Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450.

After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglobin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that haemoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of [3H]haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Our findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic 'free' haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.     

The frpB1 gene of Helicobacter pylori is regulated by iron and encodes a membrane protein capable of binding haem and haemoglobin

FrpB1 is a novel membrane protein of Helicobacter pylori that is capable of binding both haem and haemoglobin but consistently shows more affinity for haem. The mRNA levels of frpB1 were repressed by iron and lightly modulated by haem or haemoglobin. The overexpression of the frpB1 gene supported cellular growth when haem or haemoglobin were supplied as the only iron source. Three-dimensional modelling revealed the presence of motifs necessary to bind either haem or haemoglobin. Our overall results support the idea that FrpB1 is a membrane protein of H. pylori that allows this pathogen to survive in the human stomach.     

Mechanism of methaemoglobin breakdown by the lysine-specific gingipain of the periodontal pathogen Porphyromonas gingivalis

Abstract The R- and K-gingipain proteases of Porphyromonas gingivalis are involved in proteolysis of haemoglobin from which the defensive dimeric haem pigment is formed. Whilst oxyhaemoglobin is refractory towards K-gingipain, methaemoglobin is rapidly degraded. Ligation of methaemoglobin with N3-, which effectively blocks haem dissociation from the protein, prevented haemoglobin breakdown. Haem-free globin was rapidly degraded by K-gingipain. These data emphasise the need for haemoglobin oxidation which encourages haem dissociation and makes the haem-free globin susceptible to proteolytic attack.     

Structure of deoxy-quaternary haemoglobin with liganded beta subunits

We have determined the structure of a T-state haemoglobin in which the haem groups of the beta subunits have carbon monoxide bound, and the alpha subunits have nickel replacing the haem iron and are ligand-free. The structural adjustments on binding ligand in the T state are in the same direction as those associated with the quaternary transition, and a translational shift of the haem is severely restricted. We explain how these observations may account for the low ligand affinity of the beta haem of T-state haemoglobin.     

Haem disorder in reconstituted human haemoglobin

Degradation of the haem of haemoglobin (used as a chemical probe of the haem–protein relationship), suggests that reconstituted human haemoglobin contains significant haem disorder. This results from the insertion of haem into globin with an orientation 180° different from the natural orientation. Haem disorder also slowly occurs in methaemoglobin solutions.     

HmuY haemophore and gingipain proteases constitute a unique syntrophic system of haem acquisition by Porphyromonas gingivalis

Haem (iron protoporphyrin IX) is both an essential growth factor and virulence regulator for the periodontal pathogen Porphyromonas gingivalis, which acquires it mainly from haemoglobin via the sequential actions of the R- and K-specific gingipain proteases. The haem-binding lipoprotein haemophore HmuY and its cognate receptor HmuR of P. gingivalis, are responsible for capture and internalisation of haem. This study examined the role of the HmuY in acquisition of haem from haemoglobin and the cooperation between HmuY and gingipain proteases in this process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to wrest haem from immobilised methaemoglobin and deoxyhaemoglobin. Haem extraction from oxyhaemoglobin was facilitated after oxidation to methaemoglobin by pre-treatment with the P. gingivalis R-gingipain A (HRgpA). HmuY was also capable of scavenging haem from oxyhaemoglobin pre-treated with the K-gingipain (Kgp). This is the first demonstration of a haemophore working in conjunction with proteases to acquire haem from haemoglobin. In addition, HmuY was able to extract haem from methaemalbumin, and could bind haem, either free in solution or from methaemoglobin, even in the presence of serum albumin.     

Influence of anions and protons on the Adair coefficients of haemoglobins A and Cowtown (His HC3(146) beta----Leu)

We have measured the contribution of the alkaline Bohr effect of the C-terminal histidine residues of the beta-chains of haemoglobin A by comparing haemoglobin A with haemoglobin Cowtown in which those histidine residues are replaced by leucine. Oxygenation of a stripped 2.5 mM (haem) solution of haemoglobin A yielded 0.19 H+/haem, while oxygenation of a similar solution of haemoglobin Cowtown produced no change of pH. Oxygen equilibria measured at 60 microM-haem in 0.1 M-Hepes buffer gave an alkaline Bohr effect of -0.21 H+/haem for haemoglobin A and only -0.01 H+/haem for haemoglobin Cowtown, even though its Hill's coefficient was greater than 2 throughout the pH range studied. These results prove that the chloride-independent part of the alkaline Bohr effect is due to the C-terminal histidine residues of the beta-chains. Oxygen equilibria measured in 0.095 M-bis-Tris buffers with minimal chloride or with 0.1 M-chloride showed the contribution of those histidine residues to the alkaline Bohr effect to be about 0.2 H+/haem, independent of chloride concentration. Determination of the individual Adair coefficients in the three different buffers indicated that pH and chloride tend to have their greatest effects at the second or third steps of oxygenation when the change of quaternary structure is most likely to occur; between pH 7 and 9, the fourth Adair coefficient is only very slightly affected by pH and not significantly by chloride.     

