The purification,characterization and ligand-binding kinetics of hemoglobins from root nodules of the non-leguminous Casuarina glauca — Frankia symbiosis

The presence of a membrane-bound hemoglobin in aqueous extracts of nitrogen-fixing Casuarina root nodules (Davenport, H.E. (1960) Nature 186, 653–654) has been confirmed. By strictly anaerobic grinding and extraction under carbon monoxide, with inclusion of soluble polyvinylpyrrolidone and zwitterionic detergent in extraction buffer, soluble carboxyhemoglobin was obtained. This was purified by anaerobic ‘adsorption’ chromatography on Sephacryl S-200 (Pharmacia) followed by aerobic molecular exclusion chromatography on Sephadex G-75 (Pharmacia) to yield very stable oxyhemoglobin. By preparative-scale isoelectric focusing Casuarina oxyhemoglobin is separable into three major components comprising approx. 20% of applied protein, and very many minor components. Monomeric Casuarina hemoglobin is similar to other plant hemoglobins in respect of molecular weight (≈ 17 500), optical spectra, extremely rapid kinetics of binding to oxygen and carbon monoxide and high oxygen affinity (P₅₀ ≈ 0.074 torr). Hence, it is possible that this protein functions in the Casuarina symbiosis as does leghemoglobin in leguminous nitrogen-fixing symbioses. Western blot analysis showed close immunological relationships between the non-leguminous Casuarina and Parasponia hemoglobins and a weaker relationship between these two proteins and soybean leghemoglobin. It is proposed that these hemoglobins from widely separated plant orders have a common evolutionary origin.     

Formation of molten globule-like state during acid denaturation of Aspergillus niger glucoamylase

Acid denaturation of Aspergillus niger glucoamylase was studied using different conformational probes. Both far-UV CD spectral signal (MRE_222 nm) and tryptophan fluorescence remained unchanged in the pH range, 7.0–3.0 but decreased significantly below pH 3.0, whereas ANS fluorescence showed a marked increase below pH 1.5. Maximal changes in MRE_222 nm and ANS fluorescence were noticed at pH 1.0. Acid-denatured state of glucoamylase at pH 1.0 retained a significant amount of secondary structure as reflected from far-UV CD spectra but showed a deformed tertiary structure with significant exposure of nonpolar groups as well as tryptophan residues as revealed by increased ANS fluorescence, decreased tryptophan fluorescence and three-dimensional fluorescence spectral signals and increase in K_sv value in acrylamide quenching experiments. Acid-denatured state showed no significant variation in the CD spectral signal throughout the temperature range, 0–100 °C. However, a late cooperative transition was observed upon GdnHCl treatment, compared to the native enzyme. All these results suggested that the acid-denatured state of glucoamylase at pH 1.0 represented the molten globule-like state.     

The hemoglobins of phoronopsis viridis, of the primitive invertebrate phylum phoronida: characterization and subunit structure.

The primitive invertebrate, Phoronopsis viridis, of the phylum Phoronida, has intra-cellular hemoglobins composed of four unique polypeptide chains, two of which associate to form hetero- and homodimers and two which do not associate at all. The CO-derivatives of the associating chains are completely dimeric; removal of the ligand does not result in further aggregation as it does in several other invertebrate hemoglobins. Oxidation of the associating hemoglobins is accompanied by dissociation to monomers, but the cyanide derivative of the methemoglobin is dimeric. The four polypeptide chains all have molecular weights of about 16,000 as determined by iron content and gel electrophoresis with sodium dodecyl sulfate. The two associating chains form three components with isoelectric points at pH 5.6, 5.9, and 6.9 whereas those for the two monomeric chains are at pH 6.2 and 7.9. The chains have been characterized by amino acid composition, tryptic peptide patterns, and the amino acid sequence of the NH₂-terminal segment. The oxygen equilibrium of a dimeric fraction has been determined at pH 7.5 and 20 °C; the pressure of half-saturation is 2.3 mm Hg.     

A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties

The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity. The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis. Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides. The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket. Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent. The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin. Collectively, the data support the model of cyanoglobin function described by Hill et al. [(1996) J. Bacteriol. 178, 6587-6598], in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.     

The enhancement of the alkaline Bohr effect of some fish hemoglobins with adenosine triphosphate.

