Correlation between functional and structural changes of reduced and oxidized trout hemoglobins I and IV at different pHs. A circular dichroism study.

Circular dichroism (CD) spectra of two major hemoglobin components (Hb), HbI and HbIV, from Oncorhyncus mykiss (formerly Salmo irideus) trout were evaluated in the range 250-600 nm. HbI is characterized by a complete insensitivity to pH changes, while HbIV presents the Root effect. Both reduced [iron(II) or oxy] and oxidized (met) forms of the two proteins were studied at different pHs, 7.8 and 6.0, to obtain information about the pH effects on the structural features of these hemoglobins. Data obtained show that oxy and met-HbI are almost insensitive to pH decrease, remaining in the R conformational state also at low pH. On the contrary, the pH decrease induces similar structural changes, characteristics of ligand dissociation and R-->T transition, both in the reduced and in the oxidized HbIV. The structural changes, monitored by CD, are compared with the peroxidative activity of iron(II)-Hb and met-Hb forms and with the superoxide anion scavenger capacity of the proteins.     

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.     

Steady-state fluorescence and circular dichroism of trout hemoglobins I and IV interacting with tributyltin

The effect of tributyltin chloride (TBTC) on rainbow trout (Salmo irideus) hemoglobin I (HbI) and hemoglobin IV (HbIV) was characterized by the steady-state fluorescence of intrinsic and extrinsic fluorescent probes. The fluorescence emission spectrum (lambdaex 280 nm) is greatly increased in intensity by the presence of the organotin in both proteins. Circular dichroism spectra in the same samples show a small decrease in theta222, a measure correlated with the percentage of the alpha-helical content. Morever, important changes in near-UV, Soret, and visible regions of CD were induced by TBTC. The correlation of data obtained with trout hemoglobins (HbI and HbIV) with similar measurements on globins suggests that the presence of heme is necessary for the interaction of the organotin compound with the proteins.     

Structure of the Fe-heme in the homodimeric hemoglobin from Scapharca inaequivalvis and in the T72I mutant: an X-ray absorption spectroscopic study at low temperature

The Fe site structure in the recombinant wild-type and T721 mutant of the cooperative homodimeric hemoglobin (HbI) of the mollusc Scapharca itnaequivalvis has been investigated by measuring the Fe K-edge X-ray absorption near edge structure (XANES) spectra of their oxy, deoxy and carbonmonoxy derivatives, and the cryogenic photoproducts of the carbonmonoxy derivatives at T = 12 K. According to our results, the Fe site geometry in T72I HbI-CO is quite similar to that of human carbonmonoxy hemoglobin (HbA-CO), while in native HbI-CO it seems intermediate between that of HbA-CO and sperm whale MbCO. The XANES spectra of oxy and deoxy derivatives are similar to the homologous spectra of human HbA, except for T72I HbI, for which the absorption edge is blue-shifted (about + 1 eV) towards the spectrum of the oxy form. XANES spectra of the cryogenic photoproducts of HbA-CO (HbA*), HbI-CO (HbI*) and mutant HbI-CO (T72I HbI*) were acquired under continuous illumination at 12 K. The Fe-heme structures of the three photoproducts are similar; however, while in the case of HbA* and HbI* the data indicate incomplete structural relaxation of the Fe-heme towards its deoxy-like (T) form, the relaxation in T72I HbI* is almost completely towards the proposed "high affinity" Fe-heme structure of T72I HbI. This evidence suggests that minor tertiary restraints affect the Fe-heme dynamics of T72I HbI, corresponding to a reduction of the energy necessary for the T --> R structural transition, which can contribute to the observed dramatic enhancement in oxygen affinity of this hemoprotein, and the decreased cooperativity.     

Involvement of different hemoprotein thiol groups of Oncorhynchus mykiss in cadmium toxicity

