Ligand binding, reactivity and biological activity of a distal pocket mutant of neuroglobin.

We have generated the Lys67Glu mutant form of neuroglobin. Experimental spectral studies are consistent with a six coordinate heme in which the distal histidine bond is stretched compared to the wild type protein. Carbon monoxide binding to the ferrous form of the mutant follows a hyperbolic concentration dependence limiting at the histidine dissociation rate of 0.7 s(-1). Further analysis indicates a significantly lowered histidine binding constant. Oxygen binding kinetic studies confirm the higher heme ligand dissociation level and indicate a p50 value for oxygen binding<1 mmHg. The ferrous form of the protein yields an oxygenated intermediate on reaction with oxygen. The rate of oxidation, by oxygen, follows a complex concentration dependence, consistent with the presence of two distinct oxidation mechanisms. A quantitative model for the two oxidation processes has been developed, which is consistent with a lowered distal histidine binding constant in the mutant form of the protein. These data suggest that the protein structure surrounding the heme site in neuroglobin limits access to external ligands and provides an energy barrier to the structural changes following ligand binding in this protein. However, the mutation does not appear to affect reactivity with cytochrome c and the anti-apoptotic activity of the mutant in human cells of neuronal origin is increased as compared to the wild type protein.

The binding of cytochrome c to neuroglobin: a docking and surface plasmon resonance study.

It has recently been proposed that the role of neuroglobin in the protection of neurons from ischaemia induced cell death requires the formation of a transient complex with cytochrome c. No such complex has yet been isolated. Here, we present the results of soft docking calculations, which indicate one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved. surface plasmon resonance studies provide a value of 45 microM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation is associated with a small unfavourable enthalpy change (1.9 kcal mol(-1)) and a moderately large, favourable entropy change (14.8 cal mol(-1) deg(-1)). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions.

Red blood cell function and haematology in two tropical freshwater fishes from Australia.

Salmon catfish and tarpon occur in habitats that periodically become deficient in oxygen resulting in high mortalities of other fish species. The water-breathing catfish, Arius leptaspis, and the facultative air-breathing tarpon, Megalops cyprinoides, both have high haemoglobin and haematocrit, and the oxygen carrying capacity in the air-breather is exceptionally high (15.6+/-1.2 vol%). Iso-pH oxygen equilibria of the red blood cells at 25 degrees C revealed high affinity (P(50)=9 mmHg, pH 7.4) and co-operativity (n(50)>2.2, pH 7.4) in the catfish, and contrasted with low affinity (P(50)=32 mmHg, pH 7.4) and co-operativity (n(50) approximately 1) in the air-breathing tarpon. Oxygen binding was further distinguished by relative pH insensitivity (Bohr factor, Ø=Deltalog P(50)/Deltalog pH=-0.22) in the catfish, compared with a significant Bohr effect in the tarpon (Ø=-0.96). The potential for modulation of haemoglobin-oxygen affinity was indicated by a high ratio of GTP to ATP in the erythrocytes of the catfish, whereas regulation in the tarpon appeared due to ATP alone. Differences in blood respiratory functions between the two species are likely to reflect reduced opportunity for activity under extreme hypoxia in the catfish.

Cytochrome cbb(3) oxidase and bacterial microaerobic metabolism.

Cytochrome cbb(3) oxidase is a member of the haem-copper oxidase superfamily. It is characterized by its high oxygen affinity, while retaining the ability to pump protons. These attributes are central to its proposed role in bacterial microaerobic metabolism. Recent spectroscopic characterization of both the cytochrome cbb(3) oxidase complex from Pseudomonas stutzeri and the dihaem ccoP subunit expressed separately in Escherichia coli has revealed the presence of a low-spin His/His co-ordinated c-type cytochrome. The low midpoint reduction potential of this haem (E(m)<+100 mV), together with its unexpected ability to bind CO in the reduced state at the expense of the distal histidine ligand, raises questions about the role of the ccoP subunit in the delivery of electrons to the active site.

