HbII hemichrome is tetrameric when newly formed: it tends to dissociate into lower molecular weight species with the same optical properties. The rate of dissociation is relatively fast at neutral pH ($\text{t}\text{1}\text{2}$ ≈ 12 min) and markedly less at alkaline pH values.The HbI and HbII hemichromes are reduced by dithionite yielding the spectra of the native deoxygenated proteins: in the case of HbII, the tetrameric structure of the native protein is re-acquired.     

Modulation of oxygen binding to insect hemoglobins: the structure of hemoglobin from the botfly Gasterophilus intestinalis

Hemoglobins (Hbs) reversibly bind gaseous diatomic ligands (e.g., O2) as the sixth heme axial ligand of the penta-coordinate deoxygenated form. Selected members of the Hb superfamily, however, display a functionally relevant hexa-coordinate heme Fe atom in their deoxygenated state. Endogenous heme hexa-coordination is generally provided in these Hbs by the E7 residue (often His), which thus modulates accessibility to the heme distal pocket and reactivity of the heme toward exogenous ligands. Such a pivotal role of the E7 residue is prominently shown by analysis of the functional and structural properties of insect Hbs. Here, we report the 2.6 A crystal structure of oxygenated Gasterophilus intestinalis Hb1, a Hb known to display a penta-coordinate heme in the deoxygenated form. The structure is analyzed in comparison with those of Drosophila melanogaster Hb, exhibiting a hexa-coordinate heme in its deoxygenated derivative, and of Chironomus thummi thummi HbIII, which displays a penta-coordinate heme in the deoxygenated form. Despite evident structural differences in the heme distal pockets, the distinct molecular mechanisms regulating O2 binding to the three insect Hbs result in similar O(2 affinities (P50 values ranging between 0.12 torr and 0.46 torr).     

A comparison of the geminate recombination kinetics of several monomeric heme proteins

The geminate rate constants for CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide rebinding to soybean leghemoglobin and monomeric component II of Glycera dibranchiata hemoglobin were measured at pH 7, 20 degrees C using a dye laser with a 30-ns square-wave pulse. The results were compared to the corresponding parameters for sperm whale myoglobin and the isolated alpha and beta subunits of human hemoglobin (Olson, J.S., Rohlfs, R.J., and Gibson, Q.H. (1987) J. Biol. Chem., 262, 12930-12938). The rate-limiting step for O2, NO, and isonitrile binding to all five proteins is ligand migration up to the initial geminate state, and the rate of this process determines the overall bimolecular association rate constant for these ligands. In contrast, iron-ligand bond formation limits the overall bimolecular rate for CO binding. The distal pockets in leghemoglobin and in Glycera HbII are approximately 10 times more accessible kinetically to diatomic ligands than that in sperm whale myoglobin. This difference accounts for the much larger association rate constants (1-2 x 10(8) M-1 s-1) that are observed for O2 and NO binding to leghemoglobin and Glycera HbII. The rates of isonitrile migration through leghemoglobin are also very large and indicate a very fluid or open distal structure near the sixth coordination position. In contrast, there is a marked decrease in the rate of migration up to and away from the sixth coordination position in Glycera HbII with increasing ligand size. These results were also used to interpret previously published rate constants and quantum yields for the high (R) and low (T) affinity states of human hemoglobin. In contrast to the differences between the monomeric proteins, the differences between the CO-, O2-, and NO-binding parameters for R and T state hemoglobin appear to be due to a decrease in the geminate reactivity of the heme iron atom, with little or no change in the accessibility of the distal pocket.     

Dimeric and tetrameric hemoglobins from the mollusc Scapharca inaequivalvis: Structural and functional properties

The bivalve mollusc Scapharca inaequivalvis contains in the coelomic fluid erythrocytes with a dimeric (HbI) and a tetrameric (HbII) hemoglobin like the other members of the arcid family. The tetrameric protein is made up from two types of polypeptide chain, while the dimeric protein is made from a single type of chain which differs from the other two in terms of molecular weight and isoelectric point.The optical and circular dichroism spectra show that the heme environment in HbI and HbII resembles that of vertebrate hemoglobins, although distinctive features are present in the deoxygenated derivative. p]The dimeric HbI in the pH range 6 to 9 does not change its association state upon deoxygenation, while the tetrameric HbII polymerizes as indicated by the appearance of a fast peak in the sedimentation velocity patterns. The dependence of the areas and sedimentation coefficients of the fast and slow peaks on protein concentration is characteristic of a rapidly established association-dissociation equilibrium between tetramers and polymers higher than octamers. The pH, ionic strength and temperature dependence of polymer formation indicate that both hydrophobic and ionic interactions stabilize the polymers.The functional properties of HbI and HbII differ. HbI shows co-operative oxygen binding (h = 1·5) and a constant oxygen affinity ($\text{p}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7.8}\text{mm Hg}$) over the pH range 5.5 to 9.5. HbII likewise shows co-operativity in oxygen binding (h = 2·0). Its oxygen affinity at neutral and alkaline pH values is slightly lower ($\text{p}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.1}\text{mm Hg}$) than that of the dimeric protein, but becomes higher at pH values below 6.5 due to the presence of an acid Bohr effect. At high protein concentrations, under conditions of extensive polymerization of the deoxygenated derivative, the oxygen affinity is lowered and co-operativity slightly increased. Both phenomena require that the oxygen affinity of the polymer be lower than that of the tetramer, consistent with the predictions of linkage theory.     

