Helder M. Marques

Fate of haem iron in the malaria parasite Plasmodium falciparum

Chemical analysis has shown that Plasmodium falciparum trophozoites contain 61+/-2% of the iron within parasitized erythrocytes, of which 92+/-6% is located within the food vacuole. Of this, 88+/-9% is in the form of haemozoin. (57)Fe-Mössbauer spectroscopy shows that haemozoin is the only detectable iron species in trophozoites. Electron spectroscopic imaging confirms this conclusion.

Hemes and hemoproteins. 1: Preparation and analysis of the heme-containing octapeptide (microperoxidase-8) and identification of the monomeric form in aqueous solution.

The heme-octapeptide from cytochrome c, Microperoxidase-8 (MP-8), was prepared by peptic and tryptic digestion of horse heart cytochrome c and purified by gel permeation chromatography in about 50% yield. Conditions for the identification of MP-8 by TLC and analysis by HPLC are described. Study of the concentration-dependence of the absorption spectrum showed that at concentrations of less than or equal to 2.5 X 10(-5) M in aqueous solution at pH 7, 25 degrees C and mu = 0.1, MP-8 exists as an equilibrium mixture of monomers and dimers with KD = 1.17 +/- 0.02 X 10(5) M-1, decreasing to 1.21 +/- 0.02 X 10(4) M-1 and 2.16 +/- 0.21 X 10(3) M-1 in 20% and 50% (v/v) methanol:water mixtures, respectively. Comparison of the Soret region spectrum of monomeric MP-8 with other hemoproteins suggests that it is six-coordinate in aqueous solution with water and His as axial ligands.

Thermodynamic factors controlling the interaction of quinoline antimalarial drugs with ferriprotoporphyrin IX.

The interaction of a variety of quinoline antimalarial drugs as well as other quinoline derivatives with strictly monomeric ferriprotoporphyrin IX [Fe(III)PPIX] has been investigated in 40% aqueous DMSO solution. At an apparent pH of 7.5 and 25 degrees C, log K values for bonding are 5.52 +/- 0.03 (chloroquine), 5.39 +/- 0.04 (amodiaquine), 4.10 +/- 0.02 (quinine), 4.04 +/- 0.03 (9-epiquinine), and 3.90 +/- 0.08 (mefloquine). Primaquine, 8-hydroxyquinoline, 5-aminoquinoline, 6-aminoquinoline, 8-aminoquinoline, and quinoline exhibit no evidence of interaction with Fe(III)PPIX. The enthalpy and entropy changes for the interaction of quinolines with Fe(III)PPIX, as determined from the temperature dependence of the log K values, exhibit a compensation phenomenon that is suggestive of hydrophobic interaction. This is supported by the finding that the interactions of chloroquine and quinine with Fe(III)PPIX are weakened by increasing concentrations of acetonitrile. Interactions of chloroquine, quinine, and 9-epiquinine with Fe(III)PPIX are shown to remain strong at pH 5.6, the approximate pH of the food vacuole of the malaria parasite which is believed to be the locus of drug activity. Implications for the design of antimalarial drugs are briefly discussed.

Hemes and Hemoproteins. 5: Kinetics of the Peroxidatic Activity of Microperoxidase-8: Model for the Peroxidase Enzymes

The peroxidatic activity of the heme octapeptide from cytochrome c, microperoxidase-8 (MP-8), was assayed at 25 degrees C under conditions where formation of Compound I is rate limiting. In the pH range 6-9, the reaction rate increased linearly with a slope close to unity. The active form of the substrate is the hydroperoxide anion, HO2-, and an extrapolated second-order rate constant was obtained for the reaction of aquoMP-8 with HO2- of 3.7 X 10(8) M-1 sec-1, which is close to the second-order rate constants reported for reaction of the peroxidase enzymes with H2O2. Comparison with published data shows that the Fe3+ ion of MP-8 reacts as expected with simple anions, electrons, and HO2-, while the analogous reactions of the enzymes all show a requirement for one H+. We conclude that the peroxidase enzymes activate H2O2 under physiological conditions through a pH-independent, H+-coupled binding of the required H2O2-. The peroxidase activity of MP-8 can be increased more than tenfold by the presence of the guanidinium ion, which is ascribed to formation of the ion-pair GuaH+HO2-; this suggests a role for the invariant distal Arg in the enzymes.

