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.     

Erythroid differentiation in a friend erythroleukemic cell x lymphoma hybrid cell line is limited, possibly due to reduced hem levels

Induction of erythroid differentiation has been investigated in a cell hybrid formed between an inducible Friend cell and a lymphoma line (L5178Y) derived from the same strain of mouse (DBA/2). Although globin messenger RNA (mRNA) is induced by DMSO to a level similar to that in the inducible Friend cell parent (about 9 000 molecules/cell) haemoglobin does not accumulate in detectable amounts, nor do morphological changes characteristic of terminal differentiation occur. This failure to accumulate haemoglobin in response to DMSO is due to a reduced rate of globin chain synthesis (6% of total protein synthesis, compared to 25% for the parental Friend cell), and partly to inability of the globin chains synthesized to form tetrameric haemoglobin molecules. Globin chain instability is not the reason why haemoglobin does not accumulate. In comparison, treatment of the hybrid cells with haemin induces about 14% globin synthesis and about 13 000 globin mRNA molecules. These values are somewhat higher than with DMSO. Treatment of hybrid cells with haemin plus DMSO is even more effective; it induces 25% globin synthesis and about 30 000 globin mRNA molecules and terminal differentiation also occurs normally. Whether treated with DMSO or haemin or both, virtually all the globin mRNA molecules seem to be present in polysomes and are therefore presumably in the process of being translated. These results suggest that failure of differentiation in these hybrid cells is due to haem limitation which also prevents the expression of other co-ordinated erythroid functions.     

The role of neutral lipid nanospheres in Plasmodium falciparum haem crystallization

The intraerythrocytic malaria parasite constructs an intracellular haem crystal, called haemozoin, within an acidic digestive vacuole where haemoglobin is degraded. Haem crystallization is the target of the widely used antimalarial quinoline drugs. The intracellular mechanism of molecular initiation of haem crystallization, whether by proteins, polar membrane lipids or by neutral lipids, has not been fully substantiated. In the present study, we show neutral lipid predominant nanospheres, which envelop haemozoin inside Plasmodium falciparum digestive vacuoles. Subcellular fractionation of parasite-derived haemozoin through a dense 1.7 M sucrose cushion identifies monoacylglycerol and diacylglycerol neutral lipids as well as some polar lipids in close association with the purified haemozoin. Global MS lipidomics detects monopalmitic glycerol and monostearic glycerol, but not mono-oleic glycerol, closely associated with haemozoin. The complex neutral lipid mixture rapidly initiates haem crystallization, with reversible pH-dependent quinoline inhibition associated with quinoline entry into the neutral lipid microenvironment. Neutral lipid nanospheres both enable haem crystallization in the presence of high globin concentrations and protect haem from H2O2 degradation. Conceptually, the present study shifts the intracellular microenvironment of haem crystallization and quinoline inhibition from a polar aqueous location to a non-polar neutral lipid nanosphere able to exclude water for efficient haem crystallization.     

The effect of exogenous δ-aminolaevulinate on rat liver haem and cytochromes

The activity of delta-aminolaevulinate synthetase is generally regarded as rate-limiting for hepatic haem biosynthesis. It has been suggested that cytochrome synthesis may also be regulated by changes in delta-aminolaevulinate synthetase activity. This hypothesis was studied by injecting product, delta-aminolaevulinate, into adult rats over a 4-240h period. The concentrations of hepatic mitochondrial cytochromes a, b, c and c(1) were unchanged by treatment with delta-aminolaevulinate, allylisopropylacetamide or phenobarbital. In control animals, total microsomal haem content equalled the sum of cytochromes b(5) plus P-450. After delta-aminolaevulinate administration the total amount of microsomal haem, measured as the pyridine haemochromogen, exceeded these components, indicating the formation of a ;free' haem pool. Haem synthesis does not appear rate-limiting for hepatic cytochrome synthesis in the adult rat.     

