The effect of heme-linked ionizable groups on cyanide binding to methemoglobin

The pH dependence of the kinetics of the binding of cyanide ion to methemoglobins A and S and to guinea pig and pigeon methemoglobins appears to be not directly correlated with the net charges on the proteins. The kinetics can, however, be adequately explained in terms of three sets of heme-linked ionizable groups with pK1 ranging between 4.9 and 5.3, pK2 between 6.2 and 7.9, and pK3 between 8.0 and 8.5 at 20 degrees C. pK1 is assigned to carboxylic acid groups, pK2 to histidines and terminal amino groups, and pK3 to the acid-alkaline methemoglobin transition. Kinetic second order rate constants have also been determined for the binding of cyanide ion by the four sets of methemoglobin species present in solution. The pKi values and the rate constants of methemoglobin S are strikingly different from those of methemoglobin A. This result is explained in terms of different electrostatic contributions to the free energy of heme linkage arising from differences in the environments of ionizable groups at the surfaces of the two molecules.     

Structure of cyanide methemoglobin

X-ray difference Fourier analysis at 2.8 Å resolution shows that the tertiary structures of horse cyanide methemoglobin and methemoglobin differ significantly. The conformations of the heme groups and their interactions with the globin are altered. Short contacts with globin side chains affect cyanide binding to the hemes, and the changes in globin-ligand contact upon substitution of cyanide for water in turn directly affect globin structure. Although the ligand peaks lie off the heme axes, the atoms FeCN may still lie on a straight line (as they do in small iron cyanide complexes), with this line not normal to the mean heme plane. This linear binding configuration is consistent with the observed motion and deformation of the porphyrin. Although motion of the iron atoms is not directly apparent, there is evidence that some changes in tertiary structure are induced by shortening of the iron-pyrrol nitrogen bond lengths. This and other studies suggest that the structural changes responsible for co-operativity in hemoglobin may be initiated not merely by an alteration in the covalent porphyrin-proximal histidine linkage, but by changes in the noncovalent interactions of the globin with the ligand and porphyrin as well.     

Enhancement by nitrite of peroxide-induced degradation of uric acid and 3-N-ribosyluric acid

The oxidation of uric acid and 3-N-ribosyluric acid by hydrogen peroxide and methemoglobin was stimulated by the addition of sodium nitrite, which alone has no effect on the urates. The urates were not oxidized by either hydrogen peroxide alone or hydrogen peroxide and sodium nitrite unless methemoglobin was present. t-Butyl hydroperoxide also oxidized the urates in the presence of methemoglobin, but the reaction was not stimulated by sodium nitrite. The addition of either sodium azide or potassium cyanide reduced the rate of the reaction with either hydrogen peroxide or t-butyl hydroperoxide both in the presence and absence of sodium nitrite. Possible explanations for the stimulation by nitrite of peroxide-induced degradation of urates are presented.     

Formation of intermediate products in the reaction of hemoglobin with cyanide

Interaction between methemoglobin and cyanide was studied by low temperature inhibition method in combination with ESR. A new, not earlier described in literature ESR signal of low spin cyanide complex of this protein was recorded.     

Cyanide binding to the cytochrome c ferric heme octapeptide. A model for anion binding to the active site of high spin ferric heme proteins

Equilibrium constants for the binding of cyanide to the ferric heme c octapeptide in 20% ethylene glycol, 50% buffer were measured spectrophotometrically. The equilibrium constant for cyanide binding at 20 degrees C and pH 7.4 is 3.47 X 10(7), which is approximately 15-fold lower than that observed for cyanide binding to methemoglobin or metmyoglobin. Equilibrium constants at several temperatures exhibited an apparent van't Hoff relationship, yielding thermodynamic values of delta H degrees = -79,000 J/mol (-18,900 cal/mol) and delta S degrees = J/degrees K mol (-30.1 e.u.). Comparison of the ratio of equilibrium constants for cyanide ligation to methemoglobin the heme octapeptide with the ratio of equilibrium constants for azide ligation to methemoglobin and the heme octapeptide suggests that cyanide binding to the methemoproteins is much smaller than expected by comparison to azide binding. The differences in the ratios, the thermodynamic values, and the preferred binding geometries suggest that CN- ligation, like CO ligation, is sterically hindered. A comparison of these ratios to similar ratios for CO, O2, and NO binding suggests that the Fe-CN bond angle is less subject to distortion than the Fe-CO bond and/or additional binding interactions contribute to N3- but not to CN-binding to the protein.     