BackgroundCadmium is considered the seventh most toxic heavy metal as per ATSDR ranking but its mechanism of toxicity is debated. Recently, we evaluated the effects of this metal on the erythrocyte of teleost fish (Oncorhynchus mykiss) leading us to hypothesize that the pro-oxidant activity of cadmium is not linked to mitochondria but more likely to haemoglobin. In this context, the main aim of this work was to detect the ability of Cd to induce structural perturbation in haemoproteins that present different structures and thus different functional properties and to identify what sites of interaction are mainly involved.MethodsThe effect of Cd on the structural destabilization of the different haemoproteins was followed spectrophometrically through their precipitation. In addition, the sites of interaction between the different haemoproteins and bivalent cadmium ions were identified by MIB server followed by molecular docking/molecular dynamics simulations both in the dimeric and tetrameric associations.ResultsCadmium does not influence the autoxidation rate of Mb, HbA and trout HbI. However, the presence of this metal accelerates the precipitation process in trout HbIV in a dose-dependent manner. Moreover, the presence of 1−10-50−250-500−1000 μM GSH, a chelating agent, reduces the ability of cadmium to accelerate the denaturation process although it is not able to completely prevent it. In order to explain the experimental results, a computational investigations was carried out to identify the cadmium cation affinity for the studied haemoglobins and myoglobin, both in their dimeric and tetrameric forms. As a result, the highest affinity cadmium binding sites for fish HbIV are located at the interface between tetramer-tetramer association, indicating that the cation can assist supramolecular protein aggregations and induce complex precipitation. For mammalian Hb, Mb and fish HbI computational investigation did not detect any site where Cd could to induce such aggregation, in line with the experimental results.ConclusionThe present study provides new information on the mechanisms of toxicity of cadmium by specific interaction with trout O. mykiss haemoglobin component.     

Cooperativity in Scapharca dimeric hemoglobin: simulation of binding intermediates and elucidation of the role of interfacial water

Cooperative binding of ligands to proteins can serve to increase their efficiency and to regulate their activity. Thus, understanding of the mechanism of cooperativity is one of the central concerns of molecular biology. For the tetrameric human hemoglobin (HbA), the cooperative mechanism involves a reasonably well understood combination of tertiary and quaternary changes that occur during the binding process. The dimeric hemoglobin of Scapharca (HbI), which is composed of subunits with the same fold as in HbA, is also highly cooperative but the structural changes on ligand binding are small. A re-orientation of Phe97 in the binding pocket and changes in the number of interfacial water molecules have been implicated in the cooperative mechanism. To explore the role of these factors, we have investigated models of partially liganded intermediate states of HbI with molecular dynamics simulation methods. Since, unlike HbA, no structures for intermediates are available, they were constructed by combining subunits from the unliganded and liganded dimers. Two structurally distinct intermediates were examined, and it was shown that the transition between the two intermediates is directly coupled to the number of interfacial water molecules. Further, it was found that there is a well-defined water channel that connects the interface between the subunits to bulk water. The bottleneck (gate) of the channel, which can be open or closed, is made of hydrophilic residues. The implication of the present results for the cooperative mechanism of HbI is discussed.     

Formation of compound I and compound II ferryl species in the reaction of hemoglobin I from Lucina pectinata with hydrogen peroxide

The formation of ferryl heme (Fe(IV) = O) species, i.e., compound I and compound II, has been identified as the main intermediates in heme protein peroxidative reactions. We report stopped-flow kinetic measurements which illustrate that the reaction of hemoglobin I (HbI) from Lucina pectinata with hydrogen peroxide produce ferryl intermediates compound I and compound II. Compound I appears relatively stable displaying an absorption at 648 nm. The rate constant value (k'(2)) for the conversion of compound I to compound II is 3.0 x 10(-2) s(-1), more than 100 times smaller than that reported for myoglobin. The rate constant value for the oxidation of the ferric heme (k'(12) + k'(13)) is 2.0 x 10(2) M(-1) s(-1). These values suggest an alternate route for the formation of compound II (by k'(13)) avoiding the step from compound I to compound II (k'(2)). In HbI from L. pectinata the stabilization of compound I is attribute to the unusual collection of amino acids residues (Q64, F29, F43, F68) in the heme pocket active site of the protein.     

Functional Characterization of the Purified Holo Form of Hemoglobin I from Lucina pectinata Overexpressed in Escherichia coli

