The Malaria Pigment Hemozoin Comprises at Most Four Different

Article pubs.acs.org/crystal

The Malaria Pigment Hemozoin Comprises at Most Four Different Isomer Units in Two Crystalline Models: Chiral as Based on a Biochemical Hypothesis or Centrosymmetric Made of Enantiomorphous Sectors Tine Straasø,*,† Noa Marom,*,‡ Inna Solomonov,§ Lea K. Barfod,∥,⊥ Manfred Burghammer,# Robert Feidenhans’l,† Jens Als-Nielsen,*,† and Leslie Leiserowitz*,¶ †

Niels Bohr Institute, University of Copenhagen, 2100 Copenhagen, Denmark Department of Physics and Engineering Physics, Tulane University, New Orleans, Louisiana 70118, United States § Department of Biological Regulation, The Weizmann Institute of Science, 76100, Rehovot, Israel ∥ Center for Medical Parasitology, Department of International Health, Immunology and Microbiology, University of Copenhagen, 2200 Copenhagen, Denmark ⊥ Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), 2100 Copenhagen, Denmark # European Synchrotron Radiation Facility, 38043 Grenoble, France ¶ Department of Materials and Interfaces, The Weizmann Institute of Science, 76100, Rehovot, Israel ‡

S Supporting Information *

ABSTRACT: Hemozoin is a crystalline byproduct formed upon hemoglobin digestion in Plasmodium-infected blood cells. Based on X-ray powder diffraction (XRPD), hemozoin and its synthetic analogue β-hematin are very similar in structure, consisting of cyclic dimers (cd) of ferriprotoporphyrin IX [Fe(3+)-PPIX] molecules coordinated via Fe− O(propionate) bonds. Enantiofacial symmetry of Fe(3+)-PPIX implies formation of four different stereoisomeric dimers, two centrosymmetric (1̅), labeled cd1̅1 and cd1̅2, and two enantiomeric, cd2(+) and cd2(−), in which the Fe(3+)PPIX moieties are related by pseudo-2-fold symmetry. Only the cd1̅1 stereoisomer was reported as the repeat unit in the initial structural elucidation of β-hematin and refinement of hemozoin. Our recent study of β-hematin, employing a combination of XRPD and density functional theory (DFT), revealed besides the published phase, characterized in terms of a disordered cd1̅1/ cd2(±) mixture, which is diffractionally equivalent to a cd1̅1/cd1̅2 mixture, a minor phase considered to comprise mainly cd12̅ dimers. As a consequence single-phase β-hematin powders were recently reanalyzed in terms of a cd11̅ /cd1̅2 mixture, yielding an average occupancy ≅ 75:25. Here, we present evidence enhancing the biphase model of β-hematin. The primary focus is on a reexamination of the hemozoin structure in light of a biochemically based dimerization mechanism that we recently hypothesized. We suggest that upon hemoglobin degradation, the heme byproduct retains the O2 molecule bound to Fe on the Re side of the heme until Fe−O(propionate) coordination between such heme molecules occurs across their unbound Si sides yielding the cd2(+) dimer. We report Rietveld refinement of the hemozoin structure using data measured on an all-in-vacuum powder diffractometer assuming the following models: cd1̅1, cd12̅ , cd2(+), and the two mixtures cd11̅ /cd1̅2 and cd11̅ /cd2(+). The best figures of merit were obtained for the mixture cd1̅1/cd2(+) with a 50:50 occupancy, followed by the cd11̅ /cd1̅2 mixture with an occupancy ≅ 75:25, which we interpret as a structure that comprises the cd1̅1, cd2(+), cd2(−), and cd1̅2 isomers with occupancies ≅ 58:17:17:8. In this model system, the cd1̅1 “host” molecule is uniformly distributed throughout the crystal, whereas the enantiomeric molecules cd2(+) and cd2(−) are preferentially occluded in different crystalline sectors, which are thus enantiomorphous, related by overall centrosymmetric symmetry. Various arguments appear to favor the 50:50 cd11̅ /cd2(+) mixture, namely, a hemozoin crystal of overall chiral symmetry, consistent with our hypothesis. However, we cannot overrule the alternative model.

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INTRODUCTION

The malaria parasite Plasmodium falciparum detoxifies the heme byproduct of hemoglobin digestion in infected red blood cells by sequestration into submicrometer-sized hemozoin crystals.1 © 2014 American Chemical Society

Received: July 30, 2013 Revised: January 31, 2014 Published: March 17, 2014 1543

dx.doi.org/10.1021/cg401151f | Cryst. Growth Des. 2014, 14, 1543−1554

Crystal Growth & Design

Article

Table 1. The Nomenclatures10 Used To Describe the Four Stereoisomers of Hematin Anhydride

These crystals are considered an important drug target, and considerable effort has been devoted to determining the crystal structure2,3 and mapping the crystallization pathway.3 Conventional X-ray diffraction measurements on single crystals for structure characterization are hardly possible since the biogenic hemozoin crystals are too small in size, but X-ray powder diffraction (XRPD) is feasible. The synthetic form of hemozoin, named β-hematin, can be grown to somewhat larger size, albeit still not large enough for single crystal diffraction analysis. In a seminal paper, Pagola et al.2 reported the structure of βhematin as built of ferriprotoporphyrin IX (Fe(3+)-PPIX) molecules reciprocally linked via Fe−O(propionate) coordination bonds into hematin anhydride dimers. Moreover, given that several powder diffraction patterns of hemozoin4,5 appear to be quite similar to that reported by Pagola et al., it was stipulated that the biogenic crystalline form hemozoin is very similar to the synthetic compound. Indeed, based on an analysis of synchrotron radiation XRPD data of hemozoin from the blood fluke Shistosoma mansoni and the kissing bug Rhodnius prolixus, the authors concluded that “all hemozoin crystals share the same unit cell and crystal and molecular structure as βhematin”.5 In addition, a refinement of XRPD data of hemozoin extracted from red blood cells infected by P. falciparum revealed “a crystal structure very similar, within the 2.4 Å resolution limit, to that reported for β-hematin”.3 Recently, we proposed the formation of four stereoisomeric hematin anhydride dimers of Fe(3+)-PPIX in the crystallizing solution of β-hematin.6 The hypothesis was logically drawn from the enantiofacial symmetry of the Fe(3+)-PPIX monomer (Figure 1a), which upon dimerization to hematin anhydride

Straasø et al.6 CPIa vinyl sitesb

cd11̅ R/S′ α/α′

cd12̅ S/R′ β/β′

cd2(−) R/R′ α/β′

cd2(+) S/S′ β/α′

a

Taken in part from Bohle et al.,10 who applied the Cahn, Prelog, and Ingold (CPI) system to describe the handedness of Fe−O coordination to the Re or Si face of Fe(PP-IX). For convenience one of the two Fe(3+) PP-IX monomers is primed. bAn alternative nomenclature is shown by specifying the vinyl sites α and β (and α′ and β′) of the two monomer units.

Only minor energy differences exist between the four dimers, as indicated by density functional theory (DFT) calculations,7 and thus their coexistence is likely. Moreover, simultaneous existence of the different stereoisomeric dimers in solution may explain the observed submicrometer size of the crystals, since a subset of the four stereoisomers were predicted to act as “tailor-made” growth retarders,8,9 in other words a form of crystalline self-poisoners. In the characterization of the crystal structure of β-hematin, however, only the dimer cd11̅ was initially reported.2 Our hypothesis on the proposed stereoisomeric distribution of hematin anhydride dimers in hemozoin was also consistent with the presence of an additional, albeit minor, phase of βhematin.6 In our XRPD analysis of β-hematin, five Bragg peaks were noted that we attributed to a minor crystalline phase of βhematin with a crystal structure composed primarily of the centrosymmetric dimer cd12̅ complemented by the enantiomers cd2(+) and cd2(−). According to the DFT computations, this crystal structure is somewhat less stable than that of the major phase of β-hematin, which was characterized in terms of a cd11̅ /cd2(±) mixture, which is diffractionally equivalent to a cd11̅ /cd1̅2 mixture. The presence of the minor crystalline phase has been challenged, however, by Bohle et al.,10 in a comprehensive review on the structure of malaria pigment and related propanoate-linked metalloporphyrin dimers.10 We note that they referred to only three clearly resolved Bragg peaks as assignable to the minor crystalline phase. They reexamined three β-hematin XRPD data sets, including that of their published structure.2 Following the analysis by Straasø et al.,6 they treated each crystal structure as being composed of a random mixture of the centrosymmetric cd11̅ and cd1̅2 dimers, yielding molecular dimer site occupancies ranging from 83:17 to 65:35. They concluded that the same crystal lattice is able to accommodate the different stereoisomers without “serious intermolecular interference”. It would appear that stereoisomeric disorder is ubiquitous in crystal structures containing hematin anhydride dimers10,11 or Fe-PPIX monomer units,12,13 or even Fe(3+)-PPIX coordinated to halofantrine.14,15 Here, we present further evidence (vide inf ra) fortifying our original interpretation, based on additional XRPD data, as well as diffraction from single micrometer-sized powder grains. The main thrust of this paper, however, is to elucidate the crystal structure of hemozoin, in terms of its cyclic dimer stereoisomeric composition and chirality, and, as a corollary, highlight the differences between β-hematin and hemozoin. In our earlier study, based on the structural considerations detailed below, we had proposed that hemozoin is composed primarily of a mixture of the two stereoisomeric hematin anhydride dimers cd2(+) and cd1̅1 implying a chiral crystal of single handedness.6 A low-temperature X-ray structure refinement of