Investigations of cyanide as an infrared probe of hemeprotein ligand binding sites

The measurement of infrared spectra for cyanide liganded to hemeproteins and hemins has been investigated. The hemeproteins included human methemoglobin A, lamprey methemoglobin, metchlorocruorin, horse metmyoglobin, and horseradish peroxidase. The hemins were dicyanide and monopyridine monocyanide species of deuteroporphyrin IX iron(III) and its 2,4-divinyl(proto) and 2,4-diacetyl derivatives. C-N stretch bands of low intensity detected near 2100 cm-1 exhibit changes in frequency, width, intensity, and isotope shift with changes in cyanide compound structure. Infrared band parameters are particularly sensitive to a change in oxidation state (Fe2+ versus Fe3+) and are affected to a lesser extent by changes in porphyrin ring substituent, ligand trans to the cyanide, and protein structure. Evidence of multiple conformers (i.e. multiple C-N stretch bands) was found for several hemeproteins. The cyanide infrared spectra provide direct evidence for cyanide binding as a metal cyanide (Fe--C identical to N) and against HCN being the ligand in nitrile-like bonding (Fe--N identical to C--H) in all the hemeprotein and hemin cyanides studied. With the reduced horseradish peroxidase cyanide, differences between infrared spectra for D2O and H2O solutions can result from hydrogen bonding between a protein amino acid residue and the distal atom of the cyanide (Fe--C identical to N...H+--R). The binding of cyanide to reduced iron (Fe2+) of a hemeprotein was only observed in the case of the reduced peroxidase. These findings demonstrate that cyanide infrared spectra can not only determine when cyanide is bound to a metalloprotein but can also provide information on how the cyanide is bonded to metal and on characteristics of the ligand binding site.     

The production of activated oxygen species by an interaction of methemoglobin with ascorbate

Ascorbate reacts with methemoglobin to produce reactive oxygen species, most probably hydroxyl radicals. The main features of this system are: a) disappearance of ascorbate; b) consumption of oxygen with an ascorbate/O2 stoichiometry of 2:1; c) requirement of unliganded heme iron; d) formation of H2O2. The proposed mechanism involves an ascorbate-mediated interconversion of methemoglobin and oxy-hemoglobin, resulting in the production of H2O2. This product is decomposed by hemoglobin to produce hydroxyl radicals according to a Fenton-like reaction in which ascorbate recycles methemoglobin to hemoglobin. Alternative pathways of formation and of decomposition of H2O2 in this system appear to play a minor role.     

Structure of azide methemoglobin

We have compared the structures of horse azide methemoglobin and methemoglobin (MetHb) at 2.8 Å resolution by X-ray difference Fourier analysis. Of four low-spin liganded Hb derivatives (nitric oxide Hb, carbon monoxide Hb, cyanide MetHb, and azide MetHb), azide MetHb is closest in structure to MetHb. In azide MetHb the ligands are co-ordinated end-on at angles of about 125 ° to the heme axes, which is similar to the stereochemistry assumed by azide in binding to free heme. Because of its bent binding geometry, azide encounters less interference in binding and perturbs the protein structure less than carbon monoxide and cyanide, which are smaller, but prefer linear axial co-ordination to heme. Steric interactions between ligand and protein are greater on the β chain, where the E helix is pushed away from the heme relative to MetHb, than on the α chain. Iron position is the same and heme stereochemistry and position are very similar in azide MetHb and MetHb.     

The spin-state transition of the hemochrome non-equilibrium conformation in partially reduced human methemoglobin. A pulse-radiolysis study of aqueous-methanol solutions of methemoglobin.

The effect of external parameters on the relaxation process of the hemochrome-type non-equilibrium conformation in partially reduced methemoglobin has been investigated. The relaxation of the intermediate ferrous low-spin state to the high-spin equilibrium conformation of hemoglobin appears to be facilitated particularly by protons and phosphate ions. In addition to studying the spin-state transition in aquomethemoglobin we have also studied it in complexes of the heme group in methemoglobin with fluoride, azide and cyanide anions.     

Toxicology of some inorganic antihypertensive anions.