The role of haem in the activity of chloroquine and related antimalarial drugs

Abstract Advances made over the last decade indicate that the mechanism of action of important antimalarial agents, such as chloroquine, involves formation of π–π complexes between drugs and ferriprotoporphyrin IX. This process is believed to block the detoxification of host haemoglobin-derived haem in the food vacuole of the parasite. Detoxification of haem occurs via conversion to a coordination polymer involving the formation of an Fe(III)-carboxylate bond between the propionate group of one ferriprotoporphyrin IX molecule and the Fe(III) centre of the next. This compound is known as malaria pigment or haemozoin in vivo, but can also be prepared synthetically, in which case it is referred to as β-haematin. Literature relating to the structure and mechanism of formation of haemozoin/β-haematin, the mechanism of action of the drugs and thermodynamics and structures of ferriprotoporphyrin IX-drug complexes is reviewed.

The crystal structure of halofantrine–ferriprotoporphyrin IX and the mechanism of action of arylmethanol antimalarials.

The crystal structure of the complex formed between the antimalarial drug halofantrine and ferriprotoporphyrin IX (Fe(III)PPIX) has been determined by single crystal X-ray diffraction. The structure shows that halofantrine coordinates to the Fe(III) center through its alcohol functionality in addition to pi-stacking of the phenanthrene ring over the porphyrin. The length of the Fe(III)-O bond is consistent with an alkoxide and not an alcohol coordinating group. The iron porphyrin is five coordinate and monomeric. Changes in the electronic spectrum of Fe(III)PPIX upon addition of halofantrine base in acetonitrile solution are almost identical to those observed upon addition of quinidine free base in the same solvent. This suggests homologous binding. Molecular mechanics modeling of Fe(III)PPIX complexes of quinidine, quinine, 9-epiquinine and 9-epiquinidine based on this homology suggests that the antimalarially active quinidine and quinine can readily adopt conformations that permit formation of an intramolecular salt bridge between the protonated quinuclidine tertiary amino group and unprotonated heme propionate group, while the inactive epimers 9-epiquinidine and 9-epiquinine have to adopt high energy conformations in order to accommodate such salt bridge formation. We propose that salt bridge formation may interrupt formation of the hemozoin precursor dimer formed during the heme detoxification pathway and so account for the strong activity of the two active isomers.

Molecular mechanics and molecular dynamics simulations of porphyrins, metalloporphyrins, heme proteins and cobalt corrinoids

Abstract Molecular mechanics (force field) methods have become popular as an adjunct to traditional methods for the examination of molecular structure. In this review, attention is focused on the use of molecular mechanics (force field) and molecular dynamics methods for the modeling of the structure of porphyrins, metalloporphyrins and hemoproteins, and the cobalt corrinoids (derivatives of vitamin B12).

Hemes and hemeproteins: 2:The pH-dependent equilibria of microperoxidase-8 and characterization of the coordination sphere of Fe(III)

Titration of the monomeric heme octapeptide from horse heart cytochrome c, microperoxidase-8 (MP-8) from pH 1 to pH 13 in 20% (v/v) methanol-water solutions, mu = 0.1, at 25 degrees C shows three reversible concentration-independent pKs (4.43 +/- 0.09; 8.90 +/- 0.03; 10.48 +/- 0.09) which are ascribed to successive proton loss from the conjugate acid of His (and its coordination to Fe(III)), bound H2O, and from bound His to form an imidazolate complex, respectively. The equilibrium constant for coordination of imidazole between pH 5.5 and 7.0 is independent of pH (logK = 4.45) which proves that His-18 is coordinated to Fe(III) in aqueous solution.

DFT-UX3LYP studies on the coordination chemistry of Ni2+. Part 1: Six coordinate [Ni(NH3)n(H2O)(6-n)]2+ complexes.

