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.     

Electron paramagnetic resonance of Hb St Louis beta28 (B10) Leu replaced by Gln.

Hemoglobin St Louis beta28 (B10) Leu replaced by Gln is a new mutant which occurs as a natural valency hybrid (alpha2beta+2), or hemoglobin M (Cohen-Solal, M., Seligmann, M., Thillet, J. and Rosa, J. (1973) FEBS Lett. 33, 37-41). The electron paramagnetic resonance (EPR) spectrum of native Hb St Louis at pH 6.2 shows a mixture of three species. Two are high spin, one with tetragonal symmetry, like Hb+ A, the other with rhombic distortion. The third is a low-spin form corresponding to a hemichrome with the distal (E7) histidine as the sixth ligand of the ferric iron. The hemichrome is also found in red blood cells. After oxidation to the alpha+2beta+2 form, three EPR species are seen. Surprisingly, there remains only one high-spin signal, with almost tetragonal symmetry. Besides the low-spin hemichrome, a hydroxy signal is observed even at pH 6.2. These observations imply interactions between the alpha and beta hemes.     

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.     

Structural characterization of ferric hemoglobins from three antarctic fish species of the suborder notothenioidei

Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 A) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67beta in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.     

Hemoglobins of the Lucina pectinata/bacteria symbiosis. II. An electron paramagnetic resonance and optical spectral study of the ferric proteins

We report an optical and EPR spectral study of three hemoglobins, Hb I, II, and III, from the gill of the clam Lucina pectinata. Hemoglobin I reacts much more avidly with hydrogen sulfide than do Hbs II and III. The proximal ligand to the heme iron of each hemoglobin is histidyl imidazole. The acid/alkaline transition of ferric Hb I occurs with pK 9.6; those of ferric Hbs II and III with pK 6.6 and 5.9, respectively. At their acid limits each ferric hemoglobin exists as aquoferric hemoglobin. Broadening of the g = 6 resonance suggests that the bound water enjoys great positional freedom. Ferric Hb I, at the alkaline limit (pH 11), exists as ferric hemoglobin hydroxide. Ferric Hbs II and III, at their alkaline limit (pH 7.5), each exist as equal mixtures of two species. The low spin species with optical maxima near 541 and 576 nm and g values of 2.61, 2.20, and 1.82, are identified as ferric hemoglobin hydroxide. The high spin species, with optical maxima near 486 and 603 nm and g values of 6.71, 5.87, and 5.06, resemble Dicrocoelium hemoglobin and hemoglobin MSaskatoon. Here we show that Hbs II and III resemble hemoglobin MSaskatoon in which a distal tyrosinate oxygen ligated to the ferric heme iron at alkaline pH is displaced by water at acid pH. The H2S product of ferric Hb I is identified as ferric hemoglobin sulfide.     

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.     

Extensive studies of the heme coordination structure of indoleamine 2,3-dioxygenase and of tryptophan binding with magnetic and natural circular dichroism and electron paramagnetic resonance spectroscopy

In order to probe the active site of the heme protein indoleamine 2,3-dioxygenase, magnetic and natural circular dichroism (MCD and CD) and electron paramagnetic resonance (EPR) studies of the substrate (L-tryptophan)-free and substrate-bound enzyme with and without various exogenous ligands have been carried out. The MCD spectra of the ferric and ferrous derivatives are similar to those of the analogous myoglobin and horseradish peroxidase species. This provides strong support for histidine imidazole as the fifth ligand to the heme iron of indoleamine 2,3-dioxygenase. The substrate-free native ferric enzyme exhibits predominantly high-spin EPR signals (g perpendicular = 6, g parallel = 2) along with weak low-spin signals (g perpendicular = 2.86, 2.28, 1.60); similar EPR, spin-state and MCD features are found for the benzimidazole adduct of ferric myoglobin. This suggests that the substrate-free ferric enzyme has a sterically hindered histidine imidazole nitrogen donor sixth ligand. Upon substrate binding, noticeable MCD and EPR spectral changes are detected that are indicative of an increased low spin content (from 30 to over 70% at ambient temperature). Concomitantly, new low spin EPR signals (g = 2.53, 2.18, 1.86) and MCD features characteristic of hydroxide complexes of histidine-ligated heme proteins appear. For almost all of the other ferric and ferrous derivatives, only small substrate effects are observed with MCD spectroscopy, while substantial substrate effects are seen with CD spectroscopy. Thus, changes in the heme coordination structure of the ferric enzyme and in the protein conformation at the active site of the ferric and ferrous enzyme are induced by substrate binding. The observed substrate effects on the ferric enzyme may correlate with the previously observed kinetic substrate inhibition of indoleamine 2,3-dioxygenase activity, while such effects on the ferrous enzyme suggest the possibility that the substrate is activated during turnover.     