Emerging strategies in microbial haem capture

Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins. These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins. Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell. Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell. This review describes recent work on the discovery and characterization of these systems. Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded). Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined.     

Crystal structure of haemoglobin from donkey (Equus asinus) at 3A resolution

Haemoglobin from donkey was purified and crystallized in space group C2. The present donkey haemoglobin model comprises of two subunits alpha and beta. These alpha and beta subunits comprise of 141 and 146 amino acid residues, respectively, and the haem groups. The donkey haemoglobin differs from horse only in two amino acids of alpha-chain (His20 to Asn and Tyr24 to Phe) and these substitutions do not significantly change the secondary structural features of donkey haemoglobin. The haem group region and subunit contacts are closely resemble with that of horse methaemoglobin.     

Entamoeba histolytica secretes two haem-binding proteins to scavenge haem

Entamoeba histolytica is a human pathogen which can grow using different sources of iron such as free iron, lactoferrin, transferrin, ferritin or haemoglobin. In the present study, we found that E. histolytica was also capable of supporting its growth in the presence of haem as the sole iron supply. In addition, when trophozoites were maintained in cultures supplemented with haemoglobin as the only iron source, the haem was released and thus it was introduced into cells. Interestingly, the Ehhmbp26 and Ehhmbp45 proteins could be related to the mechanism of iron acquisition in this protozoan, since they were secreted to the medium under iron-starvation conditions, and presented higher binding affinity for haem than for haemoglobin. In addition, both proteins were unable to bind free iron or transferrin in the presence of haem. Taken together, our results suggest that Ehhmbp26 and Ehhmbp45 could function as haemophores, secreted by this parasite to facilitate the scavenging of haem from the host environment during the infective process.     

Structure of haemoglobin in the deoxy quaternary state with ligand bound at the alpha haems

We report the X-ray crystal structure of two analogues of human haemoglobin in the deoxy quaternary (T) state with ligand bound exclusively at the alpha haems. These models were prepared from symmetric, mixed-metal hybrid haemoglobin molecules. The structures of alpha Fe(II) beta Co(II), its carbonmonoxy derivative alpha Fe(II)CO beta Co(II), and alpha Fe(II)O2 beta Ni(II) are compared with native deoxy haemoglobin by difference Fourier syntheses at 2.8, 2.9 and 3.5 A resolution, respectively, and the refined alpha Fe(II)CO beta Co(II) structure is analysed. In both the native deoxy and liganded T molecules, the mean plane of the alpha-subunit haem is parallel with the axis of the F helix, but this plane is tilted with respect to the helix axis in the oxy-quaternary R state. The side-chains of LeuFG3 and ValFG5 sterically restrict haem tilting in the T state. We propose that strain energy develops at the contact between the haem and these residues in the liganded T-state haemoglobin, and that the strain is, in part, responsible for the low affinity of the T-state alpha haem.     

Resonance Raman scattering on the haem group of cytochrome c

Resonance Raman spectra of the haem group of 8 × 10^?5 M horse heart ferro- and ferricytochrome c solutions have been obtained. The spectra are almost identical to that of haemoglobin. The frequency of the Raman line near 1370 cm^?1, which in haemoglobin is sensitive to the position of the haem iron, indicates that the iron atom of cytochrome c lies in the plane of the porphyrin for both oxidation states.     