Transferrin-bipyridine iron transfer mediated by haemoproteins

Rabbit reticulocyte cytosol was able to mediate transferrin-bipyridine iron transfer in the presence of ATP. The cytoplasmic factor responsible for the mediation of iron transfer was identified as haemoglobin. Other cytoplasmic proteins and the membrane fraction were ineffective. Human α and β subunits and human myoglobin were over three times more effective than human haemoglobin A. Carbon monoxide strongly inhibited the mediation of iron transfer. Oxidation of haemoglobin abolished it but methaemoglobin could be reactivated with NADH, even when azide was bound to the haem iron.Neither globin nor haem alone were able to mediate iron transfer, even when NADH was present. Together, the reconstituted methaemoglobin A could be reactivated with NADH.Although the physiological significance of this phenomenon is not clear, the involvement of haemoproteins in intracellular iron metabolism seems likely.     

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.     

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.     

Haem as a multifunctional regulator

Haem has long been known as the prosthetic group of haemoproteins such as haemoglobin, catalase and the cytochromes. Its biosynthesis is regulated by feedback mechanisms that ensure its adequate production but prevent its overaccumulation, which is highly deleterious as diseases such as porphyrias attest. However, recent years have seen rapid strides in our understanding of how haem (or more accurately haemin, its oxidized form) itself acts as an intracellular regulator of a variety of other metabolic pathways for systems that utilize oxygen.     

Haemoglobin synthesis in fused cells.

When primitive erythroid cells from 5-day-old chick embryos are exposed to inactivated Sendai virus they do not undergo haemolysis but fuse with other cells by the normal process of cytoplasmic coalescence. In this way cells actively engaged in the synthesis of haemoglobin may be fused with others that are not. In heterokaryons formed by the fusion of such erythroid cells with cells from established mouse or hamster lines, haemoglobin synthesis initially continues at a high level, but then declines and ceases altogether within a period of about 60 h. This decline affects the synthesis of both haem and globin and reflects the activity of specific regulatory mechanism, for under these conditions other chick proteins continue to be synthesized. The haemoglobin synthesized in the heterokaryons is entirely chick, and not mouse or hamster, haemoglobin.     

A modeling study of the interaction and electron transfer between cytochrome b 5 and some oxidized haemoglobins

Using Brownian motion simulations we have studied the formation of docked complexes of reduced cytochrome b5 and oxidized haemoglobin. Our results indicate that the presence of molecular electrostatic fields has a significant role to play in the formation of these complexes. In contrast to previous modeling studies on this system, we clearly identify electron transfer within an ensemble of similarly docked complexes rather than the formation of a single complex. Docking involves a number of acidic residues surrounding the exposed haem edge of cytochrome b5 and a set of basic residues surrounding the exposed haem edge of the globins. Although amino acids from the partner globin proteins are involved to a small extent in the binding of some of the complexes, the reactivity of any particular globin is essentially independent of the nature of its partner globin chain within the haemoglobin molecule. Comparison of results from adult and embryonic haemoglobins indicates a significant difference in complex formation. Application of electron tunneling analysis to the complexes allows us to predict the rates of electron transfer within each ensemble of complexes. These data provide a theoretical insight into the important process of re-reduction of oxidized haemoglobins as well as explaining the general inability to produce crystalline forms of many docked electron transfer complexes.     

Haem and chlorophyll formation in etiolated and greening leaves of barley

The amounts of protochlorophyllide (P650) and protohaem were measured in ageing dark-grown barley leaves. Maximum amounts of P650 and protohaem were found in 6- to 8-day-old material after which P650 declined rapidly and protohaem more slowly. In leaves exposed to light maximum chlorophyll was produced in 6-day-old material with progressively less the older the leaves. Haem concentrations increased in seedlings of all ages exposed to light. A lag phase was observed for both chlorophyll and haem formation in leaves given a light treatment. Haem, however, showed a slight yet sig nificant decline as chlorophyll production commenced. The results indicate that chlorophyll and haem synthesis share a common pool of δ-aminolae vulinic acid (ALA). At a certain stage of development, the magnesium porphyrin pathway diverts precursors away from haem synthesis. It is only when the ALA synthesising system is well developed that the production of ALA can satisfy pathways to both haem and chlorophyll. The observed changes in haem under certain conditions suggest that, as in animal systems, haem levels may regulate porphyrin formation (chlorophylls) by controlling the supply of ALA.     