Studies on the bacterial hemoglobin fromVitreoscilla

Summary Vitreoscilla contained a homodimeric bacterial hemoglobin (VtHb). The purification of this protein yielded VtmetHb which exhibited electronic and electron paramagnetic resonance (EPR) spectra, showing that it existed predominantly in a high-spin ferric form, both axial and rhombic components being present. The preparations also contained variable amounts of low-spin components. There was no evidence that these high-spin and low-spin forms were in equilibrium. The former were reducible by NADH catalyzed by the NADH-metVtHb reductase, and the latter were not. High ionic strength and high pH led to the formation of low-spin metVtHb; both treatments were reversible. Cyanide and imidazole liganded to VtHb resulted in the conversion of high-spin to low-spin ferric heme centers, each with characteristic electronic and EPR spectra. Some preparations of VtHb exhibited EPR signals consistent with a sulfur ligand bound to the ferric site. When VtHb was treated with NADH plus the reductase in the presence of oxygen, the intensity of the high-spin EPR signals decreased significantly. No reduction occurred in the absence of oxygen, suggesting a possible role for the superoxide anion. Dithionite treatment of VtHb resulted in a slow reduction, but the main product of the reaction of dithionite-reduced VtHb with oxygen was VtmetHb, not VtHbO₂. EPR spectra of whole cells ofVitreoscilla exhibited a variety of intense signals at low and high magnetic field, theg-values being consistent with the presence of high-spin ferric heme proteins, in addition to an iron-containing superoxide dismutase (FeSOD) and iron-sulfur proteins. EPR spectra of the cytosol fraction ofVitreoscilla showed the expected resonances for VtmetHb and FeSOD.Abbreviations A absorbance - DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EPR electron paramagnetic resonance - HiPIP high-potential iron protein - SDS sodium dodecyl sulfate - SOD superoxide dismutase - VtHb Vitreoscilla hemoglobin - VtmetHb oxidizedVitreoscilla hemoglobin - VtHbO₂ oxygenatedVitreoscilla hemoglobin     

Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015

The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed.     

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 redox behavior of the heme in cystathionine beta-synthase is sensitive to pH

Human cystathionine beta-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated.     

Leghemoglobin. An electron paramagnetic resonance and optical spectral study of the free protein and its complexes with nicotinate and acetate.

Electron paramagnetic resonance (EPR) and optical spectra are used as probes of the heme and its ligands in ferric and ferrous leghemoglobin. The proximal ligand to the heme iron atom of ferric soybean leghemoglobin is identified as imidazole by comparison of the EPR of leghemoglobin hydroxide, azide, and cyanide with the corresponding derivatives of human hemoglobin. Optical spectra show that ferric soybean leghemoglobin near room temperature is almost entirely in the high spin state. At 77 K the optical spectrum is that of a low spin compound, while at 1.6 K the EPR is that of a low spin form resembling bis-imidazole heme. Acetate binds to ferric leghemoglobin to form a high spin complex as judged from the optical spectrum. The EPR of this complex is that of high spin ferric heme in a nearly axial environment. The complexes of ferrous leghemoglobin with substituted pyridines exhibit optical absorption maxima near 685 nm, whose absorption maxima and extinctions are strongly dependent on the nature of the substitutents of the pyridine ring; electron withdrawing groups on the pyridine ring shift the absorption maxima to lower energy. A crystal field analysis of the EPR of nicotinate derivatives of ferric leghemoblobin demonstrates that the pyridine nitrogen is also bound to the heme iron in the ferric state. These findings lead us to picture leghemoglobin as a somewhat flexible molecule in which the transition region between the E and F helices may act as a hinge, opening a small amount at higher temperature to a stable configuration in which the protein is high spin and can accommodate exogenous ligand molecules and closing at low temperature to a second stable configuration in which the protein is low spin and in which close approach of the E helix permits the distal histidine to become the principal sixth ligand.     

The active form of the ferric heme in neutrophil cytochrome b(558) is low-spin in the reconstituted cell-free system in the presence of amphophil.

