Inorganic and Organometallic Polymers II - ACS Publications

Chapter 37

Structural and Spectroscopic Studies of ß-Hematin (the Heme Coordination Polymer in Malaria Pigment) Downloaded by NORTH CAROLINA STATE UNIV on October 29, 2012 | http://pubs.acs.org Publication Date: November 18, 1994 | doi: 10.1021/bk-1994-0572.ch037

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D. Scott Bohle , Brenda J. Conklin , David Cox , Sara K. Madsen , Scott Paulson , Peter W. Stephens , and Gordon T. Yee 1

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Department

of Chemistry, University of Wyoming, Laramie, WY 82071-3838 Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, CO 80309-0215 Department of Physics, Brookhaven National Laboratories, Upton, New York 11973 Department of Physics, State University of New York at Stony Brook, Stony Brook, NY 11794 2

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ß-Hematin, the crystalline heme coordination polymer present in malaria pigment, is readily prepared by abstracting HCl from hemin with non-coordinating bases. Spectroscopic results(IR, Raman, and UV-Vis) suggest that polymerization occurs via Fe-O inter-heme linkage of the iron-propionate moieties. X-ray powder diffraction results indicate that ß-hematin crystallizes in a triclinic unit cell with two heme groups per cell in the space group Pī. The polymerization and aggregation of proteins, nucleic acids, carbohydrates, and fats into larger functional structures links biology at the molecular and cellular levels. As a consequence, biopolymers have diverse functional roles ranging from information storage and transport, to the compartmentalization of cellular space and extra-cellular structure. Given these diverse and pervasive roles it is surprising how few biopolymers are recognized as containing inorganic monomers. Apart from the polymeric nucleic acids, which contain a polyphosphate diester backbone, there are few well characterized inorganic biopolymers. It is convenient to define this class of compounds as those that involve inorganic elements directly within the polymeric chain. This definition excludes many biopolymers that contain metals such as the metallothionines, complexes of fulvic and humic acids, and the whole family of metalloproteins. In these cases the metals are ancillary to the polymerized functionality; indeed, it is often possible to exchange one metal or prosthetic group for another in metalloproteins. It is anticipated that inorganic biopolymers are associated with the bulk elements such as phosphorus, calcium, and magnesium, and that as our understanding of the processes involved in their biomineralization develops, new 0097-6156/94/0572-0497S08.00/0 © 1994 American Chemical Society In Inorganic and Organometallic Polymers II; Wisian-Neilson, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

Downloaded by NORTH CAROLINA STATE UNIV on October 29, 2012 | http://pubs.acs.org Publication Date: November 18, 1994 | doi: 10.1021/bk-1994-0572.ch037

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INORGANIC AND ORGANOMETALLIC POLYMERS II

examples of this general class will emerge. The subject of this chapter is the unusual heme containing biopolymer that is produced by the malaria parasite. Malaria continues to spread despite past accomplishments in drug therapy and vector control. Two main reasons for this are the rise of chloroquine resistant strains of the Plasmodium genus, and the increasing resistance of mosquitos to pesticides used in their control. Within the mammalian host the predominant portion of the parasites life cycle, shown in Figure 1, is inside red blood cells, where after the initial invasion the contents of the cell are digested and the parasite undergoes asexual schizogony to produce more merozoites. The renowned clinical symptoms of malaria, especially the periodic delirium associated with the fever/chill/sweat cycles, is due to the synchronized rupture of the red blood cells, release of the merozoites, and reinvasion of new red blood cells. Consequences of this include certain anaemias and enlargement of the spleen. In a syndrome termed cerebral malaria, which is due to especially virulent species of malaria, Plasmodium falciparum, death can result from the occlusion of the cerebral capillaries which leads to a progressive delirium, loss of consciousness, and coma (7). The digestion of red blood cells poses a significant biochemical problem for the growing merozoites in that considerable quantities of heme are liberated as the polypeptide in hemoglobin is digested. Heme is a toxic multifunction regulator which not only regulates its own biosynthesis but also the synthesis of a diverse family of proteins connected by their roles in the transport, regulation, and metabolism of oxygen (2). The degradation of heme is performed by the non-heme iron enzyme heme oxygenase, which generates a ferryl intermediate from the reductive cleavage of dioxygen, and breaks the porphyrin ring at the mewj-methylidyne position between the A and Β pyrrole rings to release carbon monoxide, ferric ion, and biliverdin, equation 1 (3). The Plasmodium parasite employs a completely different and unique metabolic pathway to detoxify the released heme, and forms an aggregated insoluble heme-based precipitate termed malaria pigment of hemozoin. It is not understood why the Plasmodium parasite aggregates the heme in this manner rather than utilizing the more ubiquitous heme oxygenase pathway, but possible rationalizations are that the large quantities of iron and carbon monoxide, which would be released by such metabolism, would pose additional toxicity problems that are avoided by heme aggregation. Furthermore, it has recently been demonstrated that merozoites require extracellular iron, bound to transferrin, and that they are unable to tap the considerable reserves of iron present within j8-hematin (4). Upon rupture of the merozoite, step A in Figure 1, the aggregated heme is deposited into the blood which then accumulates in the extensive capillary beds found in the kidney, cerebellum, spleen, and liver. The quantity of accumulated aggregated heme can be so large as to be visible by the naked eye during autopsy and its presence in victims of "marsh miasma" was noted in the eighteenth century, some 150 years before the recognition of the protozoan origin of the disease (5). The mechanism of malaria pigment's biosynthesis is unique and involves a putative heme polymerase enzyme that is associated with the aggregated heme and functions at the low pH of the food vacuole (6,7). Recent work demon strates that heme-aggregation is inhibited by the quinine family of antimalarials