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.     

Message stability in injected frog oocytes: long life of mammalian alpha and beta globin messages

The stability of messenger RNA for rabbit and mouse α and β globin has been tested by injection into living frog oocytes, which were subsequently cultured for up to two weeks. [³H]histidine was added to the culture medium at various times and incorporated into haemoglobin whose synthesis was measured by Sephadex and carboxymethylcellulose chromatography. α Globin mRNA is translated only 20% as efficiently as β globin mRNA after injection into oocytes; the same messages are translated with almost equal efficiency if tested in a reticulocyte cell-free system, or if injected into oocytes as unpurified reticulocyte polysomes. For up to two weeks, injected haemoglobin mRNA was about as stable as the mRNAs of the host oocyte. The injected mRNAs for α and β mouse globins also had a similar stability. A fall in the absolute rate of amino acid incorporation into haemoglobin was observed in oocytes that were labelled for several days, but this could be wholly accounted for by a decrease in the efficiency of the oocytes' translational system. The synthesis of β globin from each molecule of injected β mRNA takes place over a longer period of time, when the mRNA is injected into oocytes, than would have been the case if it had remained in the reticulocytes from which it was prepared. We conclude that α and β globin mRNA molecules are very stable in oocytes, and we suggest that the translational life of a message may be determined in part by the kind of cell in which it operates.     

Haem oxygenase: A functionally diverse enzyme of photosynthetic organisms and its role in phytochrome chromophore biosynthesis,cellular signalling and defence mechanisms

Haem oxygenase (HO) is a universal enzyme that catalyses stereospecific cleavage of haem to BV IX α and liberates Fe⁺² ion and CO as by‐product. Beside haem degradation, it has important functions in plants that include cellular defence, stomatal regulation, iron mobilization, phytochrome chromophore synthesis, and lateral root formation. Phytochromes are an extended family of photoreceptors with a molecular mass of 250 kDa and occur as a dimer made up of 2 equivalent subunits of 125 kDa each. Each subunit is made of two components: the chromophore, a light‐capturing pigment molecule and the apoprotein. Biosynthesis of phytochrome (phy) chromophore includes the oxidative splitting of haem to biliverdin IX by an enzyme HO, which is the decisive step in the biosynthesis. In photosynthetic organisms, BVα is reduced to 3Z PΦB by a ferredoxin‐dependent PΦB synthase that finally isomerised to PΦB. The synthesized PΦB assembles with the phytochrome apoprotein in the cytoplasm to generate holophytochrome. Thus, necessary for photomorphogenesis in plants, which has confirmed from the genetic studies, conducted on Arabidopsis thaliana and pea. Besides the phytochrome chromophore synthesis, the review also emphasises on the current advances conducted in plant HO implying its developmental and defensive role.     

Haemozoin formation in the midgut of the blood-sucking insect Rhodnius prolixus

Malaria parasites digest haemoglobin and detoxify the free haem by its sequestration into an insoluble dark-brown pigment known as haemozoin (Hz). Until recently, this pigment could be found only in Plasmodium parasites. However, we have shown that Hz is also present in the midgut of the blood-sucking insect Rhodnius prolixus [Oliveira et al. (1999) Nature 400, 517-518]. Here we show that Hz synthesis in the midgut of this insect is promoted by a particulate fraction from intestine lumen. Haem aggregation activity is heat-labile and is inhibited in vitro by chloroquine (CLQ). Inhibition of Hz formation in vivo by feeding insects with CLQ leads to increased levels of haem in the haemolymph of the insect, which resulted in increased lipid peroxidation. Taken together, these results indicate that a factor capable of promoting Hz crystallisation is present in R. prolixus midgut and that this activity represents an important physiological defence of this insect against haem toxicity.     

