Partial characterization of giant extracellular hemoglobin of Glossoscolex paulistus: a MALDI-TOF-MS study

In this work, MALDI-TOF-MS analysis was performed to obtain information on the molecular mass of the different subunits from the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy-form. Experiments were performed for the whole protein at pH 7.0, for the partially dissociated protein at pH 9.0, and for the fraction obtained from gel filtration in Sephadex G-200, at pH 9.0, corresponding to the isolated monomer d. Besides that, experiments were performed for the whole protein treated with 2-mercaptoethanol in order to monitor the effects of reduction of the disulfide bonds, which are expected to maintain the trimer (abc) in the native molecule. The results are compared to those reported for the homologous hemoglobin of Lumbricus terrestris (HbLt) and some tentative assignments are made for the observed polypeptides. The monomer d is found to exist in, at least, two major forms of identical proportions with masses of 16,355 ± 25 and 16,428 ± 24 Da, respectively. Two minor forms were also observed around 16 kDa for the monomers. Upon disulfide bonds reduction the peak associated to the trimer is absent in the mass spectrum, and new peaks assigned tentatively to the monomers a, b and c on the basis of comparison with Lumbricus terrestris hemoglobin literature data are observed. Their molecular masses were 18,258 ± 30, 16,492 ± 24 and 17,363 ± 17 Da, respectively. Two linker chains for HbGp were also observed at 25,817 ± 50 and 26,761 ± 16 Da, and this result is different from HbLt, where four linker chains were reported in the range 24–32 kDa. Finally, trimers (abc) were observed at 51–52 kDa. This partial characterization, performed for the first time, is an important step in the characterization of subunits of this giant extracellular hemoglobin.     

Interaction of giant extracellular Glossoscolex paulistus hemoglobin (HbGp) with ionic surfactants: a MALDI-TOF-MS study

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by approximately 144 subunits containing heme groups with molecular masses in the range of 16-19kDa forming a monomer (d) and a trimer (abc), and around 36 non-heme structures, named linkers (L). Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF-MS) analysis was performed recently, to obtain directly information on the molecular masses of the different subunits from HbGp in the oxy-form. This technique demonstrated structural similarity between HbGp and the widely studied hemoglobin of Lumbricus terrestris (HbLt). Indeed, two major isoforms (d(1) and d(2)) of identical proportions with masses of 16,355+/-25 and 16,428+/-24Da, respectively, and two minor isoforms (d(3) and d(4)) with masses around 16.6kDa were detected for monomer d of HbGp. In the present work, the effects of anionic sodium dodecyl sulfate (SDS) and cationic cethyltrimethylammonium chloride (CTAC) on the oligomeric structure of HbGp have been studied by MALDI-TOF-MS in order to evaluate the interaction between ionic surfactants and HbGp. The data obtained with this technique show an effective interaction of cationic surfactant CTAC with the two isoforms of monomer d, d(1) and d(2), both in the whole protein as well as in the pure isolated monomer. The results show that up to 10 molecules of CTAC are bound to each isoform of the monomer. Differently, the mass spectra obtained for SDS-HbGp system showed that the addition of the anionic surfactant SDS does not originate any mass increment of the monomeric subunits, indicating that SDS-HbGp interaction is, probably, significantly less effective as compared to CTAC-HbGp one. The acid pI of the protein around 5.5 is, probably, responsible for this behavior. The results of this work suggest also some interaction of both surfactants with linker chains as well as with trimers, as judged from observed mass increments. Our data are consistent with a recent spectroscopic study showing a strong interaction between CTAC and HbGp at physiological pH [P.S.Santiago, et al, Biochim. Biophys. Acta 1770 (2007) 506-517.].     