Sodium nitroprusside reacts with hemoglobin in vitro and in vivo to cause the formation of cyanmethemoglobin and the liberation of excess free cyanide. The latter is responsible for the typical signs of acute cyanide poisoning in mice after lethal doses of nitroprusside. Differences in the reactivity of the red cells of various species toward nitroprusside are due to differences in the permeability of the red cell membranes to nitropruside. In vivo thiocyanate results in the formation of methemoglobin in an elevation of blood cyanide levels in mice. The latter, however, does not result in cyanide poisoning since it is bound in the biologically inert form of cyanmethemoglobin. Thus, both nitroprusside and thiocyanate generate their own antidote in mice, but an excess of cyanide is released in the case of nitroprusside whereas excess methemoglobin is generated in the case of thiocyanate. Acute poisoning with thiocyanate salts apparently involves direct excitatory effects on the central nervous system. In vitro the reaction between thiocyanate and hemoglobin proceeds only in the presence of hydrogen peroxide. Chronic administration of nitroprusside results in the elevation of blood thiocyanate levels presumably because of continuous, endogenous cyanide metabolism via rhodanese (thiosulfate sulfurtransferase). When one includes these previously unrecognized effects of nitroprusside and thiocyanate, there appears to be some correlation between the ability of a chemical to oxidize hemoglobin and its ability to activate nonadrenergic receptors for the relaxation of vascular smooth muscle.     

PROTEIN COAGULATION AND ITS REVERSAL : THE IDENTITY OF NORMAL HEMOGLOBIN WITH THE HEMOGLOBIN PREPARED BY THE REVERSAL OF COAGULATION,AS DETERMINED BY SOLUBILITY TESTS

1. Methemoglobin prepared from coagulated hemoglobin by the reversal of coagulation has the same solubility within 2 per cent as normal methemoglobin. 2. Methemoglobin synthesized from hemin and the native globin prepared by the reversal of coagulation of globin likewise has the same solubility as normal methemoglobin.     

Oxidation of cyanide to the cyanyl radical by peroxidase/H2O2 systems as determined by spin trapping

The cyanyl radical was formed during the oxidation of potassium or sodium cyanide by horseradish peroxidase, lactoperoxidase, chloroperoxidase, NADH peroxidase, or methemoglobin in the presence of hydrogen peroxide. The spin adducts of the cyanyl radical with 5,5-dimethyl-1-pyrroline-N-oxide and N-tert-butyl-alpha-phenylnitrone were quite stable at neutral pH. The identity of these spin adducts could be demonstrated using 13C-labeled cyanide and by comparison with the spin adducts of the formamide radical, a hydrolysis product of the cyanyl radical adduct. The enzymatic conversion of cyanide to cyanyl radical by peroxidases should be considered in addition to its well-known role as a metal ligand. Furthermore, since cyanide is used routinely as an inhibitor of peroxidases, some consideration should be given to the biochemical consequences of this formation of the cyanyl radical by the catalytic activity of these enzymes.     

Refinement of a molecular model for lamprey hemoglobin from Petromyzon marinus

A molecular model for the protein and ambient solvent of the complex of cyanide with methemoglobin V from the sea lamprey Petromyzon marinus yields an R-factor of 0.142 against X-ray diffraction data to 2.0 A resolution. The root-mean-square discrepancies from ideal bond length and angle are, respectively, 0.014 A and 1.5 degrees. Atoms that belong to planar groups deviate by 0.012 A from planes determined by a least-squares procedure. The average standard deviation for chiral volumes, peptide torsion angle and torsion angles of side-chains are 0.150 A3, 2.0 degrees and 19.4 degrees, respectively. The root-mean-square variation in the thermal parameters of bonded atoms of the polypeptide backbone is 1.21 A2; the variation in thermal parameters for side-chain atoms is 2.13 A2. The model includes multiple conformations for 11 side-chains of the 149 amino acid residues of the protein. We identify 231 locations as sites of water molecules in full or partial occupancy. The sum of occupancy factors for these sites is approximately 154, representing 28% of the 550 molecules of water within the crystallographic asymmetric unit. The environment of the heme in the cyanide complex of lamprey methemoglobin resembles the deoxy state of the mammalian tetramer. In particular, the bond between atom NE2 of the proximal histidine and the Fe lies 5.1 degrees from the normal of the heme plane. In deoxy- and carbonmonoxyhemoglobins, the deviations from the normal to the heme plane are 7 to 8 degrees and 1 degree, respectively. Furthermore, the inequality in the distance of atom CD2 of the proximal histidine from the pyrrole nitrogen of ring-C of the heme (distance = 3.29 A) and CE1 from the pyrrole nitrogen of ring-A (distance = 3.06 A) is characteristic of deoxyhemoglobin, not carbonmonoxyhemoglobin, where these distances are equal. Finally, a hydrogen bond exists between carbonyl 111 and the hydroxyl of tyrosine 149. The corresponding hydrogen link in the mammalian tetramer is central to the T to R state transition and is present in deoxyhemoglobin but absent in carbonmonoxyhemoglobin. We suggest that the low affinity of oxygen for lamprey hemoglobin may be a consequence of these T-state geometries.     