Figure 1. (a) Molecular structure of Fe-PPIX displaying its enantiotopic Re face to the reader (the Si face is on the opposite side) and vinyl substituents at the α sites. (b) The possible vinyl sites in the dimer of hematin anhydride, marked in dark blue (α, β, α′, β′). Hydrogen atoms excluded for clarity. (c) The four possible dimeric stereoisomers, two centrosymmetric and two enantiomeric. The dimer symmetry label and nomenclature representing the occupied vinyl sites are depicted. The arrows indicate that each chiral dimer is composed of one upper and one lower part of the centrosymmetric dimers (and vice versa).

would lead to two centrosymmetric cyclic dimer (cd) stereoisomers, cd1̅1 and cd1̅2, and two enantiomeric chiral structures, cd2(+) and cd2(−), each exhibiting pseudo-2-fold symmetry. A schematic representation of the formation of the four different stereoisomers is reprinted in Figure S1 in the Supporting Information. The possible vinyl sites of the Fe(3+)PPIX dimer are depicted in Figure 1b, and all four dimers are shown in Figure 1c. Table 1 lists various nomenclatures thereof. 1544



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oxygenated hemoglobin16 reveals a structurally ordered heme molecule, so that the molecular oxygen is always coordinated to the enantiotopic Re face of the heme molecule and histidine to the opposite Si face of the heme (as depicted in the first step in Scheme 1). We note that the heme−O2 complex in myoglobin shares the same orientational ordering17 (Figure S2 in Supporting Information). We hypothesize that upon degradation of hemoglobin by the parasite during its feeding process, an appreciable number of the heme byproducts retain the O2 molecule coordinated to Fe on the Re side of the heme. On the opposite Si side, we propose that the imidazole moiety, being

part of the protein polypeptide chain, would be eventually detached from the Fe ion during the degradation process of hemoglobin and replaced by ubiquitous H2O, which binds weakly to the Fe ion. Such complexes should have a tendency to first lose water bound to the Si side of the molecule, so that Fe−O cyclic dimerization between such heme molecules across the unbound Si sides of the heme will yield the cd2(+) chiral dimers. The proposed mechanism is outlined in Scheme 1. Those heme molecules, which are free of O2, would, in principle, be able to form all four stereoisomeric dimers but would have some preference toward the more stable centrosymmetric dimer, cd1̅1.7 From stereochemical considerations, the chiral cd2(+) and the centrosymmetric cd1̅1 dimers may be accommodated within the same lattice and thus crystallize into a single but disordered phase.6,7 Here, we report Rietveld refinement of hemozoin structure using high-resolution data measured on an all-in-vacuum powder diffractometer. We present five models of the hemozoin crystal structure, the two most favorable being a distribution of the chiral cd2(+) and the centrosymmetric cd11̅ hematin anhydride dimers, thus being a crystal of overall chiral symmetry, followed by a crystal composed primarily of the three stereoisomers cd1̅1 and cd2(±) and a minor admixture of the cd1̅2 dimer. The formation of a nucleation aggregate may be envisaged via the assembly of the stereoisomeric dimers into Hbonded chains, which subsequently mesh forming close-packed bilayers that then interconnect via π−π interactions.

Scheme 1. Molecular O2 Is Bound to the Fe(2+) Ion Only at the Enantiotopic Re Face of the Heme and Histidene Is Coordinated to the Opposite Si Face of the Heme in Oxygenated Hemoglobina

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MATERIALS AND METHODS

Parasite Growth and Hemozoin Purification. Long-term, in vitro propagated P. falciparum parasites were grown in 0 Rh+ erythrocytes as described.18 The culture containing late stages was transferred to a CS MACS (manual cell separation) column placed in a VarioMACS separator and washed with phosphate-buffered saline with 2% fetal calf serum (PBS+2%FCS) until the buffer exiting the column had turned clearly transparent. This left only infected red blood cells in the column. Removal of the column from the magnetic field allowed collection of the red blood cells by washing with 20 mL of PBS+2% FCS. The infected red blood cells were then spun down at 812g for 8 min. Supernatant was removed, and the pellet was unclasped and lysed with 1 mL of distilled H2O. The lysed red blood cells were transferred back to the column and washed with a minimum 40 mL of distilled H2O. The column was removed from the magnetic field once more, and the bound hemozoin crystals were washed out with 20 mL of distilled H2O and spun down at 5200g. The crude hemozoin was purified according to the procedure of Klonis et al.3 X-ray Powder Diffraction Data Collection and Rietveld Refinement. The hemozoin powder was deposited in a 0.3 mm Lindemann glass capillary and irradiated with a monochromatic X-ray beam, λ = 1.05353 Å, extracted via a (111)-diamond monochromator. The diffraction data of the sample at room temperature were measured using an all-in-vacuum diffractometer19 installed at the wiggler source beamline 9-11 at MAX-lab. XRPD data were also measured with the sample cooled to a temperature of 80 K with a cryostat at the Swiss Light Source (SLS) at the Materials Science (MS) beamline. Due to a highly irregular background from the cryostat, only the room temperature (RT) data were used for the comprehensive Rietveld refinement, but comparison of the two data sets ensured that the thermal vibrations were correctly accounted for in the RT data analysis. The shape of the atomic displacement ellipsoid was found to be disc-like at both temperatures with very little vibration along the Hbonding axis. The Rietveld refinements were carried out using Jana2006.20 The bond lengths and angles of chemically equivalent moieties of hematin anhydride were constrained to values equal to those averaged from the low temperature crystal structure analysis of the halofantrine/ ferriprotoporphyrin IX coordination complex.14 Various interatomic

a

If the O2 remains bound to the heme at the Re side of the heme during the degradation process of hemoglobin and just prior to hematin anhydride dimerization, the dimer formed will be chiral of the cd2(+) type. The precise process of deprotonation of one of the two propionic acid groups per heme, and the corresponding one electron oxidation leading to Fe(3+) is not characterized. 1545



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Figure 2. The two figures of merit, Robs (left panel) and Rp (right panel), as a function of the percentage of molecular site disorder in the stereoisomeric mixtures cd2(+)/cd11̅ and cd1̅2/cd11̅ . The pure blue, yellow, and green colors, and mixtures thereof, correspond to pure cd2(+), cd1̅2, and cd1̅1 dimers, and dimer mixtures, respectively. Clearly the pure cd12̅ dimer model can be excluded. The most favorable models are derived in the text. Recently reported degrees of stereoisomeric cd1̅1/cd12̅ disorder in the crystal structures of β-hematin (i.e., synthetic hemozoin)10 and hematin anhydride DMSO solvate11 are included, viewed as three dotted and one full line, the latter corresponding to that found in the DMSO solvate. superposition errors (BSSE).21 We used the generalized gradient approximation (GGA) of Perdew, Burke, and Ernzerhof (PBE)24,25 combined with the Tkatchenko−Scheffler (TS) dispersion method.26 For the description of dispersively bound systems, the TS method has yielded results on par with highly accurate but computationally much more expensive methods that rely on explicit treatment of long-range correlation.27 The efficiency of the TS method enables geometry relaxations of large systems with several hundred atoms.28 The TS method has been used successfully for metal-phthalocyanine dimers, which are chemically related to heme,29 as well as for our previous study of β-hematin.6