Molecular masses and sedimentation coefficients of extracellular hemoglobin of Glossoscolex paulistus: alkaline oligomeric dissociation

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a molecular mass (M) of 3600±100 kDa and a standard sedimentation coefficient (s20,w0) of 58 S, estimated by analytical ultracentrifugation (AUC). In the present work, further AUC studies were developed for HbGp, at pH 10.0, which favors oligomeric dissociation into lower M species. The HbGp oligomer is formed by globin chains a, b, c and d plus the linker chains. The pure monomeric fraction, subunit d, and HbGp at pH 10.0, in the presence of β-mercaptoethanol, were also studied. Our results indicate that for samples of pure subunit d, besides the monomeric species with s20,w0 of 2.0 S, formation of dimer of subunit d is observed with s20,w0 of around 2.9 S. For the whole HbGp at pH 10.0 contributions from monomers, trimers and linkers are observed. No contribution from 58 S species was observed for the sample of oxy-HbGp at pH 10.0, showing its complete dissociation. For cyanomet-HbGp form a contribution of 17% is observed for the un-dissociated oligomer, consistent with data from other techniques that show the cyanomet-form is more stable as compared to oxy-HbGp. Masses of HbGp subunits, especially trimer abc and monomeric chains a, b, c and d, were also estimated from sedimentation equilibrium data, and are in agreement with the results from MALDI-TOF-MS.     

Dynamic light scattering and optical absorption spectroscopy study of pH and temperature stabilities of the extracellular hemoglobin of Glossoscolex paulistus

The extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted of subunits containing heme groups, monomers and trimers, and nonheme structures, called linkers, and the whole protein has a minimum molecular mass near 3.1 × 10⁶ Da. This and other proteins of the same family are useful model systems for developing blood substitutes due to their extracellular nature, large size, and resistance to oxidation. HbGp samples were studied by dynamic light scattering (DLS). In the pH range 6.0-8.0, HbGp is stable and has a monodisperse size distribution with a z-average hydrodynamic diameter (D_h) of 27 ± 1 nm. A more alkaline pH induced an irreversible dissociation process, resulting in a smaller D_h of 10 ± 1 nm. The decrease in D_h suggests a complete hemoglobin dissociation. Gel filtration chromatography was used to show unequivocally the oligomeric dissociation observed at alkaline pH. At pH 9.0, the dissociation kinetics is slow, taking a minimum of 24 h to be completed. Dissociation rate constants progressively increase at higher pH, becoming, at pH 10.5, not detectable by DLS. Protein temperature stability was also pH-dependent. Melting curves for HbGp showed oligomeric dissociation and protein denaturation as a function of pH. Dissociation temperatures were lower at higher pH. Kinetic studies were also performed using ultraviolet-visible absorption at the Soret band. Optical absorption monitors the hemoglobin autoxidation while DLS gives information regarding particle size changes in the process of protein dissociation. Absorption was analyzed at different pH values in the range 9.0-9.8 and at two temperatures, 25°C and 38°C. At 25°C, for pH 9.0 and 9.3, the kinetics monitored by ultraviolet-visible absorption presents a monoexponential behavior, whereas for pH 9.6 and 9.8, a biexponential behavior was observed, consistent with heme heterogeneity at more alkaline pH. The kinetics at 38°C is faster than that at 25°C and is biexponential in the whole pH range. DLS dissociation rates are faster than the autoxidation dissociation rates at 25°C. Autoxidation and dissociation processes are intimately related, so that oligomeric protein dissociation promotes the increase of autoxidation rate and vice versa. The effect of dissociation is to change the kinetic character of the autoxidation of hemes from monoexponential to biexponential, whereas the reverse change is not as effective. This work shows that DLS can be used to follow, quantitatively and in real time, the kinetics of changes in the oligomerization of biologic complex supramolecular systems. Such information is relevant for the development of mimetic systems to be used as blood substitutes.     