In Inorganic and Organometallic Polymers II; Wisian-Neilson, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

Structural and Spectroscopic Studies of β-Hematin


37. BOHLE ET AL.


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500


(6). The toxicity of the quinine drugs is attributed to the high concentrations of unaggregated heme that build-up in the merozoite. The structure and composition of malaria pigment has been the subject of considerable dispute since its identification as being a heme aggregate (8,9). Early suggestions that it was j8-hematin were refuted by elemental analyses which suggested the presence of proteinaceous material in these isolates (10). However, more recent purification protocols, which employ a magnetic field to separate the iron containing crystallites from the diamagnetic material, demonstrate that there is in fact no protein component to the pigment (11). When properly purified, malaria pigment consists solely of aggregated heme (11,12). Furthermore, reactivity, spectroscopic, and EXAFS studies have demonstrated that purified malaria pigment and β-hematin are identical (13) and that the most likely mode for aggregation involves inter-heme propionate-iron bond formation to give strands shown in Figure 2. Our work concerns 0-hematin, its synthesis, aggregation, spectroscopy, and crystallography. This chapter will describe the results of our studies to determine the details of the inter-heme interactions in 0-hematin. Synthetic Studies It was recognized very early in the development of heme chemistry that when hematin, Fe(protoporphyrin-IX)(OH), is treated with mild acids at elevated temperatures for prolonged periods, equation 2, that an insoluble material, termed jS-hematin, is produced (14). Although j3-hematin is insoluble in water at a pH less than 10, it dissolves in strong alkali to give a product with similar spectroscopic properties to that obtained when either hematin or hemin, Fe(protoporphyrin-IX)Cl, is treated with strong alkali. These results clearly indicate that 0-hematin is an aggregated heme material, and a variety of mechanisms for the inter-heme interaction, including simple electrostatic (15), oxo-bridged dimers (16,17) and carboxylate bridged units have been proposed (18). Discrimination between these proposed interactions is complicated by the fact that the material prepared from the reaction shown in equation 2 is very poorly crystalline, and contains a range of aggregated unknown phases. Furthermore, the workup of these preparations involves numerous alkaline digestions and washes with either a carbonate or phosphate buffer which invariably dissolve large quantities of heme material in the process. In order to prepare more ordered crystalline samples of 0-hematin we initially sought to improve its synthesis by reexamining the reaction of hemin with base. Although many early studies describe the complex reactions between hematins (or hemins) and nitrogenous bases, which give poorly characterized species termed hemochromes (18), these results predate the recognition of the role of the oxo-bridged dimer, 1, in this chemistry (19,20). Moreover, many of these studies were often performed in water or with hydroxide bases. Our strategy for the synthesis of 0-hematin was to treat hemin with a non-coordinating base, equation 3 (22). Methanol proved to be a suitable solvent for this preparation in that hemin has moderate solubility in this solvent and all the other reactants and products are either miscible or soluble in methanol. The key to this development is the recognition that the reaction must be performed absolutely In Inorganic and Organometallic Polymers II; Wisian-Neilson, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