The oxygen equilibria of the hemoglobins of one holostean fish, the spotted gar (Lepisosteus osculatus), and of four teleost fish, the carpsucker (Carpiodes carpio), the small mouth buffalo fish (Ictiobus bubalus), the Rio Grande cichlid (Cichlasoma cyanoguttatum), and the redear sunfish (Lepomis microlophus), have been measured as a function of pH in the presence and absence of ATP. The oxygen equilibria of the teleost hemoglobins in the presence of 200 μm ATP can be superimposed within experimental error upon the data obtained in the absence of ATP by a simple downward shift of the pH scale by 0.5 unit. Thus the effects of proton and ATP binding appear equivalent: Both can be interpreted in terms of a two-state allosteric model in which binding occurs preferentially to the low-affinity T-state. The oxygen affinities of each of the teleost hemoglobins approach asymptotically a maximal value at high pH. Although these high affinities are associated with decreased cooperativity of oxygen binding, as reflected by the Hill coefficient n, the asymptotic value of n never appears lower than 1.2 to 1.4. This indicates that the data cannot be completely described in terms of a single high-affinity R-state in alkaline solution: At least two different conformations are required. The oxygen affinity of the spotted gar hemoglobin, like that of each of the teleost hemoglobins, reaches a maximal value (minimal value of log PO₂ for half-saturation) above pH 8, but unlike teleost hemoglobins, the Hill coefficient reaches maximal values of 2.6 to 2.7 at high pH. The data in the absence of ATP are superimposable on the data in its presence by a downward shift of the pH scale by 0.25 unit. The measurement of the Bohr effect ($\text{Δlog}\text{P}{}_{30}\text{ΔpH}$) in the presence and absence of ATP shows that the Bohr effect in each of the hemoglobins is substantially enhanced by organic phosphates, as it is in mammalian hemoglobins. The extent of the enhancement of the Bohr effect by 200 μm ATP for each of the hemoglobins is approximately the same in the range pH 6.7 to 7.3 (increase in $\text{Δlog}\text{P}{}_{50}\text{ΔpH}\text{~ 0.3}$). This is a direct consequence of the equivalence of the linked-function relationship to the effects of ATP and proton binding on oxygenation.     

Gamma-glutamyltransferase activity of liver plasma membrane: induction following chronic alcohol consumption

The proton nuclear magnetic resonance spectra of soybean ferric leghemoglobin $\text{a}$ in the low-spin cyanide and nicotinate complexes have been assigned by specific deuteration of heme methyl groups. The assignments differ from those obtained solely from nuclear Overhauser enhancement measurements and are indicative of a proximal histidyl imidazole-hemin interaction which is very similar to that found in sperm whale myoglobin. The absence of a hyperfine shifted exchangeable NH peak for the distal histidine in leghemoglobin suggests either a very different orientation for this distal ligand or a significantly faster exchange rate with bulk solvent than found in myoglobin.     

15N NMR studies of the binding of 15N-labeled cyanide to various hemoglobins in intact erythrocyte.

¹⁵N-labeled cyanide binding to methemoglobins in intact erythrocytes has been studied by ¹⁵N NMR. The addition of C¹⁵N^? to human and dog hemoglobins in erythrocyte afforded hyperfine-shifted two ¹⁵N signals due to the C¹⁵N bound to ferric iron of the different heme-units. Single and three distinct signals were observed for rat and rabbit hemoglobins in erythrocyte. These C¹⁵N resonance positions are sensitive both to the structural difference in the hemoglobin subunits and to the variety of the animal sources. The C¹⁵N spectral difference between solution and intact hemoglobin cyanide is also discussed in relation to a possible change in the intra- and extracellular pH values.     

