Despite the worldwide public health impact of malaria, neither the mechanism by which the Plasmodium parasite detoxifies and sequesters haem, nor the action of current antimalarial drugs is well understood. The haem groups released from the digestion of the haemoglobin of infected red blood cells are aggregated into an insoluble material called haemozoin or malaria pigment. Synthetic beta-haematin (FeIII-protoporphyrin-IX)2 is chemically, spectroscopically and crystallographically identical to haemozoin and is believed to consist of strands of FeIII-porphyrin units, linked into a polymer by propionate oxygen-iron bonds. Here we report the crystal structure of beta-haematin determined using simulated annealing techniques to analyse powder diffraction data obtained with synchrotron radiation. The molecules are linked into dimers through reciprocal iron-carboxylate bonds to one of the propionic side chains of each porphyrin, and the dimers form chains linked by hydrogen bonds in the crystal. This result has implications for understanding the action of current antimalarial drugs and possibly for the design of new therapeutic agents.

In a process inhibited by the quinoline antimalarial drugs, Plasmodia detoxify heme released during the degradation of hemoglobin by aggregating it into malarial pigment, an insoluble crystalline heme coordination polymer. Synchrotron x-ray powder diffraction patterns for intact desiccated malarial trophozoites and synthetic ␤-hematin have been measured; both materials correspond to a single crystalline triclinic lattice with unit cell parameters a ‫؍‬ 12.2176(4), b ‫؍‬ 14.7184(5), c ‫؍‬ 8.0456(3) Å; ␣ ‫؍‬ 90.200(2), ␤ ‫؍‬ 96.806(3), ␥ ‫؍‬ 97.818(3)°a nd Z ‫؍‬ 2. These results unambiguously demonstrate that hemozoin crystallites are identical to synthetic ␤-hematin.Heme is a potent multifunction regulator whose biochemical levels and distribution are precisely controlled on both intraand extracellular levels (1). Efficient regulation of heme is particularly critical for intra-erythrocytic parasites such as plasmodia which process large quantities of heme in the postinvasion digestion of the erythrocyte's hemoglobin. Plasmodia, which lack heme oxygenases, detoxify heme by sequestering it into an insoluble heme aggregate termed malarial pigment or hemozoin (2). The quinoline-based family of antimalarials interfere with this process by an as yet unknown mechanism that has recently come under intense scrutiny as part of the effort to combat the spread of chloroquine-resistant strains of Plasmodium. A variety of spectroscopic and bioanalytical techniques indicate that hemozoin is similar to the synthetic aggregated heme phase ␤-hematin, which is thought to form strands of hemes linked by propionate oxygen-iron bonds as well as interstrand propionate hydrogen bonds, Fig. 1 (3-5). Characterization of the carboxylate stretching bands for the propionic acid side chains by IR and Raman spectroscopy provides the best evidence for the presence of iron-oxygen bonds to the propionate side chains (3-5). Unfortunately, crystallographic characterization of these heme aggregates has been hampered by the phase heterogeneity of many synthetic preparations as well as by the small size of the synthetic and natural crystallites isolated from either trophozoites and infected hosts (6). High resolution powder diffraction has been used extensively for the solution of many structural problems (7), and it can solve problems posed by diffraction from microcrystalline phases. In this communication we describe the characterization of ␤-hematin derived from both synthetic and natural sources and provide new unambiguous evidence that the heme aggregate present in late stage trophozoites is ␤-hematin. EXPERIMENTAL PROCEDURESCirca 3 ϫ 10 9 chloroquine-susceptible Plasmodium falciparum late trophozoites of the 3D7 clone (NF54 strain) were synchronized with sorbitol and allowed to achieve a 92% level of parasitemia (8). The resulting intact trophozites were isolated by rapid cooling in liquid nitrogen, and the frozen suspension of cells was lyophilized by freezedrying in vacuo at Ϫ10°C. In general this freeze-drying process required 12-14 h and res...

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