distances and angles, including the Fe−O coordination bond and the CO and C−O bond lengths and angles of the carboxylate group C(O)−O−Fe, were set to those found in the single crystal structure refinement of the hematin anhydride DMSO solvate.11 In addition, the four pyrrole groups were each kept planar. The propionic acid moiety CH2−CH2−CO was kept in a syn-planar conformation given that propionic acid derivatives X-CH2CH2CO2H almost invariably adopt such a conformation as opposed to the anti-planar counterpart.6,11 The corresponding −COOH moiety was constrained to participate in a coplanar H-bonded dimer system. The H-bonding OH···O distance was fixed by the use of a dummy atom at the center of the H-bonded dimer. Given the uncertainty in torsion angle of the different C− CHCH2 vinyl substituents, their values were determined by Rietveld refinement from a computation of the figures of merit as a function of torsional rotation. The unit cell dimensions of hemozoin were refined simultaneously with the structure. We made use of an overall anisotropic thermal displacement ellipsoid, comprising six parameters. This ellipsoidal shape is almost disc-like with very little vibration along the H-bonding axis and thus is compatible with pronounced libration of the molecular dimer about the H-bonding axis (See Table S1, Supporting Information). The reflection profile function and the overall anisotropic atomic displacement parameters of the disordered structures were kept fixed at values obtained from refinements of the pure cd1̅1 and cd2(+) models. X-ray in-House XRPD on β-Hematin. Data were obtained from an in-house measurement on a Rigaku D/MAX-200B powder diffractometer (λ = 1.541 Å) operating in the θ−2θ mode. A small angular offset of the flat plate sample holder resulted in a partial beam absorption at the lowest scattering angles. Data in the 2θ range 5°−8°, which includes the intense {100} reflection, have therefore been omitted in the refinement. X-ray Nanobeam Diffraction from Single Grains of βHematin. The nanodiffraction data were measured at the ID13 Microfocus beamline, ESRF, with a Si-NFL lens setup to obtain a focused beam of 130 nm × 180 nm (vertical × horizontal). The X-ray energy was 15.25 keV with about 109 photons per second at the sample position. The crystals were spread in a small concentration on a membrane and mounted on a magnet base attached to a piezo stage. A Maxipix detector (516 × 516 pixels, 55 μm per pixel) was used to observe diffraction spots, while a fluorescence detector set for the Fe K-edge was used to locate individual grains in scanning the sample by the piezo stage through the nanobeam. In order to keep X-ray beam damage of the β-hematin crystals to a minimum, the ω-range in the rocking curves was limited, with a rather large step-size so that reliable integrated intensities could not be calculated. Computational Details. All DFT calculations were performed with the FHI-aims code,21−23 using the tier 2 numerical atomiccentered orbital (NAO) basis set, which has been demonstrated to approach the basis set limit and be nearly free of basis set

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RESULTS AND DISCUSSION Crystal Structure Refinement of Hemozoin. Previous studies were deliberately limited to only encompass the centrosymmetric cd11̅ dimer structure in hemozoin.3,5 As outlined above, we hypothesize that hemozoin consists primarily of the enantiomeric chiral dimer cd2(+) and the centrosymmetric dimer, cd1̅1. To characterize the crystal structure, a sample of hemozoin from red blood cells infected by P. falciparum was prepared according to the procedure described in the Materials and Methods section. To ensure a high-quality XRPD data set, the room temperature data were measured on a high-resolution, all-in-vacuum powder diffractometer,19 to eliminate the background noise arising from airscattering. The crystal structure was refined assuming the following stereoisomeric dimer models: (i) the centrosymmetric cd1̅1, (ii) the chiral cd2(+), (iii) the centrosymmetric cd1̅2, (iv) a mixture of cd11̅ and cd2(+), and (v) a mixture of cd1̅1 and cd1̅2. These crystal structures were refined via the Rietveld method imposing structural constraints as outlined in the Materials and Methods section, making use of the software package Jana2006.20 The results of the refinement are summarized in Figure 2. The pure centrosymmetric cd1̅1 model structure displays a slight advantage over the chiral cd2(+) model structure (marked in green and blue, respectively) in terms of the figure of merit, Robs, but the reverse situation occurs for the figure of merit Rp. The pure centrosymmetric dimer cd1̅2 (in yellow) is the poorest structure in terms of both figures of merit. The relative molecular occupancies of the mixed stereoisomeric dimers, iv and v, were refined by combining the scattering amplitudes of the disordered stereoisomers, which implies that they are occluded in the same crystalline diffracting domains, as may be expected. Their refined figures of merit are indicated in the figure by a 1546



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proportional mixing of the colors. The lowest figures of merit (Robs and Rp) obtained for the cd1̅1/cd2(+) dimer mixture, with overall chiral symmetry, is in the occupancy range 40:60− 60:40. The corresponding occupancy range for the centrosymmetric cd1̅1/cd1̅2 mixture is 70:30−80:20. The broad minima in the figures of merit (Figure 2), of the cd11̅ /cd2(+) model lying within the 40:60−60:40 range is consistent with our biochemically based hypothesis regarding preferred formation of the cd2(+) dimer. Moreover, the derived cd1̅ 1 /cd2(+) occupancy range of 40:60−60:40 is not unreasonable given that the parasites were grown in an atmosphere similar to that found in venous blood, where ca. 75% of the heme will still be oxygenated.30 The fit between the observed and calculated powder diffraction patterns for the structural model iv is presented in Figure 3. The two dimer

50:50 chiral structure composed of the cd2(+) and cd11̅ dimers and the centrosymmetric model formally composed of the cd1̅1 and cd1̅2 dimers in a 75:25 mixture, as well as the refined model of β-hematin, shall be presented below. The Two Phases of β-Hematin Revisited. The minor phase of β-hematin is present in a few studies reported in the literature, suggestive of its formation being dependent on kinetic factors, for example, temperature and crystallization rate. In general, β-hematin is prepared via two protocols, (i) the anhydrous noncoordinating base method31 and (ii) the aqueous acid-catalyzed method.32 The minor phase has been observed using either protocol,6,33 and perhaps in the XRPD pattern of “wet” β-hematin reported by Egan et al.34 The {100} diffraction peak (Figure 3a in ref 34, reproduced in Figure S3a in Supporting Information) displays a distinct shoulder suggestive of the minor phase. In our previous study,6 five reflections of the minor phase ({100}, {011}, {001}/{020}, {031}, and {131}), whose XRPD pattern is reproduced in Figure S3b in the Supporting Information, were deemed observable, notwithstanding the small amount of this phase formed and the high degree of peak overlap in the powder pattern. In order to assign the unit cell dimensions, given the limited amount of information, we made use of, inter alia, the length of the H-bonding axis a−c of βhematin. Low and room temperature studies of synthetic and biogenic hemozoin2,4−6 reveal an H-bond axial length that is highly conserved (Table S7 in the Supporting Information). Moreover, the intensities of the minor phase reflections, although relatively weak, fitted quite well with the model assuming the structure is composed of hematin anhydride dimers in an arrangement similar to that of the major phase.6 In order to substantiate the evidence for the existence of the minor phase, we made use of XRPD data from another βhematin sample (see Figure S4 in the Supporting Information), which had been obtained in 2004 under experimental conditions already described,35 and also of single-grain nanodiffraction data from the sample used in ref 6. The XRPD pattern (Figure 5a), which had been measured in 2004, displays a larger proportion of the minor phase compared with the sample (see Figure S3b, Supporting Information) analyzed by Straasø et al.6 A simple rigid-body refinement of the major and minor phases was performed, yielding 18% of the latter. The fit between the observed and calculated XRPD patterns for both phases is very satisfactory, as shown in Figure 5c,d, which also indicates the positions of the seven Bragg peaks of the minor phase. The cell dimensions of both phases are given in Table 2. The relative differences in the unit cell dimensions of the major and minor phases, including the difference in unit cell volume of 4.6%, are substantial, except for that of the Hbonding axial length (a−c), ∼0.3%, which is almost unchanged. We rationalize the larger unit cell volume of the minor phase in terms of steric contacts; according to DFT computations (Table S6), the cd1̅1 isomer is marginally less favorably packed in the minor phase unit cell than in that of the major phase; by comparison the cd12̅ isomer is much less favorably packed in the smaller unit cell of the major phase than in that of the minor phase. Differences in density between polymorphs are normally no more than 1−2%,36 but the two structures of βhematin are not dimorphs according to our analysis but rather similar phases incorporating different concentrations of the hematin anhydride isomers, according to our analysis.