A new ATP-binding cassette protein is involved in intracellular haem trafficking in Leishmania

The characterization of LABCG5, a new intracellular ATP-binding cassette protein in Leishmania donovani, is described. Unlike other ABCG half-transporters, LABCG5 is not involved in either drug resistance or phospholipid efflux. However, we provide evidence suggesting that this protein is involved in intracellular haem trafficking. Thus, downregulation of LABCG5 function produced upon overexpression of an inactive version of the protein caused a dramatic growth arrest unless a haemin supplement was added or the mutated gene was eliminated. Supplementation with haemoglobin, an upstream metabolite normally sufficient to meet parasite haem requirements, was unable to rescue the growth defect phenotype. Haemoglobin endocytosis was not hampered in dominant-negative parasites and neither was haem uptake, a process that we show here to be dependent on a specific transporter. In contrast, LABCG5 function was required for the correct intracellular trafficking of haemoglobin-bound porphyrins to the mitochondria, not affecting the routing of free haem. Finally, LABCG5 binds haem through hydrophobic and electrostatic interactions. Altogether, these data suggest that LABCG5 is involved in the salvage of the haem released after the breakdown of internalized haemoglobin. As Leishmania is auxotrophic for haem, the pharmacological targeting of this route could represent a novel approach to control fatal visceral leishmaniasis.     

Histidine and not tyrosine is required for the peroxide-induced formation of haem to protein cross-linked myoglobin

Peroxide-induced oxidative modifications of haem proteins such as myoglobin and haemoglobin can lead to the formation of a covalent bond between the haem and globin. These haem to protein cross-linked forms of myoglobin and haemoglobin are cytotoxic and have been identified in pathological conditions in vivo. An understanding of the mechanism of haem to protein cross-link formation could provide important information on the mechanisms of the oxidative processes that lead to pathological complications associated with the formation of these altered myoglobins and haemoglobins. We have re-examined the mechanism of the formation of haem to protein cross-link to test the previously reported hypothesis that the haem forms a covalent bond to the protein via the tyrosine 103 residue (Catalano, C. E., Choe, Y. S., Ortiz de Montellano, P. R., J. Biol. Chem. 1989, 10534 - 10541). Comparison of native horse myoglobin, recombinant sperm whale myoglobin and Tyr(103) --> Phe sperm whale mutant shows that, contrary to the previously proposed mechanism of haem to protein cross-link formation, the absence of tyrosine 103 has no impact on the formation of haem to protein cross-links. In contrast, we have found that engineered myoglobins that lack the distal histidine residue either cannot generate haem to protein cross-links or show greatly suppressed levels of modified protein. Moreover, addition of a distal histidine to myoglobin from Aplysia limacina, that naturally lacks this histidine, restores the haem protein's capacity to generate haem to protein cross-links. The distal histidine is, therefore, vital for the formation of haem to protein cross-link and we explore this outcome.     

Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates

Haemoglobin-based oxygen carriers can undergo oxidation of ferrous haemoglobin into a non-functional ferric form with enhanced rates of haem loss. A recently developed human haemoglobin conjugated to maleimide-activated poly(ethylene glycol), termed MP4, has unique physicochemical properties (increased molecular radius, high oxygen affinity and low cooperativity) and lacks the typical hypertensive response observed with most cell-free haemoglobin solutions. The rate of in vitro MP4 autoxidation is higher compared with the rate for unmodified SFHb (stroma-free haemoglobin), both at room temperature (20-22 degrees C) and at 37 degrees C (P<0.001). This appears to be attributable to residual catalase activity in SFHb but not MP4. In contrast, MP4 and SFHb showed the same susceptibility to oxidation by reactive oxygen species generated by a xanthine-xanthine oxidase system. Once fully oxidized to methaemoglobin, the rate of in vitro haem loss was five times higher in MP4 compared with SFHb in the fast phase, which we assign to the beta subunits, whereas the slow phase (i.e. haem loss from alpha chains) showed similar rates for the two haemoglobins. Formation of MP4 methaemoglobin in vivo following transfusion in rats and humans was slower than predicted by its first-order in vitro autoxidation rate, and there was no appreciable accumulation of MP4 methaemoglobin in plasma before disappearing from the circulation. These results show that MP4 oxidation and haem loss characteristics observed in vitro provide information regarding the effect of poly(ethylene glycol) conjugation on the stability of the haemoglobin molecule, but do not correspond to the oxidation behaviour of MP4 in vivo.     

Formation of bile pigments by coupled oxidation of cobalt-substituted haemoglobin and myoglobin.

Treatment of cobalt-substituted haemoglobin and myoglobin with ascorbate and molecular O2 (coupled oxidation) resulted in biliverdin formation from the cobalt(II) derivatives but not from the cobalt(III) derivatives. This was apparently due to the inability of ascorbate to reduce cobalt(III) haemoproteins. Isomer analysis of the biliverdins produced from coupled oxidation of cobalt(II) oxyhaemoglobin suggested that the orientation of the cobalt protoporphyrin IX in the haem pocket differed slightly from that of the haem in native haemoglobin.     