Autoxidation studies of extracellular hemoglobin of Glossoscolex paulistus at pH 9: cyanide and hydroxyl effect

The complex oligomeric assembly of the hemoglobin subunits may influence the autoxidation rate. To understand this relation, the rate of autoxidation was studied at pH 9.0, where the Glossoscolex paulistus Hemoglobin (GpHb) dissociates. At alkaline pH, this hemoglobin is dissociated into monomers, trimers and tetramers, allowing the study of the integral protein and monomer subunit autoxidation on independent experiments. The autoxidation rate was evaluated in the presence and absence of cyanide (CN(-)), a strong field ligand to the ferric ion. The oxidation kinetic was monitored using the UV-vis absorption at 415 nm, and resulted in: i) bi-exponential kinetics for the whole hemoglobin (indicating a fast and a slow oxidative process) and ii) mono-exponential for the monomer (indicating a single process). To understand the specific characteristics of each autoxidation process, Arrhenius plots allowed the determination of the activation energy. The experimental results indicate for the whole hemoglobin in the absence of CN(-) an activation energy of 150 +/- 10 kJ mol(-1) for the fast and the slow processes. Under the same conditions the monomer displayed an activation energy of 160 +/- 10 kJ mol(-1), very close to the value obtained for the integral protein. The pseudo-second order rate constant for the whole protein autoxidation by CN(-) showed two different behaviors characterized by a rate constant k(CN1)' = 0.11 +/- 0.02 s(-1) mol(-1) L for CN(-) concentrations lower than 0.012 mol L(-1); and k(CN1)" = 0.76 +/- 0.04 s(-1) mol(-1) L at higher concentrations for the fast process, while the slow process remain constant with k(CN2) = 0.033 +/- 0.002 s(-1) mol(-1) L. The monomer has a characteristic rate constant of 0.041 +/- 0.002 s(-1) mol(-1) L for all cyanide concentrations. Comparing the results for the slow process of the whole hemoglobin and the oxidation of the monomer, it is possible to infer that the slow process has a strong contribution of the monomer in the whole hemoglobin kinetic. Moreover, as disulfide linkers sustain the trimer assembly, cooperativity may explain the higher kinetic constant for this subunit.     

The mechanism of reaction of nitrosyl with met- and oxymyoglobin: an ESR study

In this ESR work we have studied the pentacoordinate symmetry in horse, whale and sperm-whale myoglobin (Mb) in different physical states such as solution and powder. Experiments were performed in which the following parameters were varied: the sample temperature, pH, reaction time with NO, and NO concentration. The results enabled us to explain the NO reaction mechanism in the oxy and met forms of myoglobin. The study of powder samples at different degrees of hydration allowed us to identify the diamagnetic intermediate species existent in the reaction of NO with met-Mb proposed in the literature. The results presented explain adequately the pH effect and temperature dependence observed in the ESR spectra obtained using the met-Mb sample solutions from Sigma Chemical Co., which consist of a mixture (13%) of Mb-O2.     

Ferric species of the giant extracellular hemoglobin of Glossoscolex paulistus as function of pH: an EPR study on the irreversibility of the heme transitions

The present article is focused on the transitions of ferric heme species of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) induced by successive alterations in pH, involving alkaline and acid mediums. Electron paramagnetic resonance (EPR) is the spectroscopy used to evaluate the transitions that occur in the first coordination sphere of ferric ion as a consequence of ligand changes in a wide range of pH, since this tool is very sensitive to slight changes that occur in the heme pocket of paramagnetic species. This approach is adequate to obtain information regarding the reversibility/irreversibility that involves the heme transitions induced by pH, since the degree of reversibility is associated to the intensity of the changes that occur in the spatial configuration of the polypeptide chains, which is clearly associated to the first coordination sphere. The results demonstrate a significant degree of irreversibility of heme transitions, since the final species, which do not present any change after 6 h of its respective formations, are quite different of the initial species. The results denote that the more stable species are the bis-histidine (hemichrome) and pentacoordinate species, due to the properties of their ligands and to the mechanical influence of the respective subunits. EPR spectra allow to distinguish the types of hemichrome species, depending on the reciprocal orientation between the histidine axial ligands, in agreement with Walker's Classification [Walker, F.A., 1999. Magnetic spectroscopic (EPR, ESEEM, M?ssbauer, MCD and NMR) studies of low-spin ferriheme centers and their corresponding heme proteins. Coord. Chem. Rev. 185-186, 471-534]. However, these transitions are not completed, i.e., the appearance of a determined species does not mean the total consumption of its precursor species, implying the coexistence of several types of species, depending on pH. Furthermore, it is possible to conclude that a "pure" EPR spectrum of aquomet ferric species is an important indicator of a high level of conservation referent to the "native" configuration of whole hemoglobin, which is only encountered at pH 7.0. The results allow to infer important physico-chemical properties as well as to evaluate aspects of the structure-activity relationship of this hemoprotein, furnishing information with respect to the denaturation mechanism induced by drastic changes in pH. These data are very useful since HbGp has been proposed as prototype of substitute of blood, thus requiring wide knowledge about its structural and chemical properties.     