The role of beta chains in the control of the hemoglobin oxygen binding function: chimeric human/mouse proteins, structure, and function.

By using transgenic methodologies, we have produced a number of mouse/human chimeric hemoglobins containing adult mouse and human embryonic globin chains. A detailed analysis of the oxygen binding properties of these proteins identifies the dominant role played by the specific beta-type globin chains in the control of the oxygen binding characteristics. Further analysis traces the origins of these effects to alterations in the properties of the T states of these proteins. The human zeta/mouse beta chimeric protein has been crystallized, and its structure has been determined by X-ray diffraction to a resolution of 2.1 A with R (R(free)) values of 21.6% (24.9%). Close examination of the structure indicates that the subunit interfaces contain contacts which, although different from those present in either the parent human or the parent mouse proteins, retain the overall stabilizing interactions seen in other R state hemoglobins.

Oligomerization and ligand binding in a homotetrameric hemoglobin: two high-resolution crystal structures of hemoglobin Bart's (gamma(4)), a marker for alpha-thalassemia.

Hemoglobin (Hb) Bart's is present in the red blood cells of millions of people worldwide who suffer from alpha-thalassemia. alpha-Thalassemia is a disease in which there is a deletion of one or more of the four alpha-chain genes, and excess gamma and beta chains spontaneously form homotetramers. The gamma(4) homotetrameric protein known as Hb Bart's is a stable species that exhibits neither a Bohr effect nor heme-heme cooperativity. Although Hb Bart's has a higher O(2) affinity than either adult (alpha(2)beta(2)) or fetal (alpha(2)gamma(2)) Hbs, it has a lower affinity for O(2) than HbH (beta(4)). To better understand the association and ligand binding properties of the gamma(4) tetramer, we have solved the structure of Hb Bart's in two different oxidation and ligation states. The crystal structure of ferrous carbonmonoxy (CO) Hb Bart's was determined by molecular replacement and refined at 1.7 A resolution (R = 21.1%, R(free) = 24.4%), and that of ferric azide (N(3)(-)) Hb Bart's was similarly determined at 1.86 A resolution (R = 18.4%, R(free) = 22.0%). In the carbonmonoxy-Hb structure, the CO ligand is bound at an angle of 140 degrees, and with an unusually long Fe-C bond of 2.25 A. This geometry is attributed to repulsion from the distal His63 at the low pH of crystallization (4.5). In contrast, azide is bound to the oxidized heme iron in the methemoglobin crystals at an angle of 112 degrees, in a perfect orientation to accept a hydrogen bond from His63. Compared to the three known quaternary structures of human Hb (T, R, and R2), both structures most closely resemble the R state. Comparisons with the structures of adult Hb and HbH explain the association and dissociation behaviour of Hb homotetramers relative to the heterotetrameric Hbs.

Haem disorder in recombinant- and reticulocyte-derived haemoglobins: evidence for stereoselective haem insertion in eukaryotes.

We have used NMR spectroscopy to measure haem disorder in adult human haemoglobin (HbA) obtained from mature erythrocyte cells and from yeast expressing recombinant HbA. Reticulocyte-derived HbA contained much higher levels of haem disorder (11% alpha- and 28% beta-subunit disorder) than observed for HbA from mature erythrocytes (1.5% alpha- and 8% beta-subunit disorder). Thus, unlike in vitro combination of haem and apoHb, biosynthetic haem insertion is not random with respect to orientation, but appears to show stereoselectivity. Recombinant HbA isolated from yeast showed 32% alpha- and 45% beta-subunit haem disorder. These levels relaxed to their equilibrium positions after incubating the Hb in the ferric form. Recombinant embryonic human Hbs showed less haem disorder than recombinant HbA. The levels of haem disorder in embryonic Hbs zeta(2)epsilon(2) and zeta(2)gamma(2) appear to have their equilibrium values. We propose that, in eukaryotes, in vivo haem insertion occurs via both co-translational mechanisms and insertion via semiHb-beta.