The spin state of the heme in superoxide (O(2)(.)(-))-producing cytochrome b(558) purified from pig neutrophils was examined by means of room-temperature magnetic circular dichroism (MCD) under physiological conditions. Cytochrome b(558) with varying amounts of low-spin and high-spin heme was prepared by either pH adjustment or heat treatment, and the O(2)(.)(-)-forming activity in a cell-free system was found to correlate with the low-spin heme content. The possibility that the O(2)(.)(-)-forming activity results from a transient high-spin ferric heme form that is induced during activation by anionic amphophils has also been investigated. EPR spectra of cytochrome b(558) activated by either arachidonic acid or myristic acid, showed that a transient high-spin ferric species accounting for approximately 50% of the heme appeared in the presence of arachidonic acid, but not in the presence of myristic acid. Hence the appearance of a transient high-spin ferric heme species on activation with an amphophil does not afford a common activation mechanism in the NADPH oxidase system. The EPR results for cytochrome b(558) activated with arachidonic acid showed that the transient high-spin ferric heme can bind cyanide. However, the high-spin ferric heme does not contribute to the O(2)(.)(-) production of cytochrome b(558) in cell-free assays in the presence of cyanide.     

Yeast cytochrome c peroxidase. Coordination and spin states of heme prosthetic group

Electronic absorption and electron paramagnetic resonance (EPR) spectroscopic examinations revealed that a freshly prepared cytochrome c peroxidase (CCP) contains a penta-coordinated high spin ferric protoheme group. The penta-coordinated high spin state of fresh CCP is maintained in a remarkably wide range of pH (4-8). The freezing of fresh CCP induces the reversible coordination of an internal strong field ligand to the heme iron to form a hexa-coordinated low spin compound, which shows EPR extrema at gx = 2.70, gy = 2.20 and gz = 1.78. In the presence of glycerol the freezing-induced artifacts are eliminated and the fresh enzyme exhibits an EPR spectrum of rhombically distorted axial symmetry with EPR extrema at gx = 6.4, gy = 5.3, and gz = 1.97 at 10 K, characteristic of the penta-coordinated high spin enzyme. Upon aging CCP is converted to a hexa-coordinated high spin state due to the coordination of an internal weak field ligand to the heme iron. This conversion is accelerated at acidic pH values, and its reversibility varies from fully reversible to irreversible depending on the degree of enzyme aging. The aging-induced hexa-coordinated CCP is unreactive with hydrogen peroxide and exhibits an EPR spectrum of purely axial symmetry with extrema at g = 6 and g = 2 and an electronic absorption spectrum with an intensified Soret band at 408 nm (epsilon 408 nm = 120 mM-1 cm-1) and a blue-shifted charge-transfer band at 620 nm. Spectroscopic properties of different coordination and spin states of fresh and aged CCPs are compiled in order to formulate a generalized spectroscopic characterization of penta- and hexa-coordinated high spin ferric hemoproteins.     

Studies on the oxidation of hemoglobin Zurich (beta 63 E7 Arg)

Autoxidation and chemically-induced oxidation of hemoglobin Zurich (beta 63 E7 Arg) have been investigated by electron paramagnetic resonance and optical absorption spectroscopy. The results show that the replacement of the distal histidine of the hemoglobin beta chains by an arginine greatly enhances the susceptibility of the heme-iron to oxidative challenge. Both the kinetics and the products of the oxidation are pH dependent. Thus, at acidic and neutral pH, treatment of the protein with ferricyanide leads to a fast conversion of the oxy-protein to aquo-methemoglobin, which, eventually, is slowly converted to hemichromes. In contrast, the hydroxy-met derivative, formed upon chemical oxidation at high pH, is rapidly converted to hemichromes. The electron paramagnetic resonance features of the ferric derivatives of hemoglobin Zurich are somewhat singular, reflecting the modifications of the heme environment in the distal region of the abnormal chains. However, they can be related to heme complexes having their structural counterparts in oxidation products of hemoglobin A.     

Characterization of nonsymbiotic tomato hemoglobin

The nonsymbiotic tomato hemoglobin SOLly GLB1 (Solanum lycopersicon) is shown to form a homodimer of approximately 36 kDa with a high affinity for oxygen. Furthermore, our combined ultraviolet/visible, resonance Raman, and continuous wave electron paramagnetic resonance (EPR) measurements reveal that a mixture of penta- and hexacoordination of the heme iron is found in the deoxy ferrous form, whereas the ferric form shows predominantly a bis-histidine ligation (F8His-Fe(2+/3+)-E7His). This differs from the known forms of vertebrate hemoglobins and myoglobins. We have successfully applied our recently designed pulsed-EPR strategy to study the low-spin ferric form of tomato hemoglobin. These experiments reveal that, in ferric SOLly GLB1, one of the histidine planes is rotated 20 degrees (+/-10 degrees ) away from a N(heme)-Fe-N(heme) axis. Additionally, the observed g-values indicate a quasicoplanarity of the histidine ligands. From the HYSCORE (hyperfine sublevel correlation) measurements, the hyperfine and nuclear quadrupole couplings of the heme and histidine nitrogens are identified and compared with known EPR/ENDOR data of vertebrate Hbs and cytochromes. Finally, the ligand binding kinetics, which also indicate that the ferrous tomato Hb is only partially hexacoordinated, will be discussed in relation with the heme-pocket structure. The similarities and differences with other known nonsymbiotic plant hemoglobins will be highlighted.     