Measurement of the fractional oxygenation of leghemoglobin in intact detached pea nodules by reflectance spectroscopy

A method is presented for the rapid measurement of the spectral properties of detached nodules of pea (Pisum sativum L. cv “Sparkle”) by diffuse reflectance spectroscopy. After correcting the spectra for surface light scattering, the spectrum of leghemoglobin is obtained. From this, the fractional oxygenation of leghemoglobin and the internal O₂ concentration can be calculated. With this method, we determined internal O₂ while measuring nitrogenase activity (C₂H₂) in detached pea nodules over a range of external O₂ concentrations. Nitrogenase activity was maximum when leghemoglobin was 25% oxygenated, corresponding to a calculated free O₂ concentration of 45 nanomolar in infected cells. Advantages of this method over previous methods which employed transmitted light are: (a) many nodules can be assayed simultaneously, (b) nitrogenase activity (C₂H₂) can be determined at the same time as spectra are recorded, and (c) spectra can be obtained from nodules submerged in buffer containing metabolic effectors.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

A comparison of the functional properties of two lepore hemoglobins with those of hemoglobin A1.

Lepore hemoglobins result from crossovers between normal beta and delta chain genes. Structural investigation of two newly discovered examples of Lepore hemoglobins revealed one of them to be structurally identical to hemoglobin Lepore Hollandia α₂^Aδ²² -x- β⁵⁰, a rarely occurring Lepore variant, while the second had the structure of hemoglobin Lepore Boston α₂^Aδ⁸⁷ -x- β¹¹⁶. Studies of the equilibrium and kinetic properties of the liganding reactions of these two Lepore hemoglobins, which differ only in three amino acid residues, and comparison of these with the known properties of hemoglobin A₁ (α₂β₂) and hemoglobin A₂ (α₂δ₂) have been carried out. A high value of n, the Hill coefficient, indicating normal heme-heme interaction, was observed in each hemoglobin along with a normal Bohr effect. However, a slight but definite increase in oxygen affinity was observed for each Lepore hemoglobin. Furthermore, kinetic studies indicated a slight but consistently increased rate of ligand combination and a somewhat decreased rate of oxygen dissociation for hemoglobins Lepore Hollandia and Lepore Boston at pH 7 and 20 °C. Apparently, the higher oxygen affinity of these Lepore hemoglobins over those of the normal hemoglobins A₁ and A₂ reflects changes of sequence that are common to both types of hemoglobin Lepore.     

Lupine leghemoglobin I : expression in transgenic Lotus and tobacco tissues

The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the “Class II” hemoglobins.     

Functional and structural analyses on abnormal hemoglobins with impaired oxygen binding properties—To elucidate the allosteric mechanism of hemoglobin

The altered oxygen binding curves for various abnormal hemoglobins were analyzed according to a two-state allosteric model. Of three allosteric parameters computed for abnormal hemoglobins, K _R was nearly constant, but K _T and L varied with the correlation of log c=?0.4 log L, where c is K _R/K _T. This correlation indicates that the abnormal allosteric oxygen binding of hemoglobin is due to altered molecular properties of the deoxy-T state but not that of the deoxy-R state. To clarify the molecular basis of this idea, resonance Raman spectra in the low-frequency region of abnormal hemoglobins were measured under different solvent conditions. Varied frequencies of iron-histidine stretching Raman lines was found to correlate with varied oxygen affinities (K _T) of deoxy-T states. The strength of the iron-histidine bond of deoxy-T states was changed, depending upon the magnitude of the strain imposed on hemes by globin, and this bond presumably comprises an important part of the regulation mechanisms for hemoglobin oxygen binding and structure changes.     

Modification by cyanide of the aniline-binding reaction with cytochrome P-450.

Hydroxylation of aniline to p-aminophenol catalyzed by the cytochrome P-450-containing monooxygenase system of liver microsomes is inhibited by cyanide, but microsomal NADPH-cytochrome c reductase is insensitive to this inhibitor. The interaction of aniline with membrane-bound cytochrome P-450, according to spectrophotometric analyses, consists of two phases with respect to aniline concentration, and cyanide interferes differently with these two reaction phases. Noncompetitive and competitive (or mixed type) inhibitions of the aniline-binding reaction by cyanide are observed in reaction systems containing low and, high concentrations of aniline, respectively, a situation similar to the inhibitory action of cyanide on aniline hydroxylase activity. Abnormal aniline-induced difference spectra appeared when cyanide was added as the spectral modifier, and the magnitude of the spectral change in the presence of both aniline and cyanide was a nonadditive change. These results suggest the dissociation of the cytochrome P-450·cyanide complex by aniline. A similar result indicating dissociation of the complex was also obtained by epr spectroscopy. We therefore suggest that addition of a high concentration of substrate causes insensitivity of the microsomal hydroxylase system to cyanide.     