Figure 3. The observed (in red) and calculated (in blue) XRPD patterns of hemozoin, the latter corresponding to a Rietveld refinement of the room temperature crystal structure. The difference curve is shown in green.

model structures (cd2(+) and cd1̅1) superimposed on each other in the derived unit cell are shown in Figure 4. No serious

Figure 4. The packing arrangement of the two refined structures, cd2(+) and cd11̅ , in red and blue respectively, superimposed onto each other. Views along the c-axis (left) and the a-axis (right).

short contacts exist because the potentially short distance between the methyl and vinyl groups of adjacent dimers is easily circumvented by rotation of the methyl hydrogens. The unit cell dimensions are listed in Table 2, while basic information on various interatomic distances is listed in Table S1 in the Supporting Information. A detailed comparison between the refinements of the two most favorable model arrangements of hemozoin, namely, the 1547



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Table 2. Unit Cell Dimensions of Hemozoin (Hz) and of the Major and Minor β-Hematin (βH) Phases sample

a [Å]

b [Å]

c [Å]

α [deg]

β [deg]

γ [deg]

V [Å3]

a−c [Å]

a

12.1758(3) 12.086(4) 12.155(6) 12.54(2)

14.7854(5) 14.6216(5) 14.597(5) 15.30(2)

8.0407(3) 7.9942(4) 8.017(4) 7.80(2)

90.245(3) 90.758(4) 90.17(4) 100.3(1)

97.160(4) 97.093(4) 97.05(4) 95.0(1)

97.780(4) 97.060(5) 98.19(4) 95.0(1)

1423 1390 1397 1460

15.40 15.29 15.36 15.33

Hz Hzb βHmajor βHminor a

The hemozoin crystals were at RT (300 K). bThe hemozoin crystals were cooled to 80 K.

Figure 5. (a) XRPD data on β-hematin shown in red with peaks originating from the minor phase marked in blue. (b) The overall fit (blue line) related to the 2004 data. The data in the range 2θ = 5−8° have been omitted from the Rietveld analysis. (c) At low X-ray scattering angles, the {110} and the two coinciding {001}/{020} Bragg peaks are clearly observed. The {120} and {21̅0} reflections of the minor phase observed at the foot of a major counterpart were not clearly visible at first but are required for a good fit. In addition, we observe the {100} minor reflection as a shoulder to the corresponding major peak although it was omitted in the refinement. (d) At higher scattering angles, the {031} and {131} reflections of the minor phase stand out and the model fits the observed data nicely.

observation that the grains emit X-ray Fe-fluorescence. This evidence is complemented by the observation that the most intense reflection {100}, which had the highest probability of being observed given that the sample was rotated with a step size of 1°, is true for both the major and minor phases. Moreover, since the intensities of the two {100} reflections are in about the same range we may deduce, given all the other corroborating evidence, that their crystal structures are similar. Here we eliminate alternative hematin anhydride conformations and packing motifs to strengthen our assignment of the minor phase of β-hematin. The OC−O−Fe moiety of the hematin anhydride dimer may, in principle, adopt either an antiplanar conformation, as has been observed to date in the crystalline state, or a hypothetical syn-planar geometry assumed in DFT computations.37 However, these calculations did not account for dispersion interactions. In addition, the hypothetical conformation imposes a distorted propionate chain and, consequently, a H-bonding repeat incompatible with the length of the H-bonding axis a−c, and so may be precluded as a possible model. Therefore, we limit our analysis to possible ways of interlinking the observed hematin anhydride dimers. We had previously demonstrated that it is possible to form a well-packed crystal of hematin anhydride H-bonded chains

In order to further substantiate the existence of the two phases, we examined single grains and small clusters from βhematin powder samples via nanoprobe X-ray diffraction and Fe-fluorescence measurements at the ID13 beamline, ESRF. Two-dimensional scans of the sample with an X-ray beam of dimension 130 nm × 180 nm were performed for a rocking curve (ω-range) of typically 11° with a 1° step size. Software masks separating the {100} scattering events of the major and minor phases were applied to the diffraction data, which enabled us to map the diffracting grain(s). Specifically we were able to show that diffraction from the major and minor phases were only to be found in different grains, namely, they did not crystallize into a single grain. The experiment contained mesh scans of several cluster regions as well as single crystallites, but only one of the cluster mesh scans is shown in Figure 6. The measured d-spacings of the {100} and {020} reflections of the major and minor phases of β-hematin compare very well with the corresponding spacings of the powder sample shown in Figure 5, as listed in Table S8, Supporting Information, which also includes the d-spacings of the {031} and {131} reflections of the two phases. Evidence that the crystal grains, which diffract reflections corresponding to the minor phase, are composed of ferriprotoporphyrin IX moieties is provided by the 1548



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reported in ref 7, we performed DFT calculations of the binding energy per unit cell of the cd2(+) dimer in the unit cells of hemozoin and of the two phases of β-hematin (the noncentrosymmetric dimer reported in ref 7 was the cd2(−) isomer) and of the cd11̅ and cd12̅ dimers in the unit cell of hemozoin. The internal coordinates of the dimers were fully relaxed with the cell parameters determined from XRPD. The binding energies were referenced to two isolated heme molecules and a hydrogen molecule. The lattice energies were referenced to isolated dimers. The calculations were performed at the experimentally observed high spin state as well as at the intermediate spin state favored by the PBE functional. A complete account of the results is given in the Supporting Information (Tables S2−S6). We find that the cd11̅ isomer is less stable in the unit cell of the minor phase of β-hematin than in the unit cells of the major phase of β-hematin and of hemozoin. The cd12̅ isomer is less stable in the unit cell of the major phase of β-hematin than in the unit cells of the minor phase of β-hematin and of hemozoin. The cd2(+) isomer is less stable in the unit cell of the minor phase of β-hematin than in the unit cells of the major phase of β-hematin and of hemozoin with preference for the former. Moreover, the cd11̅ isomer is more stable than cd12̅ in the unit cells of hemozoin and the major phase of β-hematin, and the cd2(+) isomer is almost as stable as cd1̅1 in the unit cells of hemozoin and the major phase of β-hematin. Comparing the cd2(+) isomer with that of cd1̅2, the former is more stable in the unit cells of hemozoin and the major phase of β-hematin. These results are consistent with the major phase of β-hematin comprising the cd11̅ isomer with admixture of the cd2(+) dimer and, by symmetry, the energetically equivalent cd2(−), the minor phase of β-hematin mainly comprising the cd1̅2 isomer, and hemozoin comprising a mixture of the cd11̅ and cd2(±) isomers. We note that the unit cell parameters were not fully relaxed and the effects of temperature and of isomeric disorder were not taken into account. Comparison of the Different Model Structure Refinements of Hemozoin and β-Hematin. The structural refinement of hemozoin assuming the crystal is chiral composed of chiral cd2(+) and centrosymmetric cd1̅1 dimers yields two figures of merit (Robs and Rp), the minima of which are rather broadly centered at a cd11̅ /cd2(+) dimer occupancy of ∼50:50 (see Figure 2). Refinement assuming that the overall symmetry of hemozoin is centrosymmetric comprising the centrosymmetric cd1̅1/cd1̅2 dimer mixture yields figures of merit, Robs and Rp, whose narrow minima do not quite match each other in position, averaged at ∼75:25 (see Figure 2). The Robs minima of the chiral and centrosymmetric model structures are about the same, whereas the Rp minimum of the chiral structure is somewhat lower than that of the centrosymmetric model. The relative occupancies of the cd1̅1/cd1̅2 dimer mixture from the XRPD refinements of three β-hematin powder samples recently reported by Bohle et al.10 average at ∼75:25 (See dotted lines in Figure 2), which is indeed similar to the occupancies obtained from the hemozoin powder sample. We emphasize that a hemozoin crystal with relative occupancies (1 − Δ)/Δ of the cd1̅1 and cd1̅2 dimer mixture is diffractionally equivalent to a crystal with occupancies (1 − 2Δ)/Δ/Δ of the cd1̅ 1 , cd2(+), and cd2(−) dimers, respectively. Here we assume that the different dimers are occluded in the same crystalline diffracting domains.