The clam Lucina pectinata inhabits the sulfide-rich west coast of the island of Puerto Rico. It contains three different hemoglobins. Hemoglobin I (HbI), which is monomeric at all concentrations, carries H2S in its ferric state. Overexpression of recombinant HbI from Lucina pectinata in BL21STAR(DE3) Escherichia coli cells was performed in the presence of delta-aminolevulinic acid (delta-ALA). Purification of the protein was achieved using FPLC anion exchange and size exclusion chromatography. Functional characterization of the recombinant holo-protein was assessed by detection of the protein heme O2, CO, and H2S derivatives by UV-Vis spectroscopy, with Soret maxima at 416, 421, and 425 nm, respectively. The results indicated that the recombinant HbI binds H2S and forms a heme sulfide complex like the HbI wild-type hemoglobin. Kinetic measurements were performed to determine the H2S affinity of the recombinant HbI. The H2S dissociation and association rate constants were 0.055 x 10(-3)s(-1) and 0.068 x 10(5) M(-1)s(-1), respectively. The H2S affinity constant of the recombinant HbI (0.124 x 10(9) M(-1)) is eightfold lower than that of the native clam HbI reported earlier. This effect is attributed mostly to the first of two missense mutations [Met 61 (E4)-->Val 61 and Ile101 (FG4)-->Val 101] and additional amino acids present in our construct as demonstrated by measurements of the association rate with a new construct lacking most of the additional residues and the missense mutations. The elimination of these residues restores the similarity between the expressed and wild-type hemoglobins, as evidenced by H2S association kinetics. A pH dependence on the H2S association rate was also contributing to the overall affinity constant and was taken into account in the measurements of the functional properties of the new HbI construct.     

Temperature acclimation modulates the oxygen binding properties of the Atlantic cod (Gadus morhua L.) genotypes-HbI*1/1, HbI*1/2, and HbI*2/2-by changing the concentrations of their major hemoglobin components (results from growth studies at different temperatures)

The influence of long-term acclimation temperatures in Atlantic cod (Gadus morhua) was studied by growth experiments carried out over a total of 272 individuals. The attention focused on the structural and functional modulation of the five electrophoretically distinguishable genotypes of cod hemoglobin (HbI*1/1, HbI*1/2, HbI*2/2, HbI*1/2b, and HbI*2/2b) and on the correlation with body length/weight. The main results can be summarized as follows. (1) Acclimation to lower (4 and 8 degrees C) and higher (12 and 15 degrees C) temperatures favors the expression of, respectively, more anodic and more cathodic hemoglobin components. (2) The optimal O(2) transporting features are observed at 12 degrees C, as well as a saturation-dependent temperature dependence of O(2) binding, which furthermore is strongly dependent upon the acclimation background. (3) The optimal growth condition for the three main genotypes (HbI*1/1, HbI*1/2, and HbI*2/2) is associated with T=12 degrees C. The overall results are consistent with the idea that environmental temperatures constitute a primary factor in the aggregation of individuals physiologically more than genetically homogeneous. This is fully confirmed by careful statistical analysis carried out over a subset of individuals for which the full set of structural (isoelectric focusing), functional (O(2) binding), and growth data was available.     

A cooperative hemoglobin with directly communicating hemes. The Scapharca inaequivalvis homodimer

The unique functional properties of the homodimeric hemoglobin (HbI) extracted from the Arcid blood clam Scapharca inaequivalvis are discussed in the light of the unusual assembly of this protein. At variance with vertebrate hemoglobins, in S. inaequivalvis HbI, the heme-carrying E and F helices form the subunit interface and bring the heme groups almost into direct contact. This creates a new pathway for transferring information about the ligation state of the heme from one subunit to the other which allows cooperativity in the binding of heme ligands to be displayed by a homodimer. The tight coupling between the two subunits and the two heme groups also manifests itself in other reactions that are cooperative in S. inaequivalvis HbI, but not in human hemoglobin, namely, the cleavage of the proximal histidine-heme iron bond and the modification of specific residues located at the subunit interface.     

Expression and purification of recombinant hemoglobin I from Lucina pectinata

Hemoglobin I (HbI) from Lucina pectinata reacts with hydrogen sulfide to form the ferric sulfide complex needed to transport H₂S to the bacterial endosymbiont. To further study HbI, expression studies of this protein were performed in Escherichia coli. This is the first time that the recombinant HbI was produced using a recombinant DNA expression system. Hemoglobin I cDNA was amplified and cloned into the TOPO-P_BAD expression vector, which contains a fusion tag of six histidine residues (6XHis tag). Plasmid clone sequence analysis was carried out in order to ensure that the insert was in the correct reading frame for proper protein expression in E. coli. The expression of recombinant HbI was optimal when induced for 5 hr with 0.002% of l-arabinose as detected by Western blot analysis. The proto-porphyrin group was inserted into the recombinant HbI. Purification of the heme-bound recombinant protein was performed under native conditions by affinity chromatography using Ni-NTA and Probond resins. The sodium dithionite-reduced recombinant protein presented a shift from the Soret band at 413-435 nm, indicating the presence of the heme group in the adequate amino acid environment of HbI. These results indicate that recombinant HbI from Lucina pectinata can be successfully expressed in a prokaryotic system retaining its activity toward reduction, oxidation, and ligand binding.     

