Multi-frequency high-field epr study of iron centers in malarial pigments

Multi-Frequency High-Field EPR Study of Iron Centers in Malarial Pigments
Andrzej Sienkiewicz,§,† J. Krzystek,‡ Bertrand Vileno,† Guillaume Chatain,¶ Aaron J. Kosar,¶ D. Scott Bohle,*,¶ and La'szlo' Forro'† Institute of Physics, Polish Academy of Sciences, Al. Lotniko'w 32/46, 02-668 Warsaw, Poland, Institute of Physics of Complex Matter, EÄcole Polytechnique Fe'de'rale, CH-1015 Lausanne, Switzerland, National High Magnetic Field Laboratory, Florida State UniVersity, 1800 East Paul Dirac DriVe, Tallahassee, Florida 32310, Department of Chemistry, McGill UniVersity, 801 Sherbrooke Street West, Montreal, Quebec H3A 2K6, Canada Received January 13, 2006; E-mail: scott.bohle@mcgill.ca Malaria, whose most severe form is caused by a protozoan parasite, Plasmodium falciparum (Pf), remains the world's most
prevalent vector-borne disease. The spread of chloroquine-resistant
strains (CQR) of Pf and the absence of a suitable replacement for
this once effective antimalarial created an urgent need to understand
the biochemistry behind the drug's action.1 The intraerythrocytic
growth stage of Pf involves hemoglobin proteolysis as the primary
nutrient source with the concomitant release of free heme. The
liberated heme is detoxified by the parasite into an inert crystalline
material, called malarial pigment, or hemozoin.2,3 According to the
recent hypothesis, chloroquine inhibits heme aggregation in ring
Figure 1. The low-field (high geff) turning point in the EPR spectra of
or early-stage malaria trophozoites.4 It has been shown that hemozoin (blue) and -hematin (black) at 34 (top) and 94 GHz (bottom) at hemozoin is chemically,2,5 spectroscopically,2,3 and crystallographi- 10 K. The red traces are powder-pattern simulations using the following cally6 identical and isostructural to its synthetic phase, -hematin.
spin Hamiltonian parameters: |D| ) 5.80 cm-1, |E| ) 0.20 cm-1, g⊥ ) The magnetic susceptibility measurements and Mo¨ssbauer spec- 1.90 (top) and |D| ) 5.85 cm-1, |E| ) 0.075 cm-1, g⊥ ) 1.95 (bottom).
-hematin suggested the presence of a single high- 2, which is located 0.47 Å out of the plane of 2) iron environment of largely axial symmetry in its the porphyrin and forms a relative short bond, 1.889 Å, with one bulk phase.3,7 Recently, the crystal structure of of the oxygens of the protoporphyrin-IX propionic acid substit- solved by X-ray powder diffraction.8 The structure is surprising in uents.13 The distance between the two Fe ions within the dimer is that rather than being a coordination polymer, as widely held 9.05(1) Å, with a mean porphyrin plane separation of 4.44 Å. Due before,2,3 it is a chain of dimers formed by the FeIII-protoporphy- to their offsets, the nearest atoms in the porphyrin rings are separated rin-IX molecules through reciprocal iron-carboxylate bonds to by 5.00(1) Å, but the nearest iron neighbors are in adjacent unit one of the propionic side chains of each porphyrin. The dimers cells where the Fe-Fe separations are 7.86(1), 8.04(1), and then build chains linked by hydrogen bonds in the crystal.8 Despite the congruous nature of much of the spectroscopic data for the natural and synthetic phases, there is still considerableambiguity in interpretation of the Electron Paramagnetic Resonance(EPR) results concerning the characterization of their local Feenvironment.2,7,9-12 For example, previous work performed atconventional X-band (9.5 GHz) frequency suggested a rhombicsymmetry of the zero-field splitting (zfs) tensor, which was not inagreement with Mo¨ssbauer results.7 In this report, we present thedefinite conclusions of the spin state and properties of the groundstate of hemozoin and frequency high-field EPR (HFEPR) to simplify and fully interpret EPR spectra of hemozoin were acquired at cryogenic tempera- spectra of the ferric ion. At the same time, we will use the HFEPR tures on two Bruker Elexsys spectrometers equipped with TE011 data to find correlations between structure and magnetic properties resonators: Q-band (34 GHz) at the EPFL, and W-band (94 GHz) of both natural and synthetic malarial pigments.