Figure 6. (upper left) The Fe fluorescence signal of a mesh scan for a fixed value of ω shows a cluster of β-hematin crystals. (upper right) The complementary accumulated scattering frames obtained during the mesh reveals a partial diffraction pattern. Some low-order Debye− Scherrer rings of the major phase have been indicated as yellow lines. (lower left) The yellow patches correspond to (x,y)-positions with a {100} major scattering event, while the red patches represent a {100} minor scattering event. The {100} reflections of the two phases were separated on the detector as seen in the lower right image. By mapping out the (x,y) positions of scattering events from the major and minor phase over rocking curves of 11 degrees (not shown), there were never any coinciding (x,y)-positions, indicating that the two phases crystallize into different grains. The cutoff intensity value of the accumulated scattering frames is arbitrary but is introduced since the observed relative intensities at a fixed ω may not be representative of the relative integrated intensities.

incorporating cyclic O−H···OC acid dimers only if all the chains are parallel, implying a lattice of triclinic symmetry, but not higher.6 Moreover, the O−H···OC motif can only be achieved via a (pseudo) center of inversion rather than via (pseudo) 2-fold or 2-fold screw symmetry, as elaborated upon below in a discussion on hemozoin nucleation. Finally, the Xray diffraction pattern of the minor phase is not similar to that of the crystal structure of the β-hematin·DMSO solvate11 (vide inf ra), given that both β-hematin and the solvate crystals had been obtained from a DMSO solution, although the latter had been grown in the presence of chloroquine. The hematin anhydride dimers in the solvate stack along a 9.7 Å axis, with a pronounced π−π overlap, but stacking H-bonded chains of hematin anhydride dimers with a significant π−π overlap would yield a crystal with too low a density (vide inf ra). We had earlier proposed a model for the formation of the major and minor phases of β-hematin in terms of crystal growth kinetics.6 The rare appearance of the minor phase may be due to the difficulty discerning its presence in the XRPD pattern. There is also the possibility, albeit remote, that the minor phase, being computationally the less stable form and composed of crystals presumably smaller in size,6 dissolves over time in solution and its molecules redeposit onto crystals of the major phase, in a manner akin to Ostwald ripening.38 DFT Lattice Energy Computations of the Different Hematin Anhydride Stereoisomers. To corroborate the results of the above refinements and complete the analysis 1549



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mechanism of heme dimerization, illustrated in Scheme 1, we conducted DFT calculations of the binding energies of (a) a six-coordinated complex with the heme iron bound to the imidazole group of histidine on the Si side and O2 on the Re side, (b) a five-coordinated complex with the heme iron bound to O2 on the Re side, (c) a five-coordinated complex with the heme iron bound to a water molecule on either side, (d) a sixcoordinated complex with the heme iron bound to O2 on the Re side and a water molecule on the Si side, (e) a cd2(+) dimer, and (f) a cd2(+) dimer with O2 molecules bound on the Re side of both monomers, that is, the two Fe(3+)-PPIX units. All structures were fully relaxed. The binding energies were found to be largely insensitive to rotations of the O2 and H2O molecules on top of heme. A detailed account is given in the Supporting Information (Figures S5−S9, Tables S2, S9−S12). For the five- and six-coordinated complexes of heme with O2, namely, complexes b and a, we obtain binding energies of 24.4 and 20.3 kcal/mol, respectively. The binding energies obtained from PBE+TS are higher than the binding energies obtained for smaller heme models from high-level quantum chemistry methods41,42 and from DFT employing a different functional without a dispersion correction.43 We note that we do not expect a single-reference method, such as PBE+TS, to correctly capture the multireference character of the heme−O 2 interaction.44,45 Therefore, we interpret these results only in qualitative terms. Our calculations indicate that the binding of O2 to heme may become stronger upon cleavage of the histidine by the parasite. This is consistent with the hypothesis that O2 remains bound to the Re side of heme during the dimerization process. For the five-coordinated heme−H2O complex c, we obtain binding energies of 0.9−1.0 kcal/mol on the Re side and 0.3− 0.7 kcal/mol on the Si side. Our calculations show that the interaction of water with heme is weak and of a purely dispersive nature (without the TS dispersion correction, no binding is obtained). This is consistent with the experimental observation of water binding to myoglobin under certain conditions,46−48 as well as with an earlier computational study of water binding to smaller heme models.42 Interestingly, for the six-coordinated O2−heme−H2O complex d, we obtain an O2 binding energy of 32.4 kcal/mol, which is significantly higher than that of the five-coordinated heme−O2 complex. This is consistent with the hypothesis that the heme−O2 complex would likely bind a water molecule on the Si side. Owing to the weak nature of the heme−H2O interaction, the water molecule would be released much more easily than the oxygen molecule, “opening” the Si side of heme to dimer formation. We find that the cd2(+)/2O2 complex f has a binding energy of 17.2 kcal/mol with respect to an isolated cd2(+) dimer e and two O2 molecules. The stability of this complex means that its formation during the nucleation and growth process of hemozoin is feasible. The binding energy per O2 molecule is 8.6 kcal/mol, which is much smaller than the 24.4 kcal/mol binding energy of the molecular O2 to the heme molecule, Fe(2+)-PPIX, indicating that the hematin anhydride dimer/ 2O2 can release its two O2 molecules with relative ease for the hemozoin crystals to form. The lower binding energy of 8.6 kcal/mol is consistent with the observation that in the cd2(+) dimer/2O2 complex, the Fe−O2 coordination length of 1.93 Å is larger than the corresponding length of 1.77 Å in the heme− O2 complex (b) and the 1.73 Å in the H2O−heme−O2 complex (d).

With the above information, we address the question as to the probable relative concentrations and distribution of all four different dimers, cd11̅ , cd2(+), cd2(−), and cd12̅ , and crystal symmetry, given that the computed XRPD patterns of a hemozoin structure with occupancies 75:0:0:25 or 50:25:25:0 should be basically identical. We reject the centrosymmetric model structure composed of cd12̅ and cd1̅2 dimers with a 75:25 occupancy, since the cd1̅2 isomer cannot be easily adsorbed onto any of the three pairs of commonly observed crystal faces of hemozoin assuming cd11̅ is the “host” crystal unit.6 In contradistinction, a hemozoin crystal composed of a cd1̅1/cd2(+)/cd2(−)/cd1̅2 50:25:25:0 mixture is a more feasible model since each of the two cd2(±) enantiomers can, without impediment, selectively bind to half the enantiotopic {010}, {011}, and {100} crystal faces (the latter somewhat less easily though) and be eventually occluded within opposite sectors of the growing crystal; cd2(−) can bind to the (010), (011̅ )̅ , and (001) faces and cd2(+) to the (010̅ ), (011), and (001̅) faces.6 The growing faces of such a model crystal would occlude the centrosymmetric cd11̅ dimer but would enantioselectively occlude the cd2(±) stereoisomeric dimers of opposite handedness via opposite faces, leading to oppositely oriented chiral crystal sectors of opposite handedness but “related” by a macroscopic center of symmetry (this proposed enantiomeric distribution is also discussed in the Supporting Information). Assuming that the above hemozoin model crystal also incorporates the cd1̅2 stereoisomer with a minor concentration, of ∼5−10%, the resulting cd1̅1/cd2(+)/cd2(−)/cd12̅ occupancies would range between 55:20:20:5 and 60:15:15:10. An analogous model of enantioselective “guest” distribution within a “host centrosymmetric” crystal, leading to a reduction in crystal symmetry, has already been observed.39,40 The hemozoin model crystal with a 50:50 cd11̅ /cd2(+) dimer occupancy and thus of overall chiral symmetry holds a fundamental advantage over the cd1̅1/cd2(±)/cd12̅ model just described, despite the caveat that the cd11̅ component of the former probably includes an admixture of the cd2(−) and cd1̅2 dimers. The advantage rests on a premise that the number of the different (types of) hematin anhydride stereoisomeric dimers formed should be minimal in order that the crystals of hemozoin nucleate and grow as fast as possible. Also, the absence of the minor phase in hemozoin is no proof of a greater proportion of the cd11̅ and cd2(+) dimers than cd11̅ , cd2(±), and cd1̅2; it is nevertheless consistent with such a model dimer distribution. We note the observation that the mixture of more than one isomer is consistent with the small size of biogenic (and synthetic) hemozoin crystals because the different dimers “poison” the growing crystals. The crystal symmetry of hemozoin, chiral or centrosymmetric, cannot be resolved via analysis involving the anomalous component of the X-ray scattering term since the intensities of the (h,k,l) and (−h,−k,−l) reflection pairs in the X-ray powder diffraction pattern are superimposed. Indeed, for a model hemozoin crystal incorporating hematin anhydride cd1̅1 and cd2(+) dimers with their centrosymmetric porphyrin cores, its absolute chirality would barely be expressed even in a singlecrystal X-ray diffraction experiment since the atoms inducing molecular handedness, as in the case of the cd2(+) dimer, are all of the same element, as elaborated upon in the Supporting Information. DFT Simulations of the Dimerization Mechanism. To corroborate our biochemically based hypothesis for the 1550



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Figure 7. (a) Top view of H-bonded chain generated along the a−c axis. (b) The chain viewed edge-on revealing a relatively coplanar arrangement. (c) View as in panel a, but of two H-bonded chains stacked along the c-axis to form an a,c, that is, {010}, bilayer. One chain has been colored green for clarity. The carboxyl groups participating in interdimer OH···O bonds have been colored dark gray. (d) Edge on view of the a,c bilayer along the a−c direction, revealing a highly interleaved structure. The a and c axes overlay in this projection, as indicated by the label (a,c). (e) Stacking of the a,c bilayers along the b-axis, viewed along the a−c axis. (f) With inspiration from Klonis et al.,3 all short-range monomer−monomer interactions are shown edge on, including the three shortest Fe−Fe distances, whose lengths are listed in Table S13, Supporting Information. The black and purple monomers constitute the hematin anhydride dimer.