37. BOHLE ET AL.


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Equation 1 Biliverdin

Hematin

n{Fe(por)(OH)}-

H Q, CH CH CQ H 18hr,70°C 2

3

2

2

Hematin

2

Equation 2

por s protoporphyrin-IX

MeOH, 2,6-lutidine i2hr,22°C *

ft=*i r\tni\\ niPe(por)(CI)} Hemin n

β-Hematin + n{H 0}

nn

β-Hematin + n{2,6-lutidine-HCI}

Equation 3

o



502


waterfreein order to avoid the formation of the μ-oxohemedimer 1. In addition to improving the purity and yield of j8-hematin, the low temperature conditions employed in equation 3 allows for a greater control over the rate of polymer formation. Thus the phase which results from this preparation has a high fraction of rod shaped needles which typically measure 3 μπι in length and 0.4 μπι in width as determined by scanning electron microscopy. To our knowledge there are no reports which describe these reactions under rigorously dry conditions. To demonstrate the importance of this experimental detail we have treated hemin with 2,6-lutidine under identical conditions to those described above except that the reaction was performed in the open without predried methanol or 2,6-lutidine. In this experiment we also obtained a dark aggregated methanol insoluble product, but, on treatment with bicarbonate buffer, this completely dissolves and no β-hematin is isolated. The choice of 2,6-lutidine as the base stems from the combination of high basicity with low affinity for transition metal complexes due to the steric interference of two ortho-methyl substituents. The synthesis of 0-hematin is improved by employing dimethylsulfoxide, DMSO, as a co-solvent with methanol, equation 4. Although the aggregation process is slower, and requires 9 days at ambient conditions instead of 12 hr, the resulting phase is visibly crystalline (with crystallite dimensions typically between 0.1 mm and 0.08 mm), and the buffered workup described in the experimental section requires only a single wash rather than a more exhaustive treatment. More importantly, this preparation gives a phase which allows for the unit cell determination by X-ray powder diffraction described below. We attribute the result of this synthesis to the better coordinating ability of DMSO as contrasted with methanol. Thus, dissolution of hemin in DMSO/methanol mixtures results in the rapid equilibration of hemin with solvent to give [Fe(protoporphyrin-IX)(DMSO) ] , 2 in equation 5, which we anticipate will only slowly aggregate upon dissociation of the DMSO ligands. +

2

Spectroscopic and Magnetic Studies To establish the equivalence of the j8-hematin from the two preparations in equations 3 and 4 with prior results we have characterized these compounds by elemental analysis, infrared spectroscopy, Raman spectroscopy, magnetic susceptibility, X-ray powder diffraction, and diffuse reflectance spectroscopy. The analytical results agree within experimental error with those reported by Slayter et ai (C, 64.7; H, 5.0; Ν, 8.7%) for /Miematin prepared by the thermal dehydration reaction in equation 2 (13). These results in turn are similar to those obtained for malaria pigment isolated from P. falciparum in the late trophozoite stage (13). In Figure 3 the infrared spectra for j8-hematin and hemin are contrasted. Notable features of these spectra are a) the differences in the carboxylate stretching regions of the two samples, b) the absence of an intense v(O-H) band between 4000 and 2500 cm , and c) the absence of the strong u(Fe-0-Fe) band ca. 900 cm . These features are not only consistent with the carboxylate bridged hypothesis of Slater et al. (7), Figure 2, but also with the participation of the uncoordinated 1

as

1




37. BOHLE ET AL.

Figure 3. IR spectra for (a) 0-hematin, and (b) hemin as a potassium bromide pellet. Top panel shows the v(C-H) and p(O-H) region while the bottom panel shows the v(C0 ) region. 2