On the stability of the extracellular hemoglobin of Glossoscolex paulistus, in two iron oxidation states, in the presence of urea

The stability of the Glossoscolex paulistus hemoglobin (HbGp), in two iron oxidation states (and three forms), as monitored by optical absorption, fluorescence emission and circular dichroism (CD) spectroscopies, in the presence of the chaotropic agent urea, is studied. HbGp oligomeric dissociation, denaturation and iron oxidation are observed. CD data show that the cyanomet-HbGp is more stable than the oxy-form. Oxy- and cyanomet-HbGp show good fits on the basis of a two state model with critical urea concentrations at 220-222 nm of 5.1±0.2 and 6.1±0.1 mol/L, respectively. The three-state model was able to reveal a subtle second transition at lower urea concentration (1.0-2.0 mol/L) associated to partial oligomeric dissociation. The intermediate state for oxy- and cyanomet-HbGp is very similar to the native state. For met-HbGp, a different equilibrium, in the presence of urea, is observed. A sharp transition at 1.95±0.05 mol/L of denaturant is observed, associated to oligomeric dissociation and hemichrome formation. In this case, analysis by a three-state model reveals the great similarity between the intermediate and the unfolded states. Analysis of spectroscopic data, by two-state and three-state models, reveals consistency of obtained thermodynamic parameters for HbGp urea denaturation.     

Fluorescence properties of tryptophan residues in the monomeric d-chain of Glossoscolex paulistus hemoglobin: an interpretation based on a comparative molecular model

The primary structure of the 142 residue Glossoscolex paulistus d-chain hemoglobin has been determined from Edman degradation data of 11 endo-Glu-C peptides and 11 endo-Lys-C peptides, plus the results of Edman degradation of the intact globin. Tryptophan occupies positions 15, 33 and 129. Homology modeling allowed us to assign the positions of these Trp residues relative to the heme and its environment. The reference coordinates of the indole rings (average coordinates of the C(varepsilon2) and C(delta2) atoms) for W15 and W129 were 16.8 and 18.5 A, respectively, from the geometric center of the heme, and W33 was located in close proximity to the heme group at a distance which was approximately half of that for W15 and W129. It was possible to identify three rotamers of W33 on the basis of electrostatic and Van der Waals energy criteria. The calculated distances from the center of the heme were 8.3, 8.4 and 9.1 A for Rot1, Rot2 and Rot3, respectively. Radiationless energy transfer from the excited indole to the heme was calculated on the basis of F?rster theory. For W33, the distance was more important than the orientation factor, kappa(2), due to its proximity to the heme. However, based on kappa(2), Rot2 (kappa(2)=0.945) was more favorable for the energy transfer than Rot1 (kappa(2)=0.433) or Rot3 (kappa(2)=0.125). In contrast, despite its greater distance from the heme, the kappa(2) of W129 (2.903) established it as a candidate to be more efficiently quenched by the heme than W15 (kappa(2)=0.191). Although the F?rster approach is powerful for the evaluation of the relative efficiency of quenching, it can only explain pico- and sub-nanosecond lifetimes. With the average lifetime, =3 ns, measured for the apomonomer as the reference, the lifetimes calculated for each emitter were: W33-1 (1 ps), W33-2 (2 ps), W33-3 (18 ps), W129 (100 ps), and W15 (600 ps). Experimentally, there are four components for oxymonomers at pH 7: two long ones of 4.6 and 2.1 ns, which contribute approximately 90% of the total fluorescence, one of 300 ps (4%), and the last one of 33 ps (7.4%). It is clear that the equilibrium structure resulting from homology modeling explains the sub-nanosecond fluorescence lifetimes, while the nanosecond range lifetimes require more information about the protein in solution, since there is a significant contribution of lifetimes that resemble the apo molecule.     