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H₂O₂ using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H₂O₂ produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H₂O₂. © 1997 John Wiley & Sons, Ltd.     

NO and CO binding profiles of hemoglobin vesicles as artificial oxygen carriers

Hemoglobin vesicles (HbVs) are artificial oxygen carriers encapsulating purified and concentrated Hb solution in phospholipid vesicles (liposomes). We examined in-vitro reaction profiles of a formulation of HbV with NO and CO in anaerobic and aerobic conditions using stopped-flow spectrophotometry and a NO electrode. Reaction rate constants of NO to deoxygenated and oxygenated HbV were considerably smaller than those of cell-free Hb because of the intracellular NO-diffusion barrier. The reaction of CO with deoxygenated HbV was slightly slower than that of cell-free Hb solely because of the co-encapsulated allosteric effector, pyridoxal 5'-phosphate. The NO depletion in an aerobic condition in the presence of empty vesicles was monitored using a NO electrode, showing that the hydrophobic bilayer membrane of HbV, which might have higher gas solubility, does not markedly facilitate the O(2) and NO reaction, and that the intracellular Hb is the major component of NO depletion. In conclusion, HbV shows retarded gas reactions, providing some useful information to explain the absence of vasoconstriction and hypertension when they are intravenously injected.     

The heme pocket geometry of Lucina pectinata hemoglobin II restricts nitric oxide and peroxide entry: model of ligand control for the design of a stable oxygen carrier

Blood pressure elevation has been attributed in large part to the consumption of nitric oxide (NO) by extracellular hemoglobin (Hb) therapeutics following infusion in humans. We studied NO and hydrogen peroxide (H2O2) oxidative reaction kinetics of monomeric Hbs isolated from the clam Lucina pectinata to probe the effects of their distinctive heme pocket chemistries on ligand controls and heme oxidative stability. HbI (Phe43(CD1), Gln64(E7), Phe29(B10), and Phe68(E11)) reacted with high avidity with NO (k'(ox,NO) = 91 microM-1 s-1), whereas HbII (Phe44(CD1), Gln65(E7), Tyr30(B10), and Phe69(E11)) reacted at a much slower rate (k'(ox,NO)= 2.8 microM-1 s-1). However, replacing B10 (Phe) by Tyr in recombinant HbI (HbI PheB10Tyr) produced only a 2-fold reduction in the NO-induced oxidation rate (k'(ox,NO)= 49.9 microM-1 s-1). Among the clam Hbs, HbII exhibited the fastest NO dissociation and the slowest NO association with ferrous iron. Autoxidation, H2O2-mediated ferryl iron (FeIV) formation, and the subsequent heme degradation kinetics were much slower in HbII and HbI PheB10Tyr when compared to those of HbI. The Tyr(B10) residue appears to afford a greater heme oxidative stability advantage toward H2O2, whereas the close proximity of this residue together with Gln(E7) to the heme iron contributes largely to the distal control of NO binding. Engineering of second-generation Hb-based oxygen therapeutics that are resistant to NO/H2O2-driven oxidation may ultimately require further optimization of the heme pocket architecture to limit heme exposure to solvent.     

Cyanide binding to Lucina pectinata hemoglobin I and to sperm whale myoglobin: an x-ray crystallographic study.