Oxygen affinities of maternal and fetal hemoglobins of the viviparous seaperch,Embiotoca lateralis

Summary The striped seaperch,Embiotoca lateralis, is a viviparous teleost. The hemoglobins of adult and fetal seaperch are both tetrameric proteins which in their native state appear to be indistinguishable from one another by electrophoresis. However, differences in the subunit structure of maternal versus fetal seaperch hemoglobins can be detected by electrophoresis in urea with a reducing agent, amino acid analyses and peptide maps of the respective proteins. Furthermore, stripped adult and fetal hemoglobins have different oxygen binding affinities at all pH's tested between pH 6.8 and 8.0. Mid-gestation fetal hemoglobin has a higher oxygen affinity than late-gestation fetal hemoglobin which in turn has a higher affinity than that of the adult hemoglobin. All three stripped hemoglobins show a similar Bohr effect (=–0.9). These data suggest that a difference in oxygen affinities exists in vivo between the adult and fetal blood of the seaperchEmbiotoca lateralis and that it can be explained in part by the presence of a structurally unique fetal hemoglobin. This report is the first to provide evidence for a mechanism of maternal-fetal oxygen transfer in a teleost fish.Abbreviations A adult - LF late-gestation fetal - MF mid-gestation fetal (hemoglobins)     

A nuclear magnetic resonance study of axial ligation for the reduced states of chloroperoxidase,cytochrome P-450cam,and porphinatoiron(II) thiolate complexes

The reduced forms of cytochrome P-450_cam and chloroperoxidase were examined by proton NMR spectroscopy. The pH and temperature dependences of the proton NMR spectra of both ferrous enzymes are reported. A series of alkyl mercaptide complexes of both synthetic and natural-derivative iron(II) porphyrins was also examined. The proton NMR spectra of these complexes facilitated the assignment of resonances due to the axial ligand in the model compounds on the basis of their isotropic shifts and multiplicities. Comparison of model compound data with that for the reduced enzymes supports assignment of the methylene protons for the axial cysteinate of ferrous cytochrome P-450_cam and ferrous chloroperoxidase to proton NMR resonances at 279 and 200 ppm (pH 7.0, 298K), respectively. Differences in the active site structure of the two enzymes are further demonstrated by ¹⁵N-NMR spectroscopy of the cyanide complexes of the ferric forms.     

Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c′

Cyanide binding to Chromatium vinosum ferricytochrome c′ has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c′ appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c′ decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c′ are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c′. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.     

Role of tertiary structures on the Root effect in fish hemoglobins

Many fish hemoglobins exhibit a marked dependence of oxygen affinity and cooperativity on proton concentration, called Root effect. Both tertiary and quaternary effects have been evoked to explain the allosteric regulation brought about by protons in fish hemoglobins. However, no general rules have emerged so far. We carried out a complementary crystallographic and microspectroscopic characterization of ligand binding to crystals of deoxy-hemoglobin from the Antarctic fish Trematomus bernacchii (HbTb) at pH 6.2 and pH 8.4. At low pH ligation has negligible structural effects, correlating with low affinity and absence of cooperativity in oxygen binding. At high pH, ligation causes significant changes at the tertiary structural level, while preserving structural markers of the T state. These changes mainly consist in a marked displacement of the position of the switch region CD corner towards an R-like position. The functional data on T-state crystals validate the relevance of the crystallographic observations, revealing that, differently from mammalian Hbs, in HbTb a significant degree of cooperativity in oxygen binding is due to tertiary conformational changes, in the absence of the T–R quaternary transition. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

The effects of inositol hexaphosphate on the allosteric properties of two beta-99-substituted abnormal hemoglobins, hemoglobin Yakima and hemoglobin Kempsey.

