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

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 (1̄), labeled cd1̄₁ and cd1̄₂, and two enantiomeric, cd2(+) and cd2(-), in which the Fe(3+)PPIX moieties are related by pseudo-2-fold symmetry. Only the cd1̄₁ 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 cd1̄₁/cd2(±) mixture, which is diffractionally equivalent to a cd1̄₁/cd1̄₂ mixture, a minor phase considered to comprise mainly cd1̄₂ dimers. As a consequence single-phase β-hematin powders were recently reanalyzed in terms of a cd1̄₁/cd1̄₂ 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: cd1̄₁, cd1̄₂, cd2(+), and the two mixtures cd1̄₁/ cd1̄₂ and cd1̄₁/cd2(+). The best figures of merit were obtained for the mixture cd1̄₁/cd2(+) with a 50:50 occupancy, followed by the cd1̄₁/cd1̄₂ mixture with an occupancy ≅75:25, which we interpret as a structure that comprises the cd1̄₁, cd2(+), cd2(-), and cd1̄₂ isomers with occupancies ≅58:17:17:8. In this model system, the cd1̄₁ "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 cd1̄₁/cd2(+) mixture, namely, a hemozoin crystal of overall chiral symmetry, consistent with our hypothesis. However, we cannot overrule the alternative model.