Ligand-linked structural transitions in crystals of a cooperative dimeric hemoglobin

Cooperative ligand binding in the dimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis is mediated primarily by tertiary structural changes, but with a small quaternary rearrangement (approximately 3 degrees), based on analysis of distinct crystal forms for ligated and unligated molecules. We report here ligand transition structures in both crystal forms. Binding CO to unligated HbI crystals results in a structure that approaches, but does not attain, the full allosteric transition. In contrast, removing CO from the HbI-CO crystals results in a structure that possesses all the key low affinity attributes previously identified from analysis of HbI crystals grown in the unligated state. Subsequent binding of CO shows the reversibility of this process. The observed structural changes include the quaternary rearrangement even under the constraints of lattice interactions, demonstrating that subunit rotation is an integral component of the ligand-linked structural transition in HbI. Analysis of both crystal forms, along with data from HbI mutants, suggests that the quaternary structural change is linked to the movement of the heme group, supporting a hypothesis that the heme movement is the central event that triggers cooperative ligand binding in this hemoglobin dimer. These results show both the effects of a crystal lattice in limiting quaternary structural transitions and provide the first example of complete allosteric transitions within another crystal lattice.     

The homodimeric hemoglobin from Scapharca can be locked into new cooperative structures upon reaction of Cys92, located at the subunit interface, with organomercurials.

In the cooperative, homodimeric hemoglobin from Scapharca inaequivalvis, HbI, the subunit interface is formed by the heme-carrying E and F helices and contains the only cysteine residue of the globin chain (Cys92, F2) in an area which changes from hydrophilic to hydrophobic upon oxygenation. Binding of organomercurials to HbI is cooperative and entails major quaternary rearrangements. The reaction of Cys92 with p-chloromercuri-benzoate (PMB) and p-nitro-o-chloromercuriphenol (PN), a sensitive reporter of the cysteine microenvironment at neutral pH values, has been followed in stopped flow experiments. Kinetic evidence for the cooperativity of mercurial binding has been obtained and the rate of the corresponding conformational transition has been estimated. As expected PN, but not PMB, is able to monitor the oxygen-linked change of the cysteine microenvironment. The modification of Cys92 with PN has unique functional effects. In PN-reacted HbI cooperativity is maintained, albeit to a different extent, depending on the ligation state of the protein during mercaptide formation. It may be envisaged that PN locks the protein into new, cooperative, quaternary structures stabilized by hydrogen bonding interactions between the ionized nitrophenol moiety and the contralateral subunit.     

Structural and thermodynamic aspects of cooperativity in the homodimeric hemoglobin from Scapharca inaequivalvis

The homodimeric cooperative hemoglobin from the mollusk Scapharca inaequivalvis displays an unusual subunit assembly with respect to vertebrate hemoglobins. The intersubunit contact region is formed by the two heme-carrying E and F helices, which bring the two hemes in contact with each other. At variance with tetrameric vertebrate hemoglobins, the ligand binding is not accompanied by a significant quaternary transition. The major ligand-linked changes are tertiary and are limited to the heme pocket and subunit interface. These unique structural features of HbI are not easily reconciled with the classical thermodynamic models used to describe cooperative ligand binding in vertebrate hemoglobins. The lack of distinct quaternary states and the absence of allosteric effectors suggested that cooperativity in HbI is entirely homotropic in origin. Thereafter, high resolution X-ray crystallographic data displayed the preferential binding of water molecules at the intersubunit interface in the unliganded protein with respect to the liganded one. These ordered water molecules were thus proposed to act as heterotropic effectors in HbI. The contribution of specific water binding to the observed cooperativity in HbI is discussed in the framework of the enthalpy-entropy compensation effect emerging from previous accurate equilibrium oxygen binding measurements.     