The heme pocket of the globin domain of the globin-coupled sensor of Geobacter sulfurreducens — An EPR study

The globin-coupled sensor (GCS) of Geobacter sulfurreducens is unique amongst GCSs in that its signalling domain is a transmembrane domain with yet unknown function. In the present work we use X-band continuous-wave and pulsed electron paramagnetic resonance (EPR) to investigate the ferric form of the globin domain of the G. sulfurreducens GCS (GsGCS¹⁶²) at pH 8.5. This form shows a unique bis-histidine coordination of the heme with the F8His and E11His. In contrast with previous crystal structure data, where three conformers of the heme structure were identified, ferric GsGCS¹⁶² assumes only one conformation in frozen solution. The EPR data of ferric GsGCS162 are compared in detail with those of other bis-histidine coordinated globins, including other GCS systems.     

Interaction of cationic dodecyl‐trimethyl‐ammonium bromide with oxy‐HbGp by isothermal titration and differential scanning calorimetric studies: Effect of proximity of isoelectric point

In this work, isothermal titration and differential scanning calorimetric methods, in combination with pyrene fluorescence emission and dynamic light scattering have been used to investigate the interaction of dodecyltrimethylammonium bromide (DTAB) with the giant extracellular Glossoscolex paulistus hemoglobin (HbGp) in the oxy‐form, at pH values around the isoelectric point (pI ≈ 5.5). Our ITC results have shown that the interaction of DTAB with the hemoglobin is more intense at pH 7.0, with a smaller cac (critical aggregation concentration) value. The increase of protein concentration does not influence the cac value of the interaction, at both pH values. Therefore, the beginning of the DTAB‐oxy‐HbGp premicellar aggregates formation, in the cac region, is not affected by the increase of protein concentration. HSDSC studies show higher T_m values at pH 5.0, in the absence and presence of DTAB, when compared with pH 7.0. Furthermore, at pH 7.0, an aggregation process is observed with DTAB in the range from 0.75 to 1.5 mmol/L, noticed by the exothermic peak, and similar to that observed for pure oxy‐HbGp, at pH 5.0, and in the presence of DTAB. DLS melting curves show a decrease on the hemoglobin thermal stability for the oxy‐HbGp‐DTAB mixtures and formation of larger aggregates, at pH 7.0. Our present data, together with previous results, support the observation that the protein structural changes, at pH 7.0, occur at smaller DTAB concentrations, as compared with pH 5.0, due to the acidic pI of protein that favors the oxy‐HbGp‐cationic surfactant interaction at neutral pH. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 199–211, 2016.     

Heme spin states and peroxide-induced radical species in prostaglandin H synthase

We have examined the optical, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectra of pure ovine prostaglandin H synthase in its resting (ferric) and ferrous states and after addition of hydrogen peroxide or 15-hydroperoxyeicosatetraenoic acid. In resting synthase, the distribution of heme between high- and low-spin forms was temperature-dependent: 20% of the heme was low-spin at room temperature whereas 50% was low-spin at 12 K. Two histidine residues were coordinated to the heme iron in the low-spin species. Anaerobic reduction of the synthase with dithionite produced a high-spin ferrous species that had no EPR signals. Upon reaction with the resting synthase, both hydroperoxides quickly generated intense (20-40% of the synthase heme) and complex EPR signals around g = 2 that were accompanied by corresponding decreases in the intensity of the signals from ferric heme at g = 3 and g = 6. The signal generated by HOOH had a doublet at g = 2.003, split by 22 G, superimposed on a broad component with a peak at g = 2.085 and a trough at g = 1.95. The lipid hydroperoxide generated a singlet at g = 2.003, with a linewidth of 25 G, superimposed on a broad background with a peak at g = 2.095 and a trough around g = 1.9. These EPR signals induced by hydroperoxide may reflect synthase heme in the ferryl state complexed with a free radical derived from hydroperoxide or fragments of hydroperoxide.     

Correlation between Hemichrome Stability and the Root Effect in Tetrameric Hemoglobins

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 Å resolution. This structure reveals a 5C state of the heme iron for both the α- and β-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.