Lifetimes of the Different Heme Complexes in Scheme 1. An estimate of the computed lifetimes of the various heme complexes (Scheme 1) leading to the formation of the hematin anhydride isomer cd2(+) might help rationalize its preferred formation. The Arrhenius equation relates the lifetimes of species to their free energy. The lifetime τ of the molecular species is given by log 2/τ = A exp(−ΔG/(RT)), where A is a pre-exponential factor, ΔG is the activation energy, R is the gas constant, and T is the temperature in kelvin. The pre-exponential factor A in the Arrhenius equation has a value49 of ∼1013. Here, instead of the free energy of dissociation (ΔG) that also incorporates the entropy term, we shall make use of the binding energy (i.e., ΔG ≈ BE) to estimate the relative lifetimes of the H2O−heme−O2, heme−O2 and cd2(+)/2O2 species, whose O2 binding energies are 32.4, 24.4, and 8.6 kcal/ mol, respectively. Given that the computed binding energies may be overestimated (vide supra), we shall provide only qualitative estimates of the lifetimes of the different species (see Scheme 1). We do not account for the interaction of these

species with the aqueous environment. We expect this interaction to affect mainly the entropic contribution to the free energy, which is neglected here. There will be a constant interchange between the H2O−heme−O2 and heme−O2 species since water, given its ubiquitous presence, is attached and detached from the heme at a rapid rate. We thus propose that these two species existing alternately are sufficiently longlived to survive the hemoglobin degradation process, even though the heme−O2 complex has a much shorter computed lifetime than H2O−heme−O2. At the end of the hemoglobin degradation process, the heme−O2 species, with a computed lifetime τ on the order of an hour, will allow stereospecific formation of the cd2(+)/2O2 complex. By comparison, the binding energy of an O2 molecule to the hematin anhydride dimer corresponds to a lifetime of a fraction of a second so that the bound O2 molecules will be quickly released. Naturally, this lifetime analysis does not apply to that fraction (∼25%) of heme molecules that are not oxygenated in hemoglobin in venous type blood (vide supra). 1551



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no other H-bonding partner is present.63 However, the cyclic dimer motif is present in only 31% of all structures that contain at least one carboxylic acid functional group,63 indicating that hetero H-bonding motifs are more favorable. The carboxylic acid cyclic dimer incorporates intrinsic repulsion between the hydroxyl H atoms and between electron lone pair lobes of the carbonyl O atoms,64 which is circumvented in the catemer motif65 that interlinks carboxyl groups via 2-fold screw or glide symmetry as in the crystal structures of formic,66 acetic,67 and β-tetrolic acid.68,69 However, the constraints on molecular structure and packing preclude this motif for most systems. The repulsion inherent in carboxylic acid cyclic dimers is much reduced in the hematin anhydride DMSO solvate structure, which embodies a cyclic H-bonded heterodimer containing O− H···OS (2.5 Å) and C−H···OC (3.4 Å) bonds. This dimer is akin to that found in the crystal structure of acetic acid,67 which embodies O−H···OC and C−H···OC bonds. It is possible that the COOH···OS(CH3)2 H-bonding motif in the solvate crystal structure is generated to allow formation of the interdimer π−π arrangement. This model appears to be consistent with the recent hypothesis by Klonis et al.3 that hemozoin nucleation begins with the formation and assembly of heme monomers into π−π dimers, rather than via hematin anhydride dimers as the nucleation unit. A hypothetical crystalline layer composed of H-bonded hematin anhydride chains stacked via interdimer π−π contacts akin to what is observed in the crystal structure of hematin anhydride DMSO solvate is easily constructed. However, such layers can be juxtaposed to form a three-dimensional crystal, but with a volume ca. 40% greater than that of hemozoin (see Figure S12 in Supporting Information). Therefore, an aggregate thereof can hardly be regarded as a viable nucleus of hemozoin crystallization.

Hemozoin Crystal Nucleation. The importance of the nature of the crystal nucleating and growth unit of hemozoin cannot be overemphasized because it is relevant for the design and use of antimalarial drugs. Many different studies point toward an oriented, lipid-templated nucleation of the crystal,50−58 occurring from its {100} face both in the synthetic54,55 and in the biogenic forms,57,58 which, in the latter, occurs via the inner membrane lipid surface of the parasitic digestive vacuole in the infected red blood cell. We note, however, that hemozoin may already have formed in the early ring stage of parasitic development in predigestive vacuoles,59 in the reported absence of lipid catalysts. The complex nature of the malaria parasite hemoglobin degradation, which is mediated by the action of several proteases, has been recently reviewed by Goldberg.60 This process is required for the formation of the hematin anhydride dimers, as necessary precursors to hemozoin formation. While several questions remain,60 we are of the opinion that the following hypothesis of the hemozoin nucleation process is valid. The hematin anhydride dimers in solution are to a large extent of fixed geometry, given that the propionate moiety O C−O−Fe adopts an anti-planar geometry, as opposed to a hypothetical syn-planar geometry,37 restricting the conformational freedom of the dimer. Thus, the dimers would act as favorable units of aggregation leading to crystal nucleation. These hematin anhydride dimers may interlink stereospecifically via their propionic acid moieties to form H-bonded chains, as depicted for an ideal triplet of dimers in Figure 7a,b spanning the a−c axis of length 15.4 Å. The approximate coplanarity of this H-bonded system is achieved via the conformation of the propionic acid moieties, which allows the centers of the Hbonded acid pairs to be almost at the same level as the centers of the hematin anhydride dimers (see Figure 7b). In principle, the H-bonded motif can be achieved either via a center of inversion (1)̅ , as shown in Figure 7a,b, or via 2-fold (2) symmetry. The latter, however, would induce steric intermolecular hindrance along the chain (shown in Figure S10 in the Supporting Information).61 At the aggregation stage, the Hbonded chains may mesh and partially interleave via translation along the short 8 Å c-axis to form densely packed bilayers as shown in Figure 7c,d. The chains can mesh so as to form both π−π contacts between c-translation related porphyrin rings and C−H···O contacts between ethylene proton donors and the exposed lone pair electron lobes of the two propionate O atoms (Figure S11 in Supporting Information). The final stage of cluster formation resulting in a nucleus involves stacking of the bilayers to form interdimer π−π contacts via (pseudo) inversion symmetry, as shown in Figure 7e. This π−π contact is achieved by translation of the bilayers along the b−c axis. (see Figure 7f). Recently Gildenhuys et al.11 reported the single crystal X-ray structure of a hematin anhydride DMSO solvate, grown in the presence of the antimalarial drug chloroquine, a growth-rate inhibitor of β-hematin crystallization. The adjacent hematin anhydride dimers in this structure are stacked along a 9.7 Å axis with a high π−π overlap, unlike that in hemozoin. Such a pronounced overlap has also been found in the crystal structure of the analogous mesohematin anhydride DMSO solvate, as characterized by XRPD.62 Gildenhuys et al. have rationalized the crystallization of the hematin anhydride DMSO solvate in terms of the H-bonding properties of carboxylic acids. They referred to the propensity of carboxylic acids to form the H-bonding cyclic dimer motif if