503


504


n{Fe(por)(CI)}

M e (

e

j^ ^?^ " h

l t l t i d i r

P

β-Hematin

+

n{2,6-lutidineHCI}

Equation4

2


37. BOHLE ET AL.


propionic side chain in a hydrogen bonded network. The insolubility of βhematin in 0.1 M NaHC0 /Na2C0 , an important feature in the separation described in the experimental section, is also consistent with the propionic acid hydrogen bonding. One possible structure that combines these spectroscopic features is shown in Figure 4, and depicts a possible inter-strand hydrogen bonding. The resonance Raman spectra shown in Figure 5 also has features consistent with the formulation of jS-hematin as containing five coordinate ferric centers with ^-propionate ligation. Notably absent in Figure 5 (bottom) are bands at 890 and 416 cm which correspond to the asymmetric and symmetric Fe-O-Fe bands for the μ-οχο dimer moiety. The shift of v , assigned to the band at 1627 cm' , is consistent with the presence of high spin Fe(III) in a five coordinate environment (23,24). Furthermore, all the bands above 1450 cm" have values closest to those of hemin, Fe(protopoφhyrin-IX)Cl, a high spin five coordinate iron(III) complex. Although a variety of FeiIIIXporphyrinXV^CR) complexes have been prepared (25,26) and characterized by diffraction (27,28) there are no Raman data available for these complexes to assist in the assignment of the v(¥tO) mode. We tentatively assign the weak band at 514 cnr to this metal-ligand mode, but we note that confirmation of this assignment awaits isotopic substitu tion studies. Assignment of these vibrational spectroscopic results provides collaborating evidence for the proposed hydrogen bonding proposed in Figure 4. We suggest that the two strong carboxylate stretching bands, ^asym(C0 ) at 1712 and 1664 cnr *, are due to the hydrogen bonded and unidentate propionate respectively. Support for the first assignment stems from the similarity of the p(C0 H) band at 1712 cnr in j8-hematin to the band at 1703 cnr in hemin; The crystal structure of the latter indicates that both propionic acid groups are hydrogen bonded to adjacent porphyrins (29). Although the more intense 1664 cnr band is higher than the range reported for ^^coordination, 1645 to 1550 cnr (30), it has a lower energy than free carboxylic acids, cf. 1725 to 1700 cm . A suggested range for the asymmetric stretching bands in carboxylic acid hydrogen bonded dimers is from 1747 to 1692 cm" (31,32), and encompasses the 1712 cm" band. Apart from the positions of the bands themselves, the difference in the energy of the asymmetric and symmetric carboxylate stretching modes, Δ = ^asymiCO^ - v (C0 ), has been used as a diagnostic of the coordination mode for carboxylates, i.e. for unidentate carboxylates Δ > 200 cnr , while for bidentate carboxylates Δ < 120 cm . In hydrogen bonded carboxylic acid dimers the magnitude of the difference between the Raman and infrared allowed asym(C0 ) bands, A , has been used to diagnose dimer formation. In carboxylic acid hydrogen bonded dimers A > 40 cm , but if the hydrogen bonding is to a different functionality, A < 10 cnr . If Cerami and Slater's assignment of v(C0 ) =1211 cm for j3-hematin is verified by isotopic substitution experi ments (13), than the difference, Δ = 453 cm" , is at the long end of the range associated with unidentate carboxylates, but is nevertheless consistent with our model and assignment. On the other hand the Raman results for 0-hematin contains only a single intense band at 1627 cm , that is most likely due to a resonantly enhanced v skeletal porphyrin mode. There is no indication of a 3

3

1


505

l0

1

1

1

2

2

1

1

1

1

1

1

1

sym

2

1

1

J,

2

a

1

a

1

a

1

2

sym

1

1

l0




506

Figure 5. Raman spectra for 0-hematin obtained by the method in equation 4. Laser excitation of 647 nm was used with a sample mounted in a capillary.


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1

band ascribable to a Raman active carboxylate stretch between 1712 cm" and 1630 cm , which, following Lalancette's proposal, would support or refute propionate dimer formation (33). However, only very small or negligible enhancement of the carboxylate bands is expected for the non-iron bonded propionates since they are poorly electronically coupled to the porphyrin core by the two intervening saturated methylene groups. Finally Figure 3(top) contains features that are similar, broad bands at 2620 and 2600 cm , in both 0-hematin and hemin, which support the hydrogen bonding hypothesis. That this interheme interaction is different in the two compounds can be ascertained by the relative intensities and energy spread for the two bands, and the small feature at 3425 cm that is present in /3-hematin and not in hemin. The UV-vis transmittance spectrum shown in Figure 6 contains a Soret band at 406 nm and β-bands at 510, 538, 580, and 644 nm, the latter being especially characteristic of hemin aggregates as determined by photoacoustic spectroscopy (34) and microspectrophotometry (35). Given the number and energies of the bands in the absorption spectrum there exist manifold possibilities for resonance Raman enhancement studies. The variable temperature magnetic susceptibility of 0-hematin down to 4 Κ as determined with a SQUID magnetometer has been determined and a plot of effective magnetic moment (μ ) versus temperature is shown in Figure 7. At room temperature effective magnetic moment is 5.73 B.M. and the temperature dependence of the inverse molar magnetic susceptibility x is linear from 4 to 300 K, with C = 4.16 emuKmol" and a small Curie-Weiss constant of θ = -4.65 Κ. This result indicates that the iron atoms are only weakly coupled and that the iron is high spin, S = 5/2. Not only are these values similar to the magnetic susceptibility of other five coordinate iron (36) species such as Fe(TTP)(X) X = halide or pseudohalides, but also they are identical to results published by Brémard et al. for malaria pigment (37,38). Most notably though, this result demonstrates that the jS-hematin resulting from equation 3 and 4 is not contaminated by the μ-οχο dimer, 1. 1


