Abstract: The histidine-rich protein II (HRP II) from Plasmodium falciparum , an unusual protein composed of 40% alanine, 36% histidine, and 11% aspartate residues, has been implicated in the formation of hemozoin, a detoxified, crystalline form of ferriprotoporphyrin IX (heme) produced by the parasite. The structural basis of heme binding by HRP II and its potential relationship to hemozoin formation was studied through the use of various biochemical techniques. Heme titrations coupled with quantitative amino acid analysis showed that HRP II binds 15 heme molecules per 30 kDa monomer. Circular dichroism spectroscopy was used to probe the secondary structure of HRP II with and without bound heme and the structure changed from a random coil in the absence of heme to what most closely resembles a 3₁₀-helix upon heme binding. Coincident with this structural change, an intermolecular disulfide bond formed between HRP II monomers in the presence of heme. In vitro pulldown assays additionally revealed an interaction between monomers that was dependent upon the presence of heme. These results were incorporated into a potential model for heme binding by HRP II. The mode by which chloroquine inihibits HRP II-mediated heme polymerization was also investigated by examining the effect of chloroquine on the HRP II-heme complex through the use of resonance Raman, EPR, electronic absorption and circular dichroism spectroscopy. The results of these studies indicated that chloroquine perturbed the equilibrium between free heme and the HRP II-heme complex, creating a chloroquine-heme complex and HRP II, a conclusion in agreement with previous suggestions for the mode of action of chloroquine. Additionally, during these studies it became apparent that the expression of HRP II in E. coli produced a heterogeneous protein. Mass spectrometry revealed that the heterogeneity was due to the substitution of glutamine for some histidine residues in the repetitive HRP II sequence. The extent of the glutamine for histidine substitution was reduced 100-fold by slowing the expression rate. Mass spectral analysis of HRP II also revealed an unexpected α-amino methylation of the N-terminal alanine residue by E. coli.