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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

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journal contribution
posted on 02.04.2014, 00:00 by Tine Straasø, Noa Marom, Inna Solomonov, Lea K. Barfod, Manfred Burghammer, Robert Feidenhans’l, Jens Als-Nielsen, Leslie Leiserowitz
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 cd1̅2 dimers. As a consequence single-phase β-hematin powders were recently reanalyzed in terms of a cd1̅1/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, cd1̅2, cd2­(+), and the two mixtures cd1̅1/cd1̅2 and cd1̅1/cd2­(+). The best figures of merit were obtained for the mixture cd1̅1/cd2­(+) with a 50:50 occupancy, followed by the cd1̅1/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 cd1̅1/cd2­(+) mixture, namely, a hemozoin crystal of overall chiral symmetry, consistent with our hypothesis. However, we cannot overrule the alternative model.