The effect of quaternary structure on the state of the alpha and beta subunits within nitrosyl haemoglobin. Low temperature photodissociation and the ESR spectra.

Photodissociation of nitrosyl haemoglobin and nitrosyl hybrids, in which either the alpha or beta subunit is in the nitrosyl form has been stidued at liquid helium temperature (4.2 degrees K) by electron spin resonance and optical absorption spectroscopy. In the presence of inositol hexaphosphate, the photodissociated form of nitrosyl haemoglobin showed an anomalous absorption spectrum in the near infrared region. The experiments with nitrosyl hybrids showed that the alphaNO subunit within the T state haemoglobin is predominantly responsible for the anomalous photodissociated form and the ESR spectrum with three distinct hypefines. The ESR spectrum of alphaNO2betadeoxy2 with inositol hexaphosphate appeared to be very similar to that of the 5-coordinated NO-haem complexes but the absorption spectrum of its photodissociated form was similar to none of protoporphyrin Fe(II) derivatives so far reported. This result suggests that the anomalous photodissociated form may be attributable to some structural distortion of porphyrin or a new electronic state of the haem with different spin state from that of deoxyhaemoglobin.     

Structure of human deoxy cobalt haemoglobin

The structure of human adult deoxy cobalt haemoglobin has been compared to that of the native ferrous form by refinements based on X-ray data to 2.5 Å resolution. The two structures were refined in parallel by conventional methods and selected structural differences were measured by a novel difference refinement method applicable to closely related structures. The distance between the metal and the haem plane is 0.33 ± 0.08 Å in the cobalt derivative and 0.56 ± 0.03 Å in the native. The Co²⁺HisF8N_? bond length is about 0.1 to 0.2 Å longer than the Fe²⁺HisF8N_? bond length; the distance of N_? from the mean haem plane remains the same in the two structures and the substitution of cobalt for iron produces no significant change in globin structure. The free energy of co-operativity of cobalt haemoglobin is known to be about one-half of that of the native haemoglobin; the reason for this reduction is not evident from the structure of cobalt deoxyhaemoglobin.     

Variable-temperature magnetic-circular-dichroism spectra of cytochrome c oxidase and its derivatives

A detailed study of the effect of temperature on the m.c.d. (magnetic circular dichroism) spectra of cytochrome c oxidase and some of its derivatives was undertaken to characterize the spin states of haem a and a(3). The fully reduced enzyme contains haem a(3) (2+) in its high-spin form and haem a(2+) in the low-spin state. This conclusion is reached by comparing the spectrum with that of the mixed-valence CO derivatives and its photolysis product. The cyanide derivative of the fully reduced enzyme contains both haem a and a(3) in the low-spin ferrous form. The m.c.d. spectra of the fully oxidized derivatives are consistent with the presence of one low-spin ferric haem group, assigned to a, which remains unaltered in the presence of ligands. Haem a(3) is high spin in the resting enzyme and the fluoride derivatives, and low spin in the cyanide form. The partially reduced formate and cyanide derivatives have temperature-dependent m.c.d. spectra due to the presence of high- and low-spin haem a(3) (3+) respectively. Haem a is low-spin ferrous in both. A comparison of the magnitude of the temperature-dependence of haem a(3) (3+) in the fully oxidized and partially reduced forms shows a marked difference which is tentatively ascribed to the presence of anti-ferromagnetic coupling in the fully oxidized form of the enzyme, and to its absence from the partially reduced derivatives, owing to the reduction of both Cu(2+) ions.