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SUMMARY AND CONCLUSION We investigated the structure of the malaria pigment, hemozoin, and its synthetic form, β-hematin, with respect to the presence of four possible Fe(3+)-PPIX stereoisomeric dimers. The absence of a minor phase in biogenic hemozoin, in contrast to β-hematin, triggered us to envisage a fundamental difference in the distribution of the stereoisomeric hematin anhydride dimer units in β-hematin and hemozoin crystals. We also relied on the premise that for the crystals of hemozoin to nucleate and grow as fast as possible, the number of the dif ferent (types of) hematin anhydride stereoisomeric dimers formed should be minimal. We postulated that the dimer distribution in the structure of hemozoin is primarily a consequence of the heme molecules remaining coordinated to molecular oxygen in the same chiral configuration after degradation of hemoglobin, in accordance with DFT computations (vide supra). We correlated this statement with the observation that the parasites were grown in an atmosphere similar to that found in venous blood, where ca. 75% of the heme was still oxygenated. The analysis of the XRPD data of hemozoin described here yielded two primary models of the hemozoin structure. The first model is chiral in symmetry of single handedness, whose mixed molecular sites comprise the chiral cd2(+) and centrosymmetric cd11̅ dimers in about a 1:1 ratio, perhaps including a minor admixture of cd2(−) and cd1̅2 isomers. The alternative model is composed of the four stereoisomers, cd1̅1, cd2(+), cd2(−), and cd1̅2 with estimated relative concentrations ≅ 58:17:17:8. The centrosymmetric cd1̅1 “host” molecule is uniformly distributed throughout the crystal, 1552


Crystal Growth & Design whereas the enantiomeric molecules cd2(+) and cd2(−) are preferentially occluded in different enantiomorphous crystalline sectors of opposite handedness, which has overall centrosymmetric symmetry. This model is akin to that of the major phase of β-hematin, reported by Straasø et al.6 As for the phase composition of β-hematin, studied previously by XRPD refinement6 complemented by DFT analysis,7 we had characterized the major phase as composed mainly of the cd11̅ dimer and the minor phase as composed mainly of the cd12̅ dimer. We had suggested, based on stereochemical considerations, that the enantiomeric cd2(+) and cd2(−) dimers may be occluded in both phases. Here, we have analyzed XRPD data of an additional β-hematin sample containing a larger fraction of the minor phase and exhibiting seven resolvable Bragg peaks associated with the minor phase (as opposed to five in our earlier work). Moreover, we examined single grains and small clusters from β-hematin powder samples via nanoprobe X-ray diffraction and Fefluorescence measurements. The diffraction from the major and minor phases were only to be found in different grains, namely, they did not cocrystallize into a single grain. In addition, we infer from the Fe X-ray fluorescnce signal arising from the minor phase that it is composed of Fe-PPIX moieties. Thus, the presence of all the denoted peaks of the minor phase has been substantiated, and the agreement between the proposed structural model and the observed X-ray powder diffraction data is more than satisfactory. Also, we have eliminated various alternative hematin anhydride structures, crystal lattices, and packing motifs for the minor phase. We proposed, based on the XRPD data analysis and DFT lattice energy calculations, that the hematin anhydride dimers of the minor phase are mainly of the cd12̅ type, in order to rationalize its presence. Regarding the nucleation process, it most likely begins with the hematin anhydride dimer formation, rather than assembly of heme monomers.3 Indeed, the stereoisomeric dimer distribution in the crystals of β-hematin,10 hemozoin, and βhematin·DMSO solvate11 strengthens the model that hematin anhydride dimers constitute the nucleation and growth unit of both the synthetic and biogenic forms of hemozoin crystals. The hematin anhydride dimer formation is followed by stereospecific H-bond linkage between the dimers into chains (along the a−c axis). The chains may then interleave via C− H···O (propionate) and π−π contacts (along the c-axis), yielding densely packed bilayers. Stacking of these bilayers (along the b-axis) via π−π interactions completes formation of a nucleation aggregate. In conclusion, we have provided evidence and arguments that hemozoin may be a crystal of overall chiral symmetry, as a consequence for the preference of the chiral dimer cd2(+) as a dominant structural unit. However, we cannot overrule the alternative possibility that the overall crystal structure is centrosymmetric, comprising the four hematin anhydride stereoisomeric dimers. The most dominant structural unit is the centrosymmetric cd11̅ dimer, followed by the enantiomers cd2(±) occluded in enantiomorphous crystalline sectors. Both structural models of hemozoin incorporate stereoisomeric disorder of their vinyl (CHCH2) and methyl (CH3) substituents. By taking such disorder into account, it should be possible to calculate more correctly the crystal surface binding energies of antimalarial drugs to the principal faces of hemozoin.8

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

Article

S Supporting Information *

Reprints of figures previously published to promote understanding of the present work, structural information regarding the crystal structures, detailed schemes containing the results of the DFT calculations, the hypothetical molecular structure of an H-bonded “chain” of hematin anhydride dimers connected by 2-fold symmetry, the hypothetical packing arrangement of hematin anhydride dimers assuming π−π packing, and crystallographic information on the three anhydride dimers of hemozoin. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Authors

*E-mail: *E-mail: *E-mail: *E-mail:

[email protected]. [email protected]. [email protected]. [email protected].

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Keld Teodor at beamline 9-11, MAX-lab, has provided support during the installation of the all-in-vacuum diffractometer, while the purification of hemozoin not could have been done without the facilities provided by Katrine Nørgaard Toft, Department of Drug Design and Pharmacology at University of Copenhagen. We thank Isabella Weissbuch who worked with Inna Solomonov during her Ph.D. studies at the Weizmann Institute of Science, when I.S. measured the data set on β-hematin in 2004. In addition, we acknowledge Ellen Wachtal for helpful insight concerning the in-house data acquisition. We are indebted to Vaclav Petricek for his advice on the program Jana2006. We gratefully acknowledge invaluable input from Michael McBride. We are thankful for the beamtime at ESRF to perform the nanodiffraction experiment and at SLS to perform low temperature XRPD studies. Finally we thank Danscatt for financial support.

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REFERENCES

(1) Slater, A. F. G.; Swiggard, W. J.; Orton, B. R.; Flitter, W. D.; Goldberg, D. E.; Cerami, A.; Henderson, G. B. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 325. (2) Pagola, S.; Stephens, P. W.; Bohle, D. S.; Kosar, A. D.; Madsen, S. K. Nature 2000, 404, 307. (3) Klonis, N.; Dilanian, R.; Hanssen, E.; Darmanin, C.; Streltsov, V.; Deed, S.; Quiney, H.; Tilley, L. Biochemistry 2010, 49, 6804. (4) Bohle, D. S.; Dinnebier, R. E.; Madsen, S. K.; Stephens, P. W. J. Biol. Chem. 1997, 272, 713. (5) Oliveira, M. F.; Kycia, S. W.; Gomez, A.; Kosar, A. J.; Bohle, D. S.; Hempelmann, E.; Menezes, D.; Vannier-Santos, M. A.; Oliveira, P. L.; Ferreira, S. T. FEBS Lett. 2005, 579, 6010. (6) Straasø, T.; Kapishnikov, S.; Kato, K.; Takata, M.; Als-Nielsen, J.; Leiserowitz, L. Cryst. Growth Des. 2011, 11, 3342. (7) Marom, N.; Tkatchenko, A.; Kapishnikov, S.; Kronik, L.; Leiserowitz, L. Cryst. Growth Des. 2011, 11, 3332. (8) Buller, R.; Peterson, M. L.; Almarsson, O.; Leiserowitz, L. Cryst. Growth Des. 2002, 2, 553. (9) Weissbuch, I.; Addadi, L.; Leiserowitz, L. Science 1991, 253, 637.