Subunit dissociation and reassociation leads to preferential crystallization of haemoglobin Bart's (gamma4) from solutions of human embryonic haemoglobin Portland (zeta2gamma2) at low pH.

A variety of human haemoglobins (Hbs) are produced at different stages of human development, including three embryonic Hbs, foetal Hb and adult Hb. All are heterotetramers. During crystallization experiments on human embryonic Hb Portland (zeta(2)gamma(2)), it was discovered by crystallographic and biochemical analysis that the homotetramer Hb Bart's (gamma(4)) preferentially crystallizes from zeta(2)gamma(2) solutions below pH 5. This results from dissociation of Hb Portland into gamma(2) dimers and zeta monomers and has interesting implications for subunit interactions and tetramer stability in Hbs. It also makes possible a full crystallographic analysis of Hb Bart's, which is of considerable medical significance because of its presence in the red blood cells of millions of people worldwide who suffer from alpha-thalassaemia.

The ferric form of the three human embryonic hemoglobins and their reactions with azide ions.

The ferric forms of the three human embryonic hemoglobins exhibit pH titration's with pKa values in the range 8.25-8.6, which correlate with the reduced proteins affinity for oxygen. Azide ions bind to each ferric protein in a process consisting of two separate binding steps. The equilibrium constants for this process are in the 10-30 microM range and can be assigned to the individual chain within the proteins as reactivity appears independent of the nature of the partner chain. The kinetics of the azide binding reactions occur on the second time scale at mM ligand concentrations and also indicate the presence of two distinct processes that have been assigned to each of the two chains within the proteins. Non of these processes indicate any evidence of heme-heme interactions within the ferric form of the proteins. These findings are discussed in comparison with previously reported properties of adult ferric hemoglobin.

Functional protection of human haemoglobin against protein dissociation.

The spectroscopic and functional properties of human adult haemoglobin are clearly disrupted by concentrations of urea > 0.4 M. This disruption of structure and function is completely obviated by the presence of 0.2 M trimethylamine N-oxide (TMAO). Spectroscopic data suggest that TMAO prevents urea-induced production of high-spin haem. Functional analysis shows that TMAO exerts its influence by counteracting urea-induced destabilisation of the T state of the haemoglobin protein. Further studies show, however, that TMAO is not able to exert any such stabilising influences in the presence of high concentrations of typical organic solvent denaturants.

The role of amino acid alpha38 in the control of oxygen binding to human adult and embryonic haemoglobin Portland.

The role of the amino acid at position alpha(38) in haemoglobin has been probed using site-directed mutagenesis. When the Thr residue at position alpha(38) (which is totally conserved in all mammals) is changed to a Gln, the equilibrium properties of the protein are significantly altered. Equilibrium and kinetic data show that the R-state properties of the protein are essentially unaffected by the mutation whilst the allosteric equilibrium and T-state properties are changed. Mutation of the naturally occurring Gln(38) of the human embryonic haemoglobin zeta-chain (the only known non-Thr containing globin) to a Thr residue shows the converse change in properties produced by the adult mutation, although in this case the situation is complicated by significant chain heterogeneity in the T state. An extension of the two-state model of co-operativity is presented to describe quantitatively the equilibrium ligand binding in the presence of T-state chain heterogeneity. A molecular model is described in which the putative interaction of alphaGln(38) and betaTyr(145) is identified which make a significant contribution to the previously reported unusual ligand-binding properties of the zeta-chain containing human embryonic haemoglobins.

Origin of the suppression of chloride ion sensitivity in human embryonic hemoglobin Gower II.

The molecular origin of the observed reduction of chloride ion sensitivity in the oxygen binding characteristics of the human embryonic hemoglobin Gower II has been probed by using site-directed mutant forms of the protein. Separate mutation of the three amino acids previously identified as candidates for the suppression of chloride sensitivity in the epsilon chain allowed us to unambiguously identify the His beta 77-->epsilon Asn as the origin of the lower sensitivity towards chloride ions in hemoglobin Gower II, which allows oxygen exchange from the mother to the late embryo under physiological conditions.