-Hematin was investigated in a wide range of Malarial pigment (hemozoin) was isolated from a K-1 chloro- frequencies (27-500 GHz) in transmission-type single-pass spec- quine-resistant strain of Plasmodium falciparum. The pigment was trometers at the NHMFL.16 At each frequency, the isolated from late-stage trophozoites following nonproteolytic spectrum consists of a strong turning point at high effective geff ∼ methods.14 Homogeneous single-phase microcrystalline powders of 4.3-5.5, depending on frequency, and a weak turning point at geff -hematin were prepared by anhydrous dehydrohaloge- 2. At low frequencies (27-94 GHz), the high geff absorption nation of hemin.15 The structure of a building block of the malarial line is partly split into two components (Figure 1). In this frequency range, the spectra of hemozoin are nearly identical to those of 2 dimer, 1, has a five-coordinate
-hematin. At higher frequencies, the splitting disappears, and a single line is observed (Figure S1 in the Supporting Information).
† EÄcole Polytechnique Fe'de'rale de Lausanne.
‡ At high frequencies (above ca. 270 GHz), a new turning point is detected in the -hematin spectra (Figure S1), appearing at yet 4534 9 J. AM. CHEM. SOC. 2006, 128, 4534-4535
10.1021/ja058420h CCC: $33.50 2006 American Chemical Society
C O M M U N I C A T I O N S
troscopy as 7.5 cm-1.19 As for the small but measurable rhombicparameter E, its presence was detected both in Fe(DmePP-IX)-(N3)18 and in the above-mentioned complex with an axial acetategroup,19 and apparently depends on the symmetry of the liganditself. The propionate linker between the two porphyrins in -hematin may be conducive to producing such a small rhombic distortion. An explanation of the effect of averaging of the E valuewith increasing EPR frequency remains outside the scope of thepresent work, but we mention here the analogy with averaging the Figure 2. Resonance field versus quantum energy (or frequency) depen-
dipolar broadening of EPR resonances in solids with increasing dence of turning points in the powdered sample of -hematin. Experimental observation frequency in the intermediate exchange regime.20 This points are marked by squares. Below 100 GHz, the average position of the analogy seems to be particularly attractive since the averaged partly split low-field resonance was taken. Lines were simulated using best- perpendicular turning point at high frequencies actually gets fitted spin Hamiltonian parameters as in text. Blue lines, parallel turningpoints; red lines, perpendicular turning points. For clarity, only those narrower with increasing frequency and field in the solid, unlike transition branches are plotted that are actually observed experimentally.
the situation existing in a magnetically diluted system, such as The broken line corresponds to the frequency (378.2 GHz) at which the bottom trace in Figure S1 was recorded.
Acknowledgment. This work was partially supported by the
lower fields than the previously described high g Polish KBN Grant #2-PO3B-090-19, by the IHP-Contracts HPRI- new signal approaches zero field at ca. 350 GHz and starts CT-2001-00140, by G1MA-CI-2002-4017 (CEPHEUS) of the increasing in field again at higher frequencies. Figure 2 summarizes European Commission (A.S.), by the Burroughs-Wellcome Fund (P.W.S. and D.S.B.), by a CIHR Traineeship grant (C.G.), and by the Swiss National Science Foundation (B.V. and L.F.). We thank The HFEPR spectra obtained for hemozoin and -hematin can Dr. A. Ozarowski (NHMFL) for his simulation software, and Peter only be interpreted as originating from high-spin FeIII (S ) 5/ W. Stephens for his valuable assistance.
described by a nearly axial spin Hamiltonian of the standard form, Supporting Information Available: EPR spectra of -hematin in
comprising both Zeeman and zfs terms, H ) BgS +D[S 2 -
the high-frequency regime at T ) 10 K are shown in Figure S1 (1 page, print PDF). This material is available free of charge via the Sy ). An observation of the zero-field transition at 350 GHz, which, in the axial case, is equal to 2|D|, yields the value of |D| ) 5.83 cm-1. To our best knowledge, this is the first References
successful EPR detection of an inter-Kramers transition in a heme- like molecule. A complete set of intrinsic spin Hamiltonian parameters is delivered through a simultaneous fit to all the observed (2) Slater, A. F. G.; Swiggard, W. J.; Orton, B. R.; Flitter, W. D.; Goldberg, D. E.; Cerami, A.; Henderson, G. B. Proc. Natl. Acad. Sci. U.S.A. 1991,
resonances, as previously described:17 D ) +5.85(1) cm-1, E ) 0, g⊥ ) 1.95(1), g ) 2.00(1) (the actual positive sign of D is given (3) Bohle, D. S.; Conklin, B. J.; Cox, D.; Madsen, S. K.; Paulson, S.; Stephens, P. W.; Yee, G. T. In Inorganic and Organometallic Polymers II, AdVanced by simulations of single-frequency spectra shown in Figure S1).
Materials and Intermediates; Wisian-Neilson, P., Allcock, H. R., Wynne, The splitting in the perpendicular turning point of the intra-Kramers K. J., Eds.; American Chemical Society: Washington, DC, 1994; pp 497- transition at low frequencies indicates, however, that the zfs tensor (4) Dorn, A.; Stoffel, R.; Matile, H.; Bubendorf, A.; Ridley, R. G. Nature may not be entirely axial, as suggested by HFEPR. A fit to the 1995, 374, 269-271.