The x-ray crystal structures of the cyanide derivative of Lucina pectinata monomeric hemoglobin I (L. pectinata HbI) and sperm whale (Physeter catodon) myoglobin (Mb), generally taken as reference models for monomeric hemoproteins carrying hydrogen sulfide and oxygen, respectively, have been determined at 1.9 A (R-factor = 0. 184), and 1.8 A (R-factor = 0.181) resolution, respectively, at room temperature (lambda = 1.542 A). Moreover, the x-ray crystal structure of the L. pectinata HbI:cyanide derivative has been studied at 1.4-A resolution (R-factor = 0.118) and 100 K (on a synchrotron source lambda = 0.998 A). At room temperature, the cyanide ligand is roughly parallel to the heme plane of L. pectinata HbI, being located approximately 2.5 A from the iron atom. On the other hand, the crystal structure of the L. pectinata HbI:cyanide derivative at 100 K shows that the diatomic ligand is coordinated to the iron atom in an orientation almost perpendicular to the heme (the Fe-C distance being 1.95 A), adopting a coordination geometry strictly reminescent of that observed in sperm whale Mb, at room temperature. The unusual cyanide distal site orientation observed in L. pectinata HbI, at room temperature, may reflect reduction of the heme Fe(III) atom induced by free radical species during x-ray data collection using Cu Kalpha radiation.     

Evidence for nonhydrogen bonded compound II in cyclic reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide

Studies that elucidate the behavior of the hemoglobins (Hbs) and myoglobins upon reaction with hydrogen peroxide are essential to the development of oxygen carrier substitutes. Stopped-flow kinetics and resonance Raman data show that the reaction between hydrogen peroxide and oxygenated and deoxygenated ferric Hb I (oxy- and deoxy-HbI) from Lucina pectinata produce compound I and compound II ferryl species. The rate constants ratio (k23/k41) between the formation of compound II from compound I (k23) and the oxidation of the ferrous HbI (k41, i.e., 25 M(-1) s(-1)) of 12 x 10(-4) M suggests that HbI has a peroxidative capacity for removing H2O2 from solution. Resonance Raman presents the formation of both, met-aquo-HbI and compound II ferryl species in the cyclic reaction of HbI with H2O2. The ferric HbI species is maintained by the presence of H2O2; it can produce HbI compound I, or it can be reduced to a deoxy-HbI derivative to form HbI compound II upon reaction with H2O2. The compound II ferryl vibration frequency appears at 805 and 769 cm(-1) for HbIFe(IV)=(16)O and HbIFe(IV)=(18)O species, respectively. This ferryl mode indicates the absence of hydrogen bonding between the carbonyl group of the distal Q64 and the HbIFe(IV)=O ferryl moiety. The observation suggests that both the trans-ligand effect and the polarizabilty of the HbI heme pocket are responsible for the observed ferryl oxo vibrational energy. The vibrational mode also suggests that the carbonyl group of the distal Q64 is oriented toward the iron of the heme group, increasing the distal pocket electron density.     

Structure-volume relationships in hemoglobin. A densitometric and dilatometric study of the oxy leads to deoxy transformation.

Partial volume measurements have been performed for human hemoglobin, both on the oxygenated and deoxygenated forms. Density measurements (by pycnometry) give vHbO2 = 0.752 +/- 0.002 and vHb =0.753 +/- 0.006 for the partial specific volume and do not distinguish between the two different structures. Differential measurements, by dilatometry, however, show a signigicantly higher molal volume (of about 50 cm3/mol hemoglobin tetramer) for the deoxy over the oxygenated from at pH 7. The same reaction, at pH 9, gives a much smaller increase or even a decrease of volume. The different volume changes at pH 7 and at pH 9 are not due to the so-called Bohr ionization but to the weakening, at pH 9 compared to pH 7, of stabilising salt linkages in the deoxy structure.     

Quaternary structure of <Emphasis Type="Italic">Artemia</Emphasis> haemoglobin II: analysis of T and C polymer alignment and interpolymer interface

Background  

The brine shrimp Artemia expresses four different types of haemoglobin subunits namely C1, C2, T1 and T2. Two of these four subunits dimerize in different combinations to produce the three isoforms of the heterodimeric Artemia haemoglobin: HbI (C1 and C2), HbII (C1 and T2) and HbIII (T1 and T2). Previous biochemical, biophysical and computational analyses demonstrate that the T and C polymers are rings of nine concatenated globin domains, which are covalently joined by interdomain linkers. Two such rings stacked coaxially give the functional molecule. This research aimed to construct a quaternary structural model of Artemia HbII that shows the interpolymer interface and domain-domain alignment, using the MS3D (mass spectrometry for three dimensional analysis) approach. This involved introducing chemical crosslinks between the two polymers, cleaving with trypsin and analyzing the resulting products by mass spectrometry. This was followed by computational analysis of the mass spectrometry data using the program SearchXlinks to identify putatively crosslinked peptides.     

Molecular basis of the Bohr effect in arthropod hemocyanin

Flash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, k(on), rather than changes in k(off). In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observed for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure (Blackburn, N. J., Strange, R. W., Reedijk, J., Volbeda, A., Farooq, A., Karlin, K. D., and Zubieta, J. (1989) Inorg. Chem., 28, 1349-1357). A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s --> 4p(z) transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs.     

