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

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 cd1̅ 1 and cd1̅ 2 , and two enantiomeric, cd2(+) and cd2(−), in which the Fe(3+)PPIX moieties are related by pseudo-2-fold symmetry. Only the cd1̅ 1 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 cd1̅ 1 / cd2(±) mixture, which is diffractionally equivalent to a cd1̅ 1 /cd1̅ 2 mixture, a minor phase considered to comprise mainly cd1̅ 2 dimers. As a consequence single-phase β-hematin powders were recently reanalyzed in terms of a cd1̅ 1 /cd1̅ 2 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 O 2 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: cd1̅ 1 , cd1̅ 2 , cd2(+), and the two mixtures cd1̅ 1 /cd1̅ 2 and cd1̅ 1 /cd2(+). The best figures of merit were obtained for the mixture cd1̅ 1 /cd2(+) with a 50:50 occupancy, followed by the cd1̅ 1 /cd1̅ 2 mixture with an occupancy ≅ 75:25, which we interpret as a structure that comprises the cd1̅ 1 , cd2(+), cd2(−), and cd1̅ 2 isomers with occupancies ≅ 58:17:17:8. In this model system, the cd1̅ 1 "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 cd1̅ 1 /cd2(+) mixture, namely, a hemozoin crystal of overall chiral symmetry, consistent with our hypothesis. However, we cannot overrule the alternative model.

Nanocrystallography measurements of early stage synthetic malaria pigment

“…The SFX XRPD pattern exhibits a much stronger scattering of X-rays within the 0.21-0.22 Å À1 range, as well as a significant reduction of the intensities of the 031 and 131 Bragg reflections, which are usually much stronger than the 001 Bragg reflection. This diffraction pattern for -hematin has not been reported in any previous study (Straasø et al, 2011(Straasø et al, , 2014Solomonov et al, 2007;Pagola et al, 2000;Klonis et al, 2010;Bohle et al, 1997). In the discussion that follows we examine the possible origin for the significant differences between the measured SFX and synchrotron XRPD patterns.…”

Structure and Morphology of Light-Reflecting Synthetic and Biogenic Polymorphs of Isoxanthopterin: A Comparison

“…82 A plausible binding motif between the DMSO solvent and the isoxanthopterin molecule would involve the formation of heterocyclic H-bonded dimers, as shown in Figure 8, akin to that observed in the crystal structure of the β-hematin-DMSO solvate. [83][84][85] These interactions may occur at the surface of the (0kl) faces, thus rationalizing the morphology of the elongated crystals of isoxanthopterin in terms of an interplay between the internal crystal structure and solventsurface interactions. In contrast to the synthetic α-polymorph, the biogenic crystals of isoxanthopterin develop as thin {100} plates, although the theoretical growth morphology (Figure 6E(i)) is bulky and prismatic, as is the synthetic polymorph.…”

Unraveling the Electronic Structure, Spin States, Optical and Vibrational Spectra of Malaria Pigment

“…Recently,S traasø et al [52,53] and Marom et al [11] appliedX RPD and van der Waals-corrected density functional theory (DFT + vdW) and identified four different stereoisomers of b-hematin. Twoo ft hem are centrosymmetric and two of them are chiral isomersw ith opposite handedness symmetries.…”