Striped mullet (<Emphasis Type="Italic">Mugil cephalus)</Emphasis> hemoglobin system: multiplicity and functional properties

The most frequent (90%) phenotype of the hemoglobin system of M. cephalus presented two major hemoglobins, the more anodal HbI accounting for approximately 70% of the total. The two hemoglobin components separated by ion-exchange chromatography were analyzed by reverse-phase HPLC and electrospray ionization-mass spectrometry which revealed a more complex pattern: HbI consists in four different globins, two β (named β1 and β3) and two co-eluting α chains (α1 and α2); HbII consists in three globins, one β chain (named β2) and the same α1 and α2 present in HbI. The oxygen-binding properties of both hemoglobin components purified by DEAE cellulose were almost identical to those of the hemolysate: stripped hemoglobin showed a large Bohr effect which was enhanced by chloride ions and, at a larger extent, by organic phosphates which, at acidic pH values gave rise to the Root effect. A series of oxygen-binding experiments at increasing GTP concentrations was carried out in order to compare GTP-binding activities in the absence and presence of physiological amounts of chloride. The results indicated that hemoglobin do have two sites for GTP binding. In the absence of chloride, the two sites cannot be discriminated, whereas in the presence of chloride, a competition between the two anions occurred for both GTP-binding sites. The presence of multiple hemoglobin components with identical properties confirms that hemoglobin heterogeneity that often occurs in fish cannot be only explained as an evolutionary response to the physiological and/or environmental needs of the species.     

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.     

Sol-gel trapping of functional intermediates of hemoglobin: geminate and bimolecular recombination studies

The encapsulation of proteins in porous sol-gels is a promising technique for generating, trapping, and probing functionally significant nonequilibrium protein species. An essential step needed in the pursuit of that goal is establishing the degree to which the sol-gel limits conformational change upon adding or removing substrates. In the present study, geminate recombination and solvent phase bimolecular recombination of CO to human adult hemoglobin (HbA) are used as sensitive probes of the degree of conformational constraint within the sol-gel. Two forms of CO saturated encapsulated HbA are generated. In one case, designated [COHbA], the equilibrium form of COHbA is directly encapsulated. In the second case, designated as [deoxyHbA] + CO, the equilibrium form of deoxyHbA is encapsulated and only after the sample has aged is CO introduced to the HbA through the porous sol-gel matrix. Three different preparative protocols are used to generate the sol-gels for each of the two forms of encapsulated COHbA. The kinetic traces obtained from these encapsulated samples allow for an easy evaluation of the extent to which the sol-gel is locking in the initial tertiary/quaternary structure. The results show that the sol-gel encapsulated samples can be used with pulsed laser sources and that one of the tested encapsulation protocols is far superior with respect to conformational locking. This protocol is used to trap and probe nonequilibrium forms such as the liganded T state of HbA, a species whose properties are needed to fully explore allostery in HbA.     

Determination of H(2)S solubility via the reaction with ferric hemoglobin I from the bivalve mollusc Lucina pectinata

A new, simple and fast spectrophotometric method for the determination of the H(2)S concentration is reported. This method, based on the 1:1 reaction between H(2)S and the ferric derivative of hemoglobin I (HbI) from the bivalve mollusc Lucina pectinata, allows the quantitative determination of H(2)S dissolved in a given solution even at concentrations as low as 1 x 10(-6) M. Note that L. pectinata HbI is considered the physiological receptor of H(2)S.     

Properties of normal and glycated human hemoglobin in presence and absence of antioxidant

We present a novel approach to study properties of normal (HbA) and nonenzymatically glycated (HbA(Ic), HbA(Ia+b)) human hemoglobin using absorption spectroscopy and differential scanning calorimetry. The effect of the presence of the antioxidant fisetin on glycation of HbA is studied. Here, absorption spectroscopy has been fruitfully exploited to observe the formation of the glycated hemoglobin. With the differential scanning calorimetry, we studied the thermal unfolding of the protein hemoglobin at various conditions. The thermogram of the pure HbA showed two transition regions, with the occurrence of a partially unfolded intermediate state (the formation of which is mainly reversible) prior to complete denaturation (irreversible process). The denaturation temperature of HbA was found to be strongly dependent on the heating rate. Furthermore, there is a significant cooperativity between the two transition regions in pure HbA. The overall denaturation for the glycated hemoglobin takes place at a lower temperature, suggesting a decrease in the stability of the protein when it is glycated. In presence of fisetin, glycation is inhibited to a certain extent and the thermograms match well with that of normal HbA. Implications of the results are discussed.