Proximal and distal effects on the coordination chemistry of ferric Scapharca homodimeric hemoglobin as revealed by heme pocket mutants

The ferric form of the homodimeric hemoglobin from Scapharca inaequivalvis (HbI) displays a unique pH-dependent behavior involving the interconversion among a monomeric low-spin hemichrome, a dimeric high-spin aquomet six-coordinate derivative, and a dimeric high-spin five-coordinate species that prevail at acidic, neutral, and alkaline pH values, respectively. In the five-coordinate derivative, the iron atom is bound to a hydroxyl group on the distal side since the proximal Fe-histidine bond is broken, possibly due to the packing strain exerted by the Phe97 residue on the imidazole ring [Das, T. K., Boffi, A., Chiancone, E. and Rousseau, D. L. (1999) J. Biol. Chem. 274, 2916-2919]. To determine the proximal and distal effects on the coordination and spin state of the iron atom and on the association state, two heme pocket mutants have been investigated by means of optical absorption, resonance Raman spectroscopy, and analytical ultracentrifugation. Mutation of the distal histidine to an apolar valine causes dramatic changes in the coordination and spin state of the iron atom that lead to the formation of a five-coordinate derivative, in which the proximal Fe-histidine bond is retained, at acidic pH values and a high-spin, hydroxyl-bound six-coordinate derivative at neutral and alkaline pH values. At variance with native HbI, the His69 --> Val mutant is always high-spin and does not undergo dissociation into monomers at acidic pH values. The Phe97 --> Leu mutant, like the native protein, forms a monomeric hemichrome species at acidic pH values. However, at alkaline pH, it does not give rise to the unusual hydroxyl-bound five-coordinate derivative but forms a six-coordinate derivative with the proximal His and distal hydroxyl as iron ligands.     

The 2.7 A crystal structure of deoxygenated hemoglobin from the sea lamprey (Petromyzon marinus): structural basis for a lowered oxygen affinity and Bohr effect.

BACKGROUND: The hemoglobins of the sea lamprey are unusual in that cooperativity and sensitivity to pH arise from an equilibrium between a high-affinity monomer and a low-affinity oligomer. Although the crystal structure of the monomeric cyanide derivative has previously been determined, the manner by which oligomerization acts to lower the oxygen affinity and confer a strong Bohr effect has, until now, been speculative. RESULTS: We have determined the crystal structure of deoxygenated lamprey hemoglobin V by molecular replacement to 2.7 A resolution, in a crystal form with twelve protomers in the asymmetric unit. The subunits are arranged as six essentially identical dimers, with a novel subunit interface formed by the E helices and the AB corner using the standard hemoglobin helical designations. In addition to nonpolar interactions, the interface includes a striking cluster of four glutamate residues. The proximity of the interface to ligand-binding sites implicates a direct effect on ligand affinity. CONCLUSIONS: Comparison of the deoxy structure with that of the cyanide derivative revealed conformational changes that appear to be linked to the functional behavior. Oligomerization is coupled with a movement of the first half of the E helix by up to 1.0 A towards the heme, resulting in steric interference of ligand binding to the deoxy structure. The Bohr effect seems to result from proton uptake by glutamate residues as they are buried in the interface. Unlike human and mollusc hemoglobins, in which modulation of function is due to primarily proximal effects, regulation of oxygen affinity in lamprey hemoglobin V seems to depend on changes at the distal (ligand-binding) side of the heme group.     

Crystalline ligand transitions in lamprey hemoglobin. Structural evidence for the regulation of oxygen affinity

The hemoglobins of the Sea Lamprey (Petromyzon marinus) exist in an equilibrium between low affinity oligomers, stabilized by proton binding, and higher affinity monomers, stabilized by oxygen binding. Recent crystallographic analysis revealed that dimerization is coupled with key changes at the ligand binding site with the distal histidine sterically restricting ligand binding in the deoxy dimer but with no significant structural rearrangements on the proximal side. These structural insights led to the hypothesis that oxygen affinity of lamprey hemoglobin is distally regulated. Here we present the 2.9-A crystal structure of deoxygenated lamprey hemoglobin in an orthorhombic crystal form along with the structure of these crystals exposed to carbon monoxide. The hexameric assemblage in this crystal form is very similar to those observed in the previous deoxy structure. Whereas the hydrogen bonding network and packing contacts formed in the dimeric interface of lamprey hemoglobin are largely unaffected by ligand binding, the binding of carbon monoxide induces the distal histidine to swing to positions that would preclude the formation of a stabilizing hydrogen bond with the bound ligand. These results suggest a dual role for the distal histidine and strongly support the hypothesis that ligand affinity in lamprey hemoglobin is distally regulated.     