Q-band spectra, where the observed splitting is the largest, yields (5) Fitch, C. D.; Kanjananggulpan, P. J. Biol. Chem. 1987, 262, 15552-
the |E| value of 0.2 cm-1, that is, a rhombicity of the zfs tensor (6) Bohle, D. S.; Dinnebier, R. E.; Madsen, S. K.; Stephens, P. W. J. Biol. |E/D| equal to 0.035. This is in very good agreement with the Chem. 1997, 272, 713-716.
(7) Bohle, D. S.; Debrunner, P.; Jordan, P. A.; Madsen, S. K.; Schultz, C. E.
previous Mo¨ssbauer spectroscopy conclusions.7 The effect of the J. Am. Chem. Soc. 1998, 120, 8255-8256.
splitting decreasing and finally vanishing at higher frequencies is, (8) Pagola, S.; Stephens, P. W.; Bohle, D. S.; Kosar, A. D.; Madsen, S. K.
Nature 2000, 404, 307-310.
however, puzzling since simulations show that it should increase (9) Schoffa, G. Nature 1964, 203, 640-641.
rather than decrease. We see spin exchange as a tentative explana- (10) Arese, P.; Schwarzer, E. Ann. Trop. Med. Parasitol. 1997, 91, 501-516.
(11) Cammack, R.; Patil, D. S.; Linstead, D. J. Chem. Soc., Fraday Trans.
tion of this phenomenon (see below).
1994, 90, 3409-3410.
In general, however, our results show that magnetic exchange (12) Bremard, C.; Girerd, J. J.; Kowalewski, P.; Merlin, J. C.; Moreau, S. Appl. within each dimer is negligible, even weaker than suggested by Spectrosc. 1993, 47, 1837-1842.
(13) Bohle, D. S.; Kosar, A. D.; Stephens, P. W. Acta Crystallogr. 2002, D58,
previous susceptibility measurements.3 Otherwise, the ground spin state of the dimer would be zero or an integer number, and not, as (14) Ashong, J. O.; Blench, I. P.; Warhurst, D. C. Trans. R. Soc. Trop. Med. Hyg. 1989, 83, 167-172.
observed by us, S ) 5/2. Apparently, the significant distance between (15) Bohle, D. S.; Helms, J. B. Biochem. Biophys. Res. Commun. 1993, 193,
the two Fe centers and the number of chemical bonds between them (16) (a) Hassan, A. K.; Pardi, L. A.; Krzystek, J.; Sienkiewicz, A.; Goy, P.; make magnetic exchange very inefficient.
Rohrer, M.; Brunel, L.-C. J. Magn. Reson. 2000, 142, 300-312. (b)
The spin Hamiltonian parameters obtained for the first time with Zvyagin, S. A.; Krzystek, J.; van Loosdrecht, P. H. M.; Dhalenne, G.;
Revcolevschi, A. Physica B 2004, 346-347, 1-5.
high accuracy for a heme-like system are within the range observed (17) Krzystek, J.; Zvyagin, S. A.; Ozarowski, A.; Trofimenko, S.; Telser, J. J. in similar mononuclear Fe centers. It is known that the zfs parameter Magn. Reson. 2006, 178, 174-183.
(18) Brackett, G. C.; Richards, P. L.; Caughey, W. S. J. Chem. Phys. 1971,
D in five-coordinated Fe(III) complexes depends on the nature of the axial ligand. Thus, D as measured by us is very similar in value (19) Bominaar, E. L.; Ding, X. Q.; Gismelseed, A.; Bill, E.; Winkler, H.; to that determined for Fe in protoporphyrin-IX dimethyl ester with Trautwein, A. X.; Nasri, H.; Fischer, J.; Weiss, R. Inorg. Chem. 1992,
31, 1845-1854.
a fluoride axial ligand (5.0 cm-1) as measured by far-IR magnetic (20) Krzystek, J.; Sienkiewicz, A.; Pardi, L.; Brunel, L. C. J. Magn. Reson. spectroscopy.18 A more relevant comparison can be made with 1997, 125, 207-211.
(21) van Kan, P. J. M.; van der Horst, E.; Reijerse, E. J.; van Bentum, P. J.
another porphyrinic Fe(III) complex, with Fe axially ligated by an M.; Hagen, W. R. J. Chem. Soc., Faraday Trans. 1998, 94, 2975-2978.
acetate residue, whereas D was determined by Mo¨ssbauer spec- J. AM. CHEM. SOC. 9 VOL. 128, NO. 14, 2006 4535

Source: http://www.magnet.fsu.edu/library/publications/NHMFL_Publication-2936.pdf