Pentacoordinate and hexacoordinate ferric hemes in acid medium: EPR, UV-Vis and CD studies of the giant extracellular hemoglobin of Glossoscolex paulistus

The equilibrium complexity involving different axially coordinated hemes is peculiar to hemoglobins. The pH dependence of the spontaneous exchange of ligands in the extracellular hemoglobin from Glossoscolex paulistus was studied using UV-Vis, EPR, and CD spectroscopies. This protein has a complex oligomeric assembly with molecular weight of 3.1 MDa that presents an important cooperative effect. A complex coexistence of different species was observed in almost all pH values, except pH 7.0, where just aquomet species is present. Four new species were formed and coexist with the aquomethemoglobin upon acidification: (i) a "pure" low-spin hemichrome (Type II), also called hemichrome B, with an usual spin state (d(xy))(2)(d(xz),d(yz))(3); (ii) a strong g(max) hemichrome (Type I), also showing an usual spin state (d(xy))(2)(d(xz),d(yz))(3); (iii) a hemichrome with unusual spin state (d(xz),d(yz))(4)(d(xy))(1) (Type III); (iv) and a high-spin pentacoordinate species. CD measurements suggest that the mechanism of species formation could be related with an initial process of acid denaturation. However, it is worth mentioning that based on EPR the aquomet species remains even at acidic pH, indicating that the transitions are not complete. The "pure" low-spin hemichrome presents a parallel orientation of the imidazole ring planes but the strong g(max) hemichrome is a HALS (highly anisotropic low-spin) species indicating a reciprocally perpendicular orientation of the imidazole ring planes. The hemichromes and pentacoordinate formation mechanisms are discussed in detail.     

Ferric species equilibrium of the giant extracellular hemoglobin of Glossoscolex paulistus in alkaline medium: HALS hemichrome as a precursor of pentacoordinate species

The present work is focused on the complex ferric heme species equilibrium of the giant extracellular hemoglobin from Glossoscolex paulistus (HbGp) in alkaline medium. EPR, UV-vis and CD spectroscopies were used in order to characterize the ferric heme species formed as a consequence of the medium alkalization as well as the oligomeric changes occurring simultaneously with heme transitions. EPR experiments allowed us to characterize the different hemichrome species in equilibrium, illustrating the small difference in spin state of this species and the complexity of the equilibira involving hemoglobin ferric species. The results emphasize the importance of the alkaline oligomeric dissociation, which is decisive to promote the heme ferric species transition as function of the increase in water accessibility to the heme pocket. In fact, the oligomeric dissociation in alkaline medium is a consequence of the intense electrostatic repulsion between anionic charges on the protein surface, since the isoelectric point (pI) of this hemoglobin is acid. This explains the more drastic aquomet-hemichrome-pentacoordinate species transition in alkaline medium as compared with the acid medium. However, these heme species transitions are not completed, i.e., the appearance of new species does not mean the total consumption of the precursor species. This equilibrium complexity is associated to the effective influence of oligomeric arrangement of this whole hemoglobin, which present 144 molecular subunits. The acid pI is probably an important factor to the structure-activity relationship of the giant extracellular hemoglobins.     