Effects of active site mutations in haemoglobin I from Lucina pectinata: a molecular dynamic study

Haemoglobin I from Lucina pectinata is a monomeric protein consisting of 142 amino acids. Its active site contains a peculiar arrangement of phenylalanine residues (PheB10, PheCD1 and PheE11) and a distal Gln at position E7. Active site mutations at positions B10, E7 and E11 were performed in deoxy haemoglobin I (HbI), followed by 10 ns molecular dynamic simulations. The results showed that the mutations induced changes in domains far from the active site producing more flexible structures than the native HbI. Distance analyses revealed that the heme pocket amino acids at positions E7 and B10 are extremely sensitive to any heme pocket residue mutation. The high flexibility observed by the E7 position suggests an important role in the ligand binding kinetics in ferrous HbI, while both positions play a major role in the ligand stabilisation processes. Furthermore, our results showed that E11Phe plays a pivotal role in protein stability.     

Tyrosine B10 and heme-ligand interactions of Lucina pectinata hemoglobin II: control of heme reactivity

The distal pocket of hemoglobin II (HbII) from Lucina pectinata is characterized by the presence of a GlnE7 and a TyrB10. To elucidate the functional properties of HbII, biophysical studies were conducted on HbII and a HbI PheB10Tyr site-directed mutant. The pH titration data at neutral conditions showed visible bands at 486, 541, 577 and 605 nm for both proteins. This suggests the possible existence of a conformational equilibrium between an open and closed configuration due to the interactions of the TyrB10, ligand, and heme iron. The kinetic behavior for the reaction of both ferric proteins with H2O2 indicates that the rate for the formation of the ferryl intermediates species varies with pH, suggesting that the reaction is strongly dependent on the conformational states. At basic pH values, the barrier for the reaction increases as the tyrosine adopts a closed conformation and the ferric hydroxyl replaces the met-aquo species. The existence of these conformers is further supported by resonance Raman (RR) data, which indicate that in a neutral environment, the ferric HbII species is present as a possible mixture of coordination and spin states, with values at 1558 and 1580 cm(-1) for the nu2 marker, and 1479, 1492, and 1503 cm(-1) for the nu3 mode. Moreover, the presence of the A3 and A(o) conformers at 1924 and 1964 cm(-1) in the HbII-CO infrared spectra confirms the existence of an open and closed conformation due to the orientation of the TyrB10 with respect to the heme active center.     

Hydrogen-bonding conformations of tyrosine B10 tailor the hemeprotein reactivity of ferryl species

Ferryl compounds [Fe(IV)=O] in living organisms play an essential role in the radical catalytic cycle and degradation processes of hemeproteins. We studied the reactions between H₂O₂ and hemoglobin II (HbII) (GlnE7, TyrB10, PheCD1, PheE11), recombinant hemoglobin I (HbI) (GlnE7, PheB10, PheCD1, PheE11), and the HbI PheB10Tyr mutant of L. pectinata. We found that the tyrosine residue in the B10 position tailors, in two very distinct ways, the reactivity of the ferryl species, compounds I and II. First, increasing the reaction pH from 4.86 to 7.50, and then to 11.2, caused the the second-order rate constant for HbII to decrease from 141.60 to 77.78 M⁻¹ s⁻¹, and to 2.96 M⁻¹ s⁻¹, respectively. This pH dependence is associated with the disruption of the heme–tyrosine (603 nm) protein moiety, which controls the access of the H₂O₂ to the hemeprotein active center, thus regulating the formation of the ferryl species. Second, the presence of compound I was evident in the UV–vis spectra (648-nm band) in the reactions of HbI and recombinant HbI with H₂O₂, This band, however, is completely absent in the analogous reaction with HbII and the HbI PheB10Tyr mutant. Therefore, the existence of a hydrogen-bonding network between the heme pocket amino acids (i.e., TyrB10) and the ferryl compound I created a path much faster than 3.0×10⁻² s⁻¹ for the decay of compound I to compound II. Furthermore, the decay of the heme ferryl compound I to compound II was independent of the proximal HisF8 trans-ligand strength. Thus, the pH dependence of the heme–tyrosine moiety complex determined the overall reaction rate of the oxidative reaction limiting the interaction with H₂O₂ at neutral pH. The hydrogen-bonding strength between the TyrB10 and the heme ferryl species suggests the presence of a cycle where the ferryl consumption by the ferric heme increases significantly the pseudoperoxidase activity of these hemeproteins.