Electron addition to the (FeO2) unit of oxyhaemoglobin Glycera

Exposure of glassy solutions containing the monomeric fraction of the oxyhaemoglobin of the polychaete annelid Glycera dibranchiata to 60Co gamma-rays at 77 K resulted in electron addition to the (FeO2) moiety. The form of the g tensor components obtained from the ESR spectrum indicates that the spin-density on oxygen is much greater than that observed for similar paramagnetic centres formed in haemoglobin A or myoglobin. A major difference between these monomer haem units and normal haem units is that the distal histidine (E7 58) is replaced by leucine. We therefore postulate that the oxygen in the (FeO2)- units formed in haemoglobin A and myoglobin is hydrogen-bonded to the NH group of the distal histidine, whilst that of the (FeO2)- units in haemoglobin Glycera are not hydrogen-bonded. However, on annealing to approx. 160 K the spectrum changed irreversibly into one resembling those for (FeO2)- units in haemoglobin A and myoglobin. We postulate that this is caused by hydrogen-bonding to a water molecule in the haem pocket. Exposure of the polymeric fractions of haemoglobin Glycera to gamma-rays gave an (FeO2)- unit with an ESR spectrum remarkably similar to that obtained from oxymyoglobin. The X-ray structure of this protein is unknown but we suggest that our results could indicate the presence of a distal histidine in this material.     

Haemoglobins of invertebrate tissues. Nerve haemoglobins of Aphrodite, Aplysia and Halosydna

1. The occurrence of haemoglobin in invertebrate nerves is surveyed. Haemoglobin was observed in the nerves and ganglia of the marine nematode Amphiporus sp. and of the polychaet annelid Halosydna sp. 2. Haemoglobins from the nerve and ganglia of the polychaet annelid Aphrodite aculeata L. and from the nerve of the gastropod mollusc Aplysia californica have been partially purified. The haem in each case was identified as iron protoporphyrin IX. 3. The minimum molecular weight of Aphrodite nerve haemoglobin deduced from the haem content and amino acid analysis is 17090, in agreement with the molecular weight 15600+/-1000 determined by sedimentation equilibrium. 4. The molecular weight of Aplysia nerve haemoglobin was determined by sedimentation equilibrium to be 16400+/-1000. 5. The oxygen dissociation curves are hyperbolic. Half-saturation is achieved at 1.1mm. Hg for Aphrodite nerve haemoglobin and at 4.0mm. Hg for Aplysia nerve haemoglobin. The coefficients for partition between carbon monoxide and oxygen are: Aphrodite nerve haemoglobin, 167; Aplysia nerve haemoglobin, 116. 6. The ferrous haemoglobins combine with cyanide. 7. We conclude that the intracellular haemoglobins of muscle and nerve are similar.     

Unusual flexibility of distal and proximal histidine residues in the haem pocket of Drosophila melanogaster haemoglobin

Several pH-dependent low-spin ferric haem forms are identified in a frozen solution of the ferric 121Cys→Ser mutant of Drosophila melanogaster haemoglobin (DmHb1*) using electron paramagnetic resonance (EPR) techniques. Different forms with EPR parameters typical of bis-histidine coordinated haem iron centers were observed. Strong pH-dependent changes in the EPR signatures were observed related to changes in the haem pocket. The pulsed EPR data indicate that both the distal and proximal histidine exhibit a large libration around the Fe-N(His) axis. The resonance Raman spectra of the CO-ligated ferrous form of Drosophila melanogaster haemoglobin are typical of an open conformation, with little stabilization of the CO ligand by the surrounding amino-acid residues. The EPR data of the cyanide-ligated ferric DmHb1* indicates a close similarity with cyanide-ligated ferric myoglobin. The structural characteristics of DmHb1* are found to clearly differ from those of other bis-histidine-coordinated globins.     

Control of haem synthesis by feedback inhibition on human-erythrocyte δ-aminolaevulate dehydratase

The action of haem, haemoglobin and other haemoproteins on crude and purified δ-aminolaevulate dehydratase of human erythrocyte is described. The results show a feedback inhibition by haem of porphyrin synthesis at the level of erythrocyte δ-aminolaevulate dehydratase. Some kinetic characteristics of this inhibition are described.