The Role of the Four Stereoisomers of the Heme Fe–O Cyclic Dimer in the Crystalline Phase Behavior of Synthetic Hemozoin: Relevance to Native Hemozoin Crystallization

Abstract

Hemozoin is a crystalline byproduct formed upon host hemoglobin digestion in malaria-infected blood cells, crucial for parasitic survival. On the basis of published spectroscopic and X-ray powder diffraction (XRPD) data, hemozoin is believed to be very similar to the synthetic compound β-hematin, which consists of cyclic centrosymmetric dimers of ferriprotoporphyrin IX [Fe(3+) PPIX] molecules coordinated via Fe–O bonds. The enantio-facial symmetry of Fe(3+) PPIX implies, however, that four different Fe–O cyclic stereoisomers, two centrosymmetric and two chiral, of opposite handedness, should be formed in the crystallizing solution of β-hematin. A low-temperature XRPD study of β-hematin, i.e. synthetic hemozoin, revealed the presence, not only of the published phase ( Pagola S. Stephens P. W. Bohle D. S. Kosar A. D. Madsen S. K. Pagola S. Stephens P. W. Bohle D. S. Kosar A. D. Madsen S. K. Nature2000404307) but also of a minor phase. We propose, based on Rietveld refinement and DFT+vdW computations (companion manuscript, DOI: 10.1021/cg200409d), that the minor phase consists mainly of the second centrosymmetric isomeric type in a crystal structure similar to that of the major phase. The enantiomeric chiral isomers may, on symmetry grounds, be enantioselectively occluded into the growing crystals, introducing disorder. The chiral dimers, on being first adsorbed on the crystal faces, would act as tailor-made additives, retarding crystal growth, which also explains the crystalline micrometer size. The existence of two phases in β-hematin may be crucial for a fuller understanding and more complete determination of the crystal structure of hemozoin, of which only one phase has crystallized according to published data.

Structure and Formation of Synthetic Hemozoin: Insights From First-Principles Calculations

“…The H-bonds in the minor phase unit cell are somewhat shorter than in the major phase unit cell. These results are in excellent agreement with the predominant hydrogen bond length of 2.65 Å, obtained from an analysis of about 5000 species extracted from the Cambridge Database 18 On the basis of the results of our calculations regarding the energetics of the different stereoisomers of the Fe(3+) PPIX dimer, we suggest that β-hematin nucleation begins with dimerization via the formation of ironÀcarboxylate bonds between two Fe(3+) PPIX monomers. At this stage, the most stable cd1 1 stereoisomer forms predominantly, but because the three isomers are close in energy, the cd1 2 and cd2 stereoisomers may also form in smaller quantities.…”

“…This is reflected in the deviation from planarity. In this respect, the geometry of the dimers in the crystal is in better agreement with the refinement of Straasø et al, 18 where the structure of the monomer is based on that of a recently reported complex of Fe(3+) PPIX with halofantrine, 75 as compared to the refinement of Pagola et al, 6 where the structure of the monomer is based on that of R-chlorohemin. 20 The optimized OH 3 3 3 O distances (in the H-bonds between adjacent dimers) range from 2.62 to 2.68 Å with some variation between the different stereoisomers and the different spin states.…”

“…First, we focus on assessing the contribution of weak interactions, that is, dispersion and hydrogen bonds, to the stabilization of the crystal. For this purpose, we use the refined structure of the major phase of β-hematin, cd1 1 , as obtained by Straasø et al, 18 with the lattice parameters a = 12.089 Å, b = 14.474 Å, c = 7.989 Å, R = 90.82°, β = 96.8°, and γ = 97.64°. 73 The lattice parameters were changed manually, one at a time, around the experimental values to obtain binding energy curves along the different crystal axes.…”

“…74 From stereochemical 18 and thermodynamic considerations, the cd2 stereoisomer is likely occluded into the major phase. Table 4 shows some structural parameters of the relaxed β-hematin stereoisomers in both unit cells, as compared to the refined structure of the major phase as obtained by Pagola et al 6 and to the refined structures of the major and minor phases obtained by Straasø et al 18 Figure 5 shows the relaxed structures of the cd1 1 stereoisomer as an isolated dimer and in the unit cell of the major phase. The main difference between the structures of the isolated dimers and the structures of the dimers in the crystal is that in the crystal the bridging propionates are fully extended.…”

“…This gives rise to the question of the possible presence of stereoisomers other than the observed cd1 1 . A clue to the presence of a second phase of β-hematin has been observed by Bohle et al, based on powder diffraction data, 14 but only now, Straasø et al 18 have succeeded in identifying and determining the structure of a minor phase of β-hematin, which Received: April 1, 2011 ABSTRACT: Malaria, an infectious disease once considered eradicated, has reemerged in recent years, primarily due to parasite resistance to commonly used synthetic antimalarial drugs. These drugs act by inhibiting crystallization of the malaria pigment, hemozoin (HZ).…”