DFT calculations with the UX3LYP hybrid functional and a medium-sized 6-311++G(d,p) basis set were performed to examine the gas-phase structure of paramagnetic (S = 1) six-coordinate complexes [Ni(NH3)n(H2O)(6-n)](2+), 0 < or = n < or = 6. Significant interligand hydrogen bonding was found in [Ni(H2O)6](2+), but this becomes much less significant as NH3 replaces H2O in the coordination sphere of the metal. Bond angles and bond lengths obtained from these calculations compare reasonably well with available crystallographic data. The mean calculated Ni-O bond length in [Ni(H2O)6](2+) is 2.093 A, which is 0.038 A longer than the mean of the crystallographically observed values (2.056(22) A, 108 structures) but within 2sigma of the experimental values. The mean calculated Ni-N bond length in [Ni(NH3)6](2+) is 2.205(3) A, also longer (by 0.070 A) than the crystallographically observed mean (2.135(18) A, 7 structures). Valence bond angles are reproduced within 1 degree. The successive replacement of H2O by NH3 as ligands results in an increase in the stabilization energy by 7 +/- 2 kcal mol(-1) per additional NH3 ligand. The experimentally observed increase in the lability of H2O in Ni(II) as NH3 replaces H2O in the coordination sphere is explained by an increase in the Ni-OH2 bond length. It was found from a natural population analysis that complexes with the highest stabilization energies are associated with the greatest extent of ligand-to-metal charge transfer, and the transferred electron density is largely accommodated in the metal 4s and 3d orbitals. An examination of the charge density rho bcp and the Laplacian of the charge density nabla(2)rho(bcp) at the metal-ligand bond critical points (bcp) in the series show a linear correlation with the charge transferred to the metal. Values of nabla(2)rho(bcp) are positive, indicative of a predominantly closed-shell interaction. The charge transferred to the metal increases as n, the number of NH3 ligands in the complex, increases. This lowers the polarizing ability of the metal on the ligand donors and the average metal-ligand bond length increases, resulting in a direct correlation between the dissociation energy of the complexes and the reciprocal of the average metal-ligand bond length. There is a strong correlation between the charge transferred to the metal and experimental DeltaH values for successive replacement of H2O by NH3, but a correlation with stability constants (log beta values) breaks when n = 5 and 6, probably because of entropic effects in solution. Nevertheless, DFT calculations may be a useful way of estimating the stability constants of metal-ligand systems.

Hemepeptide models for hemoproteins: the behavior of N-acetylmicroperoxidase-11 in aqueous solution.

The acetylation of the hemeundecapeptide prepared by proteolysis of cytochrome c yields a species di(N-acetyl)-microperoxidase-11, NAcMP11, that is monomeric in aqueous solution at least for concentrations below 20 microM, in contrast to MP11 itself, which aggregates because of intermolecular coordination of Fe(III) by the N-terminal amino group or the amino group of the side chain of Lys-13. The present report complements a report by Peterson and co-workers on the preparation and properties of NAcMP11 (Inorg. Chem. 35 (1996) 6885). We show that NAcMP11 has six spectroscopically observable pH-dependent transitions at 1.90 +/- 0.03, 3.37 +/- 0.01, 4.6 +/- 0.1, 5.4 +/- 0.03, 9.56 +/- 0.01 and 12.4 +/- 0.03. The first is probably due to displacement of one of two H2O molecules from the coordination sphere of Fe(III) by the C-terminal Glu-21 carboxylate (giving the axial ligand combination RCOO-/H2O); as the pH is raised, His-18 is deprotonated and coordinates the metal (His/H2O). The next two transitions are due to ionization of heme propionic acid groups; the penultimate is caused by the ionization of Fe(III)-bound H2O (His/OH-); and the final transition is from ionization of His-18 to form a histidinate (His-/OH-). The EPR spectrum of NAcMP11 at pH 0.7 is consistent with a mixture of a di(aqua) and a mono(aqua) species. Both the aqua complex of NAcMP11 (at pH 7.6) and the hydroxo complex (at pH 11.0) are in equilibrium between a quantum-mechanically admixed spin state (S = 3/2, 5/2) and a low-spin state (S = 1/2). The crystal field parameters of the two complexes (which are similar) as derived from the EPR spectrum are reported. The EPR spectrum at pH 13.8 shows that the hydroxo-histidinate complex of NAcMP11 undergoes a slow reaction, possibly to form a di(hydroxo) complex with displacement of the histidinate ligand, or a dimerization with the histidinate acting as bridging ligand. The coordinated H2O molecule in NAcMP11 is readily replaced by an exogenous ligand, and binding constants for coordination of cyanide, imidazole, azide and chloride are reported. NAcMP-11 is shown to display similar physical and chemical properties to the analogous octapeptide, NAcMP-8, but is easier to prepare; this makes NAcMP-11 a useful alternative model for the hemoproteins.