Small angle x-ray scattering (SAXS) study of the extracellular Hemoglobin of Glossoscolex paulistus: effect of pH, iron oxidation state, and interaction with anionic SDS surfactant

pH effects on the oligomeric structure of giant Glossoscolex paulistus extracellular hemoglobin in the oxyand met-forms have been studied as well as effects of the addition of anionic sodium dodecyl sulfate surfactant. A radius of gyration of 110 A is observed for a macromolecule. At 2 mm surfactant, the radius of gyration diminishes slightly for the oxy-form. However, the extrapolated initial scattering intensity (I0) decreases a factor of 2.5, indicating protein dissociation. At 20 mm surfactant, further I0 decrease is observed, with a reduction of radius of gyration to approximately 30 A consistent with dissociation into smaller subunits. At pH 9.0, the scattering curves are similar to that obtained for the protein in the presence of 20 mm surfactant at pH 7.0. A radius of gyration of approximately 35 A shows that the giant hemoglobin dissociation into small subunits also occurs at alkaline pH. From the I0 value, one can suggest that the tetramer is the main scatter at pH 9.0. At pH 7.0, the met-form dissociates to a larger extent at 2 mm surfactant as compared with the oxy-form, and the main scatters seem to be the 1/12 subunit. At pH 9.0, for the oxy-form, the addition of surfactant does not modify the scattering curve and a radius of gyration approximately 30 A is obtained, while for the met-form some kind of aggregation is observed. Our results give support to conclude that the iron oxidation state is an important factor modulating the oligomeric dissociation.     

Probing axial ligands in ferric haemoproteins: an ESR study of myoglobin and horseradish peroxidase in H217O.

Substitution of H₂¹⁶O by H₂¹⁷O induces a substantial broadening of the high-field line in the electron-spin resonance spectrum of ferric myoglobin due to the presence of H₂¹⁷O at the axial ligand-site. Computer simulations of the experimental spectra yielded the values of the reciprocal relaxation time T₂^?1 = 7.8 G and the ¹⁷O-hyperfine coupling constant A = 18 ± 1 G. Under identical experimental conditions no effect of H₂¹⁷O was observed in horseradish peroxidase. The latter finding excludes the possibility that a water molecule is liganded to the peroxidase haem-iron and supports either the idea that both axial ligands are amino acid residues or that the haem in ferric horseradish peroxidase is pentacoordinate.     

An ESR study of the nitroxide radical of pentastarch-conjugated deferoxamine

At higher concentrations, deferoxamine (DFO) reacts with hydroxyl radicals to produce a stable nitroxide free radical. Formation and decay of this nitroxide radical was investigated and compared with a novel modified pentastarch conjugate of DFO (MPS-DFO). Photolytic generation of hydroxyl radicals from H2O2 in the presence of free DFO produced a nitroxide radical with coupling constants of aN = 8.0 G and aH = 6.5 G. Under the same experimental conditions, equimolar concentrations of MPS-DFO produced an ESR signal of reduced intensity while iron-saturated MPS-DFO produced no signal. Incubation of free DFO with pentastarch (i.e., without conjugation) greatly decreased the intensity of the nitroxide radical signal. Using a spin-trapping technique with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), the pentastarch vehicle was shown to inhibit the DMPO-OH adduct formation. The decay of the DFO nitroxide radical decayed with a second-order rate constant while that of MPS-DFO decayed with a first-order rate constant. Thus, a novel derivative of DFO may provide some additional benefit in limiting DFO nitroxide radical formation and might explain the reported reduced in vivo toxicity of MPS-DFO relative to free DFO.     

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein-surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein-surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein-surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein-surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

Proton electron nuclear double resonance from nitrosyl horse heart myoglobin: the role of His-E7 and Val-E11

Electron nuclear double resonance (ENDOR) spectroscopy has been used to study protons in nitrosyl horse heart myoglobin (MbNO). (1)H ENDOR spectra were recorded for different settings of the magnetic field. Detailed analysis of the ENDOR powder spectra, using computer simulation, based on the "orientation-selection" principle, leads to the identification of the available protons in the heme pocket. We observe hyperfine interactions of the N(HisF8)-Fe(2+)-N(NO) complex with five protons in axial and with eight protons in the rhombic symmetry along different orientations, including those of the principal axes of the g-tensor. Protons from His-E7 and Val-E11 residues are identified in the two symmetries, rhombic and axial, exhibited by MbNO. Our results indicate that both residues are present inside the heme pocket and help to stabilize one particular conformation.