Microspectrophotometric and scanning microphotometric studies of carp (Cyprinus carpio L.) erythrocytes

Carp (Cyprinus carpio) hemoglobin readily autoxidizes in blood smears. Quantification of Soret-band absorbance in individual erythrocytes by means of scanning cytophotometry therefore requires more elaborate methods of preparation of blood samples. Of the fixatives that have been tested, suspension of whole blood in isotonic salt solutions containing glutaraldehyde was most suitable. Glutaraldehyde-fixed red blood cells are totally resistant to hemolysis. In the course of fixation, hemoglobin is transformed to methemoglobin. Spectrophotometry indicated extensive similarities between glutaraldehyde-fixed carp methemoglobin and human methemoglobin. In aqueous solutions, the intensity of the Soret-peak was pH-dependent. The allosteric modifier organic polyphosphate caused an R----T transition, resulting in increased molar extinctions. Dried preparations showed Soret-spectra that were not influenced from either pH or organic polyphosphate concentration of the aqueous suspensions in which the erythrocytes had been stored. The same was true for slide preparations of cyanomethemoglobin, easily derived from methemoglobin on addition of potassium cyanide. In the absence of oxygen fresh blood cells from carp slowly transform their hemoglobin into deoxyhemoglobin. Spectra of the intermediate stages of deoxygenation, Hb4(O2)3, Hb4(O2)2 and Hb4(O2), as well as mixtures of these intermediates, could be monitored.     

Conversion of oxyhemoglobin to methemoglobin by organic and inorganic reductants

Human oxyhemoglobin is converted to methemoglobin by a wide array of organic and inorganic reductants. Depending upon the concentration and nature of the reductant, varying amounts of deoxyhemoglobin are produced. The general overall sequence is: FeO2 leads to (1) FeIII leads to (2) FeII. The intermediacy of methemoglobin can be demonstrated by direct spectral observation and by cyanide trapping. For organic reductants, the second-order rate constants for (1) vary from greater than 300 (phenylhydroxylamine) to 1.4 X 10(-4) M-1 s-1 (malononitrile). Generally the rates parallel the ease of hydrogen abstraction by iron-bound oxygen from the substrate, and simply hydrocarbons are reactive. Rates for these processes have been ascertained with recrystallized protein, lysed cells, and intact human erythrocytes. At room temperature oxyhemoglobin quantitatively converts benzaldehyde to benzoic acid and hydroquinone to benzoquinone. Rates for inorganic species (process 1) range from greater than 7 X 10(3) (chromous ion) to 0.015 M-1 s-1 (ferrocyanide). Ferrous ion rapidly deoxygenates oxyhemoglobin by direct attack on the oxy complex but methemoglobin is not an intermediate with this reagent. Taken together the results support the theoretical prediction that reductants should oxidize oxyhemoglobin, and they demonstrate at least some degree of radical character to the oxy complex.     

Up-regulation of alpha1-microglobulin by hemoglobin and reactive oxygen species in hepatoma and blood cell lines

alpha(1)-Microglobulin is a 26-kDa glycoprotein synthesized in the liver, secreted to the blood, and rapidly distributed to the extravascular compartment of all tissues. Recent results show that alpha(1)-microglobulin has heme-binding and heme-degrading properties and it has been suggested that the protein is involved in the defense against oxidation by heme and reactive oxygen species. In the present study the influence of hemoglobin and reactive oxygen species (ROS) on the cellular expression of alpha(1)-microglobulin was investigated. Oxy- and methemoglobin, free heme, and Fenton reaction-induced hydroxyl radicals induced a dose-dependent up-regulation of alpha(1)-microglobulin on both mRNA and protein levels in hepatoma cells and an increased secretion of alpha(1)-microglobulin. The up-regulation was reversed by the addition of catalase and ascorbate, and by reacting hemoglobin with cyanide which prevents redox reactions. Furthermore, the blood cell lines U937 and K562 expressed alpha(1)-microglobulin at low levels, and this expression increased up to 11-fold by the addition of hemoglobin. These results suggest that alpha(1)-microglobulin expression is induced by ROS, arising from redox reactions of hemoglobin or from other sources and are consistent with the hypothesis that alpha(1)-microglobulin participates in the defense against oxidation by hemoglobin, heme, and reactive oxygen species.     