Partitioning of oxygen and carbon monoxide in the three human embryonic hemoglobins.

The ratio of oxygen to carbon monoxide binding to the three fully saturated human embryonic hemoglobins has been determined. The embryonic hemoglobins exhibit significantly lower values of carbon monoxide binding than any previously reported mammalian fetal or adult hemoglobins. The possible protective role of this with regards to carbon monoxide intoxication is discussed. These data are combined with previous parameters yielding a significant correlation between oxygen affinity and carbon monoxide binding. A possible molecular origin of this correlation is discussed.

Crystal structure of a human embryonic haemoglobin: the carbonmonoxy form of gower II (alpha2 epsilon2) haemoglobin at 2.9 A resolution.

The production of recombinant embryonic haemoglobins via a yeast expression system has enabled structural and functional studies to be conducted on these proteins. As part of a programme aimed at understanding the properties of the embryonic haemoglobins we have crystallized the human alpha2 epsilon2 (Gower II) embryonic haemoglobin in its carbonmonoxy form, and determined its structure by X-ray crystallography. The structure was solved by molecular replacement and refined at 2.9 A to give a final model with R-factor=0.185 and Rfree=0.235. The Gower II hemoglobin tetramer is intermediate between the adult R and R2 states, though closer to R2. The tertiary structure of the conserved alpha subunit is essentially identical when compared to that found in the adult (alpha2 beta2) and fetal (alpha2 gamma2) hemoglobins. The embryonic epsilon subunit has a structure very similar to that of the homologous adult beta and fetal gamma subunits, although with small differences at the N terminus and in the A helix. Amino acid substitutions can be identified that may play a role in the altered response of the Gower II haemoglobin to allosteric effectors, in particular chloride ions. The reduced chloride effect is thought to be the primary cause of the higher affinity of this embryonic hemoglobin in comparison to the adult molecule.

Non-synergistic interactions between strong allosteric effectors and human embryonic and adult haemoglobins.

The binding of two strong allosteric effectors (2,3 Diphosphoglycerate D.P.G., and Bezafibrate, Bzf) to both adult and the three human embryonic haemoglobins, either individually or in combination, have been studied in detail. The adult protein exhibits one binding site for D.P.G and two for Bzf. When both effectors are present simultaneously their effects are simply additive. The same qualitative pattern of binding is observed in the case of the three human embryonic haemoglobins, although with different binding constants. The lack of synergism between these effectors and the different binding affinity expressed by these proteins are discussed in terms of the known amino acid sequence differences.

A conserved glutamic acid in helix VI of cytochrome bo3 influences a key step in oxygen reduction.

We have compared the reactions with dioxygen of wild-type cytochrome bo3 and a mutant in which a conserved glutamic acid at position-286 of subunit I has been changed to an alanine. Flow-flash experiments reveal that oxygen binding and the rate of heme-heme electron transfer are unaffected by the mutation. Reaction of the fully (3-electron) reduced mutant cytochrome bo3 with dioxygen yields a binuclear center which is substantially in the P (peroxy) state, not the well-characterized F (oxyferryl) state which is the product of the reaction of the fully reduced wild-type enzyme with dioxygen [Puustinen, A., et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1545-1548]. These results confirm that proton uptake is important in controlling the later stages of dioxygen reduction in heme-copper oxidases and show that E286 is an important component of the channel that delivers these protons to the active site.

A two-state analysis of co-operative oxygen binding in the three human embryonic haemoglobins.