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(10) Bohle, D. S.; Dodd, E. L.; Stephens, P. Chem. Biodivers. 2012, 9, 1891. (11) Gildenhuys, J.; le Roex, T.; Egan, T. J.; de Villiers, K. A. J. Am. Chem. Soc. 2013, 135, 1037. (12) Hoard, J. L.; Hamor, M. J.; Hamor, T. A.; Caughey, W. S. J. Am. Chem. Soc. 1965, 87, 2312. (13) Koenig, D. F. Acta Crystallogr. 1965, 18, 663. (14) de Villiers, K. A.; Marques, H. M.; Egan, T. J. J. Inorg. Biochem. 2008, 102, 1660. (15) The large atomic displacement factors and the short 1.25 Å C C bond lengths of the vinyl groups reported in ref 14 are suggestive of disorder of the pyrrole rings B and C involving vinyl and methyl subsituents. (16) Royer, W. E. J. Mol. Biol. 1994, 235, 657. (17) Vojtechovsky, J.; Chu, K.; Berendzen, J.; Sweet, R. M.; Schlichting, I. Biophys. J. 1999, 77, 2153. (18) Barfod, L.; et al. Mol. Microbiol. 2007, 63, 335. (19) Straasø, T.; Becker, J.; Iversen, B. B.; Als-Nielsen, J. J. Synchrotron Radiat. 2013, 20, 98. (20) Petricek, V.; Dusek, M.; Palatinus, L. Jana2006, Institute of Physics, Praha, Czech Republic, 2006. (21) Blum, V.; Gehrke, R.; Hanke, F.; Havu, P.; Havu, V.; Ren, X.; Reuter, K.; Scheffler, M. Comput. Phys. Commun. 2009, 180, 2175. (22) Havu, V.; Blum, V.; Havu, V.; Scheffler, M. J. Comput. Phys. 2009, 228, 8367. (23) Auckenthaler, T.; Blum, V.; Bungartz, H.-J.; Huckle, T.; Johanni, R.; Krämer, L.; Lang, B.; Lederer, H.; Willems, P. R. Parallel Comput. 2011, 37, 783. (24) Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. Rev. Lett. 1996, 77, 3865. (25) Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. Rev. Lett. 1997, 78, 1396 (E). (26) Tkatchenko, A.; Scheffler, M. Phys. Rev. Lett. 2009, 102, No. 073005. (27) Marom, N.; Tkatchenko, A.; Rossi, M.; Gobre, V. V.; Hod, O.; Scheffer, M.; Kronik, L. J. Chem. Theory Comput. 2011, 7, 3944. (28) Marom, N.; Moussa, J. E.; Ren, X.; Tkatchenko, A.; Chelikowsky, J. R. Phys. Rev. B 2011, 84, No. 245115. (29) Marom, N.; Tkachenko, A.; Scheffler, M.; Kronik, L. J. Chem. Theory Comput. 2010, 6, 81. (30) Barratt-Boyes, B. G.; Wood, E. H. J. Lab. Clin. Med. 1957, 50, 93. (31) Bohle, D. S.; Helms, J. B. Biochem. Biophys. Res. Commun. 1993, 193, 504. (32) Slater, A. F. G.; Swiggard, W. J.; Orton, B. R.; Flitter, W. D.; Goldberg, D. E.; Cerami, A.; Henderson, G. B. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 325. (33) Bohle, D. S.; Kosar, A. D.; Stephens, P. W. Acta Crystallogr. 2002, D58, 1752. (34) Egan, T. J.; Mavuso, W. W.; Ncokazi, K. K. Biochemistry 2001, 40, 204. (35) Solomonov, I.; Osipova, M.; Feldman, Y.; Baehtz, C.; Kjaer, K.; Robinson, I. K.; Webster, G. T.; McNaughton, D.; Wood, B. R.; Weissbuch, I.; Leiserowitz, L. J. Am. Chem. Soc. 2007, 129, 2615. (36) Bernstein, J. Polymorphism in Molecular Crystals; Clarendon Press: Oxford, U.K., 2002; p 147. (37) Charkin, O. P.; Klimenko, N. M.; Charkin, D. O.; Chang, H. C.; Lin, S. H. J. Phys. Chem. A 2007, 111, 9207. (38) Ostwald, W. F. Z. Phys. Chem. 1897, 22, 289. (39) For example, R and S α-amino acid additives present in a supersaturated aqueous glycine solution are enantioselectively adsorbed and occluded at the opposite {010} faces of the growing “centrosymmetric” glycine crystal to yield two crystal halves of reduced chiral symmetry.40 (40) Weissbuch, I.; Popovitz Biro, R.; Lahav, M.; Leiserowitz, L. Acta Crystallogr.. 1995, B51, 115. (41) Radon, M.; Pierloot, K. J. Phys. Chem. A 2008, 112, 11824. (42) Strickland, N.; Harvey, J. N. J. Phys. Chem. B 2007, 111, 841.

(43) Ali, M. E.; Sanyal, B.; Oppeneer, P. M. J. Phys. Chem. B 2012, 116, 5849. (44) Jensen, K. P.; Ryde, U. J. Biol. Chem. 2004, 279, 14561. (45) Jensen, K. P.; Roos, B. O.; Ryde, U. J. Inorg. Biochem. 2005, 99, 45. (46) Lamb, D. C.; Ostermann, A.; Prusakov, V. E.; Parak, F. G. Eur. Biophys. J. 1998, 27, 113. (47) Lamb, D. C.; Prusakov, V.; Engler, N.; Ostermann, A.; Schellenberg, P.; Parak, F. G.; Nienhaus, G. U. J. Am. Chem. Soc. 1998, 120, 2981. (48) Lamb, D. C.; Kriegl, J.; Kastens, K.; Nienhaus, G. U. J. Phys. Org. Chem. 2000, 13, 659. (49) Benson, S. W. The Foundations of Chemical Kinetics; McGrawHill: New York, 1960; p 249. (50) Goldberg, D. E.; Slater, A. F. G.; Cerami, A.; Henderson, G. B. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 2931. (51) Jackson, K. E.; Klonis, N.; Ferguson, D. J. P.; Adisa, A.; Dogovski, C.; Tilley, L. Mol. Microbiol. 2004, 54, 109. (52) Pisciotta, J. M.; Coppens, I.; Tripathi, A. K.; Scholl, P. F.; Schuman, J.; Bajad, S.; Shulaev, V.; Sullivan, D. J. Biochem. J. 2007, 402, 197. (53) Egan, T. J.; Chen, J. Y. J.; de Villiers, K. A.; Mabotha, T. E.; Naidoo, K. J.; Ncokazi, K. K.; Langford, S. J.; McNaughton, D.; Pandiancherri, S.; Wood, B. R. FEBS Lett. 2006, 580, 5105. (54) de Villiers, K. A.; Osipova, M.; Mabotha, T. E.; Solomonov, I.; Feldman, Y.; Kjaer, K.; Weissbuch, I.; Egan, T. J.; Leiserowitz, L. Cryst. Growth Des. 2009, 9, 626. (55) Wang, X.; Ingall, E.; Lai, B.; Stack, A. G. Cryst. Growth Des. 2010, 10, 798. (56) Hoang, A. N.; Ncokazi, K. K.; de Villiers, K. A.; Wright, D. W.; Egan, T. J. Dalton Trans. 2010, 39, 1235. (57) Kapishnikov, S.; Weiner, A.; Shimoni, E.; Guttmann, P.; Schneider, G.; Dahan-Pasternak, N.; Dzikowski, R.; Leiserowitz, L.; Elbaum, M. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 11188. (58) Kapishnikov, S.; Berthing, T.; Hviid, L.; Dierolf, M.; Menzel, A.; Pfeiffer, F.; Als-Nielsen, J.; Leiserowitz, L. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 11184. (59) Hanssen, E.; Knoechel, C.; Klonis, N.; Abu-Bakar, N.; Deed, S.; LeGros, M.; Larabell, C.; Tilley, L. J. Struct. Biol. 2011, 173, 161. (60) Goldberg, D. E. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 5283. (61) On a more general note, the 2-fold symmetry as an element generating cyclic carboxylic H-bonded pairs is rarely observed, if at all, since it would induce poor intermolecular packing (62) Bohle, D. S.; Dodd, E. L.; Kosar, A. J.; Sharma, L.; Stephens, P. W.; Suarez, L.; Tazoo, D. Angew. Chem., Int. Ed. 2011, 50, 6151. (63) Shattock, T. R.; Arora, K. K.; Vishweshwar, P.; Zaworotko, M. J. Cryst. Growth Dess 2008, 8, 4533. (64) Bernstein, J.; Etter, M.; Leiserowitz, L. In Structure Correlation; Dunitz, J. D., Bergi, H. B., Eds.; VCH: Weinheim, Germany, 1994; Vol. 2, p 431. (65) Leiserowitz, L. Acta Crystallogr. 1976, B32, 775. (66) Nahringbauer, I.; Larsson, G. Ark. Kemi 1969, 30, 91. (67) Jönsson, P.-G. Acta Crystallogr. 1971, B27, 893. (68) Benghiat, V.; Leiserowitz, L. J. Chem. Soc., Perkin Trans. 2 1972, 1763. (69) N. Marom, N.; DiStasio, R. A., Jr.; Atalla, V.; Levchenko, S.; Reilly, A. M.; Chelikowsky, J. R.; Leiserowitz, L.; Tkatchenko, A. Angew. Chem., Int. Ed. 2013, 52, 6629.

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