EXPERIMENTALLY INDUCED CHROMOSOME ABERRATIONS IN PLANTS : II. THE EFFECT OF CYANIDE AND OTHER HEAVY METAL COMPLEXING AGENTS ON THE PRODUCTION OF CHROMOSOME ABERRATIONS BY X-RAYS

The discovery of Lilly and Thoday, that the presence of potassium cyanide (KCN) increases the production of chromosome aberrations by x-rays in anoxia, but has no effect on the production of chromosome aberrations by x-rays in air, was confirmed. In the presence of cyanide, the effect of a given dose of x-rays in nitrogen was found to be even greater than the effect of the same dose of x-rays in air. The cyanide effect on x-ray breakage in nitrogen was obtained at cyanide concentrations as low as 2 x 10^–5 M. The breakage obtained after the combined x-ray-cyanide treatments was of the x-ray type, as evidenced by the distribution of breaks within and between the chromosomes. A number of other heavy metal complexing agents as well as some other compounds were tested for their ability to increase x-ray breakage in nitrogen and air. Of these compounds only cupferron proved to be effective. The results are discussed and it is concluded that the increased x-ray breakage in the presence of cyanide or cupferron cannot be due to an accumulation of peroxides. Instead it is suggested that the cyanide effect may be due to a complex formation between the active agents and heavy metals, presumably iron, within the chromosomes. The consequences of this hypothesis on the concept of the "oxygen effect," are discussed.     

Conformation and spin state in methemoglobin.

The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.     

Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide

Human neutrophils incubated with phorbol myristate acetate oxidized hemoglobin within the intact erythrocyte by a mechanism dependent on cell-cell contact but independent of phagocytosis. Spectrophotometric examination of the erythrocyte lysates revealed that the major component formed was methemoglobin along with small amounts of a species with spectral characteristics similar to choleglobin. Methemoglobin formation was directly related to the neutrophil concentration and the time of incubation. The addition of superoxide dismutase or catalase modestly inhibited the formation of methemoglobin, while a combination of the enzymes provided the most dramatic protection. Methemoglobin of hydroxyl radical or hypochlorous acid scavengers. Apparently, either O2.- or H2O2 alone was capable of mediating methemoglobin formation in the intact erythrocyte. Maintenance of the intraerythrocytic hemoglobin in its oxygenated state or its derivatization to carbon monoxyhemoglobin markedly inhibited methemoglobin formation. Blockade of the anion channels in the intact erythrocyte with sulfonated stilbenes inhibited O2.- but not H2O2 from oxidizing intracellular hemoglobin. It appears that neutrophil-derived O2.- and H2O2 can cross the erythrocyte membrane through the anion channel or diffuse directly into the intracellular space and react with oxyhemoglobin or deoxyhemoglobin to form a mixture of hemoglobin oxidation products within the intact cell.     

Dormancy in Cereal Seeds: I. THE EFFECTS OF OXYGEN AND RESPIRATORY INHIBITORS

A wide spectrum of respiratory inhibitors has been found tostimulate the breaking of dormancy in barley. These includecarbon monoxide, cyanide, azide, hydrogen sulphide, sodium sulphide,hydroxylamine, diethyldithiocarbamate (DIECA), fluoride, iodoacetate,malonate, monofluoroacetate, and 2,4-dinitrophenol (DNP). Inrice, only the first six of these have been shown to be effective.Apart from CO, all the above inhibitors were tested on winteroats, but in this material only cyanide, azide, and hydroxylaminewere found to increase the germination of dormant seeds. Allthe terminal-oxidase inhibitors except CO were tested on perennialryegrass, but in this case only cyanide was found to break dormancy. As compared with air, an atmosphere of 96 per cent oxygen appliedto barley during the first 24 h after the seeds have been setto germinate stimulates the breaking of dormancy. When appliedat later stages, this high oxygen tension inhibits the germinationof dormant seeds although it has no effect on nondormant seeds.Paradoxically, the stimulatory effects of respiratory inhibitorsapplied during the initial stages of germination are relatedto their ability to inhibit oxygen uptake. Thus cyanide, azide,malonate, and monofluoroacetate, while stimulating the breakingof dormancy in barley, also inhibit oxygen uptake. In rice,cyanide and azide had similar effects, but fluoride, which hadno effect on dormancy, also had no effect on the oxygen uptakeof dormant seeds. These results are compatible with the hypothesis that some oxidationreaction is necessary for germination. This oxidation is notpart of the normal respiratory pathway, and does not proceedsatisfactorily in dormant seeds. It may be stimulated, however,by increasing the oxygen tension or by reducing normal respiratorycompetition with respiratory inhibitors.