The binding of oxygen to the three human embryonic haemoglobins, at pH 7.4, has been shown to occur as a co-operative process. Analysis of oxygen-binding curves obtained in the absence of organic phosphate allosteric effectors shows that the process can be described quite accurately by the two-state model of allosteric action. In the presence of organic phosphates, the binding affinity for oxygen to the T-state of the alpha 2 epsilon 2 and zeta 2 epsilon 2 haemoglobins is significantly lowered. The values of the best-fit two-state parameters determined for each of the embryonic haemoglobins together with the temperature-dependence of the overall equilibrium binding process are discussed in terms of oxygen transfer from the maternal blood supply. Fast-reaction studies have been used to determine the rate constants of the oxygen association and dissociation processes occurring in the R-state and the rate of the allosteric R > T conformational transition. Analysis of these data suggests a likely reason for the high affinity and low co-operativity of the embryonic proteins and identifies the origins of the inability of equilibrium measurements to identify chain non-equivalence in the R-state.

Reaction of variant sperm-whale myoglobins with hydrogen peroxide: the effects of mutating a histidine residue in the haem distal pocket.

The reaction of hydrogen peroxide with a number of variants of sperm-whale myoglobin in which the distal pocket histidine residue (His64) had been mutated was studied with a combination of stopped-flow spectroscopy and freeze-quench EPR. The rate of the initial bimolecular reaction with hydrogen peroxide in all the proteins studied was found to depend on the polarity of the amino acid side chain at position 64. In wild-type myoglobin there were no significant optical changes subsequent to this reaction, suggesting the rapid formation of the well-characterized oxyferryl species. This conclusion was supported by freeze-quench EPR data, which were consistent with the pattern of reactivity previously reported [King and Winfield (1963) J. Biol. Chem. 238, 1520-1528]. In those myoglobins bearing a mutation at position 64, the initial bimolecular reaction with hydrogen peroxide yielded an intermediate species that subsequently decayed via a second hydrogen peroxide-dependent step leading to modification or destruction of the haem. In the mutant His64-->Gln the calculated electronic absorption spectrum of the intermediate was not that of an oxyferryl species but seemed to be that of a low-spin ferric haem. Freeze-quench EPR studies of this mutant and the apolar mutant (His64-->Val) revealed the accumulation of a novel intermediate after the first hydrogen peroxide-dependent reaction. The unusual EPR characteristics of this species are provisionally assigned to a low-spin ferric haem with bound peroxide as the distal ligand. These results are interpreted in terms of a reaction scheme in which the polarity of the distal pocket governs the rate of binding of hydrogen peroxide to the haem iron and the residue at position 64 governs both the rate of heterolytic oxygen scission and the stability of the oxyferryl product.

The control of oxygen affinity in the three human embryonic haemoglobins by respiration linked metabolites.

The effect of CO2, ATP and lactate ions on the oxygen affinity of the three human embryonic haemoglobins have been studied. CO2 lowers the affinity of both the adult and embryonic haemoglobins for oxygen, as does ATP. The ATP effect follows a simple binding process with an equilibrium constant in the mM range. Lactate ions have no effect on the oxygen equilibrium process, over the concentration range studied. These findings are discussed in terms of the likely physiological conditions experienced by human embryonic red blood cells.

The reaction of Escherichia coli cytochrome bo with H2O2: evidence for the formation of an oxyferryl species by two distinct routes.

We have re-examined the reaction of fast oxidised cytochrome bo with H202 in a stopped-flow spectrophotometer. Monitoring the reaction at 582 nm allows us to observe the formation and decay of a spectroscopically distinct intermediate which accumulates transiently prior to the formation of an oxyferryl species previously characterised in this laboratory (Watmough, N.J., Cheesman, M.R., Greenwood, C. and Thomson, A.J. (1994) Biochem. J. 300, 469-475 [1]). The reaction shows three distinct phases of which the fast and intermediate phases are bimolecular and show a marked pH dependence. Initially these results appeared incompatible with the report that only one equivalent of H202 is required to generate the oxyferryl species (Moody, A.J. and Rich, P.R. (1994) Eur. J. Biochem. 226, 731-737 [21]. However, these data can be reconciled by a branched reaction mechanism whose contributions differ according to the peroxide concentration used.