Hemoglobins (Hb) Yakima and Kempsey were purified from patients' blood with diethylaminoethyl cellulose column chromatography. The oxygen equilibrium curves of the two hemoglobins and the effects of organic phosphates on the function were investigated. In 0.1 M phosphate buffer, Hill's constants n for Hb Yakima and Hb Kempsey were 1.0 to 1.1 at the pH range for 6.5 to 8.0 and the oxygen affinities of both the mutant hemoglobins were about 15 to 20 times that of Hb A at pH 7.0. The Bohr effect was normal in Hb Yakima and one-fourth normal in Hb Kempsey. In the presence of inositol hexaphosphate, the oxygen affinities to Hb Yakima and Hb Kempsey were greatly decreased, and an interesting result revealed that these hemoglobins showed clear cooperativity in oxygen binding. Hill's constant n in the presence of inositol hexaphosphate was 1.9 for Hb Kempsey and 2.3 for Hb Yakima at pH 7.0. The cooperativities of these mutant hemoglobins were pH-dependent, and Hb Kempsey showed high cooperativity at low pH (n equal 2.1 at pH 6.6) and low cooperativity at high pH (n equal 1.0 at pH 8.0). Hb Yakima showed similar pH dependence in cooperativity. In the presence of inositol hexaphosphate, Hb A showed a pH-dependent cooperativity different from those of Hb Yakima and Hb Kempsey, namely, Hill's n was the highest in alkaline pH (n equal 3.0 at pH 8.0) and decreased at lower pH (n equal 1.5 at pH 6.5). 2,3Diphosphoglycerate bound with the deoxygenated Hb Yakima and Hb Kempsey, however, had no effect on the oxygen binding of these abnormal hemoglobin. The pH-dependent cooperativity of alpha1beta2 contact anomalous hemoglobin and normal hemoglobin was explained by the shifts in the equilibrium between the high and low ligand affinity forms.     

Heme-linked ionization and chloride binding in intestinal peroxidase and lactoperoxidase.

Hog intestinal peroxidase and bovine lactoperoxidase exhibited similar spectral shifts upon pH alteration. From spectrophotometric titrations, it was found that there are hemelinked ionizations of pK_a = 4.75 in intestinal peroxidase and pK_a = 3.5 in lactoperoxidase. The apparent pK_a (pK_a′) increased with the increase in chloride concentration. The pK_a′ vs log[Cl^?] plots showed that the chloride forms complex with the acid forms of these enzymes with a dissociation constant (pK = 2.7). Although the dissociation constant (K_d) of the peroxidase-cyanide complexes is nearly independent of pH, cyanide competed with chloride in the acidic pH region. The slopes of logK_d vs log[Cl^?] were 1.0 for intestinal peroxidase and 0.5 for lactoperoxidase. The reaction of hydrogen peroxide with these peroxidases was also affected by chloride, similarly as the reaction with cyanide was. The results were explained by assuming that protonation occurs at the distal base and destroys the hydrogen bond between the base and a water molecule at the sixth coordinate position of the heme iron.     

Preparation,spectroscopic characterization and anion binding studies of a mononuclear Co(II) derivative of carcinus maenas hemocyanin

The binuclear copper in the active site of Carcinus maenas hemocyanin has been substituted with one EDTA-resistant Co(II) per 75 000 M_r by reconstitution of the apo protein. Specific cobalt substitution at the copper binding site is demonstrated from the optical spectral changes directly correlated with the amount of Co(II) bound to the protein, the ellipticity in CD spectra in the near UVVis region, and the efficiency of tryptophan fluorescence quenching. The optical absorption spectrum of the cobalt-substituted protein is characterized by a band pattern attributable to d-d transitions of the metal ion. Both the position of the wavelength maximum (568 nm) and the molar extinction coefficient (≅300 M^-1 cm^-1) are typical of a four-coordinate, pseudo-tetrahedral Co(II) center.Optical titrations indicate that Cl^-, Br^-, N₃^-, SCN^-, and CN^- bind to Co(II)Hc, each with a stoichiometry of 1:1 per metal center. The apparent stability constants determined from Hill plots of titration data decrease in the order CN^- » N₃^- ≅ SCN^- >Cl^->Br^-. Low temperature EPR studies demonstrate that at pH 7, the cobalt is high spin both in the presence and absence of anionic ligands. A low spin species is formed at pH 9 in the presence of cyanide. The spectrum of this latter complex exhibits superhyperfine structure indicative of metal ligation to ¹⁴N supplied by the protein. Direct ligation of cyanide to cobalt is demonstrated by additional spectral splitting observed when this complex is formed using ¹³C-labelled CN^-.