1

-1

Β

_1

m

1

Diffraction Results Native malaria pigment is a highly ordered crystalline substance. This order is shown in the transmission electron micrograph image shown in Figure 8 (39), which indicates that there is a distinct preferred axis for growth and that the lattice planes are separated by ca. 9 Â. Characterization of 0-hematin by powder X-ray diffraction techniques with both conventional and synchrotron radiation indicates that the phase which results from equations 3 and 4 are sufficiently crystalline to give strong diffraction peaks out to 45° in 20 with Cu K (X = 1.540598 À) radiation. The pattern obtained with synchrotron radiation (λ = 1.7492 Â) on beam line X7A at the National Synchrotron Light Source at the Brookhaven National Laboratory is shown in Figure 9. It has been possible to index this diffraction pattern with the TREOR90 program (21) and we have obtained a triclinic unit cell with dimensions a = al




508

Figure 6. Transmittance UV-vis spectrum for j8-hematin measured as a potassium bromide pellet.



Ο

100

150

Τ (Κ)

200

250

Figure 7. SQUID Magnetic susceptibility results for 0-hematin as a polycrystalline solid. Identical results are also found for a suspension of β-hematin in nujol.

50

300


350



Figure 8. Transmission electron micrograph image of native malaria pigment crystals extracted from Plasmodium berghei showing the direct resolution of the lattice planes separated by - 8 A. (Reproduced with permission from reference 39. Copyright 1974 School of Tropical Medicine.)


37. BOHLE ET AL.


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12.19, b = 14.69, c = 8.05 À, and a = 90.25, β = 96.92, and y = 98.37°, V = 1416 Â , and rf = 1.393(3) g m l . This cell has a high de Wolff figure of merit, Af(20) = 32, and all lines are successfully indexed. The experimental ly determined density and volume can be used to calculate the mass of the contents of the unit cell, which in this instance corresponds to two Fe(protoporphyrin-IX) units per cell. Based on the value of the mean of the | E*E-11 for the integrated intensities of the indexed reflections, 1.034, we suggest that the unit cell is centrosymmetric and the space group is uniquely defined as P i . This space group is coincidentally found for the hemin, Fe(protoporphyrin-IX)Cl, which has a slightly larger unit cell (29). Studies are currently underway to use the integrated intensities obtained from high resolution synchrotron radiation powder diffraction pattern to determine the orientation of the hemes within the unit cell of jS-hematin. These results will be described elsewhere. 3

1


exper

Heme Aggregation The digestion of hemoglobin and heme aggregation leading to the formation of 0-hematin is a highly ordered process which does not involve unassociated free heme (40). Our knowledge of heme transport is limited, and perhaps the best understood heme transport protein is hemoplexin which may or may not be present in the food vacuole of Plasmodium (41). Above all else, the inhibition of heme polymerization by the quinine antimalarials is a mechanistic question. In order to understand heme polymerase and the biosynthesis of 0-hematin, we need a detailed understanding of heme aggregation in general. Prior studies have used UV-visible spectroscopy (42,43), ultracentrifugation (44), and *H NMR (45) to study hematin aggregation. As a result of these studies it is recognized that in alkaline solutions there is a rapid dimerization, to give a μ-οχο bridged species, 1, (44) followed by a slower aggregation process which leads to micelles with an upper limit of 45-50 heme units, distributed around a critical size. Hydrodynamic measurements by ultracentrifugation sedimentation techniques suggest that these micelles are spherically shaped and Blauer proposed the arrangement shown schematically in Figure 10, with the carboxylate groups pointing outward (44). By implication, the vinyl groups are oriented inward, and they may therefore be involved in the thermodynamic stabilization of the micelle.

Conclusion The heme coordination polymer which results from the heme polymerase activity of the Plasmodium parasite is an insoluble crystalline solid with propionate interheme links. How the quinine antimalarials inhibit heme polymerase and how this enzyme is associated with j8-hematin remain important, albeit poorly understood, aspects of this biochemistry. Furthermore, heme detoxification by malaria is the only currently recognized example of iron excretion. The homeostasis of iron is usually srictly controlled, and excess is usually stored in ferritin for subsequent



Figure 10. Schematic depiction of Blauer's proposed micelles formed from 40-50 units of the oxo bridged dimer 1.


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use. j8-Hematin represents an intriguing and unique type of inorganic biopolymer. It can be anticipated that other inorganic biopolymers will be discovered as our knowledge of the bulk inorganic elements develops; undoubtedly an understanding of their structures and functions will follow from many of the unique types of observations that have led to our current knowledge of j3-hematin.


Experimental Water and oxygen sensitive reactions and materials were handled and stored in a Vacuum Atmospheres inert atmosphere box with an atmosphere maintained at

Inorganic and Organometallic Polymers II - ACS Publications

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