4480 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Znorg. Ckem. zyxwvut 1993, 32, 4480-4482 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK Reaction of Hydrogen Sulfide with Native Horse Spleen Ferritin T. C. St. Pierre, W. Chua-anusorn, P. Sips,' I. Krob2 and J. Webb' zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA School of Mathematical and Physical Sciences, Murdoch University, Perth, WA 6150, Australia Received February 3, I993 Introduction Ferritin is a protein that consists of 24 subunits that self- assemble to form a roughly spherical shell with external and internal diameters of approximately 12 and 8 nm, re~pectively.~ Hydrophilicand hydrophobicchannels connect the internal cavity with the exterior space. These channels are thought to facilitate the passage of ions into and out from the central cavity of the protein. Different forms of ferritin are found in all five living kingdoms and function variously as iron-storage, -detoxification, -regulation, and -transport protein^.^" Iron is usually bound in the form of an inorganicsolid phase of hydrated iron oxyhydroxide or phosphate within the central cavity of the m~lecule.~ The composition, size, and structure of the inorganic phase vary depending on the source and history of the ferritin. Recently it was proposed that the supramolecular protein structure of ferritin could be used as a reaction cage to engineer novel inorganic nanospace structures with structural and optical properties that may find applicationin catalysisand optoelectronic de~ices.~,~ Ferritin has already been used to synthesize discrete inorganic nanospace structures containing iron sulfides, man- ganese oxides, uranium oxides! and ferrimagnetic iron oxides.1° It was shown using transmission electron microscopy (TEM) and energy-dispersive X-ray analysis (EDXA) that iron and sulfur were present within ferritin cages after reaction of native ferritin (containingferrihydrite (5Fe203*9HzO)-like cores) with hydrogen sulfide gas.8 The aim of the present work is to use Mdssbauer spectroscopy (i) to determine the extent of the conversion of ferrihydrite to iron sulfide phases and (ii) to determine the nature of the iron sulfide phase(s) generated. Materials and Methods Native horsespleen ferritin solution was obtained fromSigma Chemical zyxwvuts Co. Synthetic ferrihydrite was prepared by adding potassium hydroxide solution to iron(II1) nitrate solution such that the final pH was between 7 and 8." The precipitate was dialyzed with distilled water for 2 weeks and was then freeze-dried. Hydrogen sulfide gas was prepared by reacting zyxwvut HCI with pyrite (FeS2). (1) On leave from Department of Inorganic and Analytical Chemistry, A. Jbsef University, H-6701 Szeged, P.O. Box 440, Hungary. (2) On leave from the Institute of Experimental Medicine, P. J. Safirik University, tr SNP 1, 040 66 Kdice, Czechoslovakia. (3) Ford, G. C.; Harrison, P. M.; Rice, D. W.; Smith, J. M. A.; Treffry, A.; White, J. L.; Yariv, J. Phil. Trans. R. SOC. London 1984, 8304, 551- 565. (4) Harrison, P. M.; Andrew, S. C.; Artymiuk, P. J.; Ford, G. C.; Guest, J. R.; Hirzmann, J.; Lawson, D. M.; Livingstone, J. C.; Smith, J. M. A.; Treffry, A.; Yewdall, S. J. Adu. Inorg. Chem. 1991, 36, 449486. (5) Thiel, E. C. Annu. Rev. Biochem. 1987, 56, 289-315. (6) Webb, J.; St. Pierre, T. G.; Macey, D. J. In Iron Biominerals; Frankel, R. B., Blakemore, R. P., Eds.; Plenum Press: New York, 1990 pp 193- 220. (7) St. Pierre, T. G.; Webb, J.; Mann, S. In Biomineralization: chemical and biochemical perspectives; Mann, S., Webb, J., Williams, R. J. P., Eds.; VCH: Weinheim, Germany, 1989; pp 295-344. (8) Meldrum, F. zyxwvutsrqpo C.; Wade, V. J.; Nimmo, D. L.; Heywd, B. R.; Mann, S. Nature 1991, 349, 684-687. (9) Mann, S.; Meldrum, F. C. Adv. Mater. 1991, 3, 316-318. (IO) Meldrum F. C.; Heywood, B. R.; Mann, S. Science 1992,257,522-523. (1 1) Schwertmann, U.; Cornell, R. M. Iron Oxides in the Laboratory; VCH: Weinheim, Germany, 1991; pp 90-94. 0020-166919311332-4480$04.00/0 The ferritin solution (500 pL) was buffered with 100 pL of 0.1 M Trizma base (Sigma) at pH 8.5. A suspension of the synthetic ferrihydrite was prepared by adding 200 mg of synthetic ferrihydrite to 5 mL of 0.1 M Trizma base at pH 8.5. The ferritin solution and the ferrihydrite suspension were then deaerated by bubbling nitrogen gas through for 15 min. Hydrogen sulfide gas was then bubbled through for 90 s. Both the ferritin solution and the ferrihydrite suspension turned black within 20 s, as has been reported previously.8 This procedure was carried out on nine samples of horse spleen ferritin from two different stock solutions with similar results each time. One sample (0.6 mL) each of the black ferritin solution and the ferrihydrite reaction products were then immediately transferred to 19 mm diameter nylon sample holders and frozen in liquid nitrogen ready for Mbssbauer s p e c t r m p i c measurements. Mbsbauer spectra were recorded and analyzed using a-Fe as a calibration reference as described elsewhere.12 ReSults Reaction with Ferritin. The reaction of hydrogen sulfide gas with clear reddish brown horse spleen ferritin solution at pH 8.5 produced a black solution with no bulk precipitation. This suggested that most of the black reaction product was bound within ferritin molecules. However, after a few hours, a greenish tinge was noted, and eventually a cloudy white precipitate was also observed. Miissbauer spectra of the sample of reaction product that was immediately frozen are shown in Figures 1 and 2. Spectral parameters derived from fitting Lorentzian peaks to the data are shown in Table I. Figure 1 shows the Miissbauer spectra recorded over the velocity range -13 to +13 mm/s with the sample at temperatures between 13.5 and 78 K. With a sample temperature of 78 K, the spectrum appears to consist of two doublets. As the temperature is lowered, a sextet of broad peaks due to magnetic hyperfine field (&f) splitting appears in the wingsof thespectrum. At 40 K, this component has Bhf = 45.4 zyx i 0.5 T and continues to grow in intensity at the expenseof doublet 1 as the temperature is lowered. By 13.5 K, &f has increased to 48.2 f 0.5 T. Between 40 and 20 K, a second sextet of broad peaks appears with a lower value of &f (Table I). The appearance of sextet 2 also appears to be at the expense of doublet 1, since the relative intensity of doublet 2 appears to be invariant with temperature (Table I). The appearance of two discrete magnetic hyperfine field components from doublet 1 implies that doublet 1 has a more detailed structure. Indeed, at least four doublets were needed to fit the spectrum of the sample at 78 K when recorded over the range -4 to +4 mm/s (Figure 2, Table I). Reaction with Ferrihydrite. The reaction of hydrogen sulfide gas with the cloudy brown ferrihydrite suspension at pH 8.5 produced a cloudy black suspension (in contrast to the reaction with ferritin solution). A greenish tinge and cloudy white precipitate were also seen after a few hours. Mbssbauer spectra of the sample of reaction product that was immediately frozen are shown in Figure 3. Spectral parameters derived from fitting Lorentzian peaks to the data are shown in Table I. The spectra at 78 and 13.5 K are qualititatively similar to those of the horse spleen ferritin/hydrogen sulfide reaction product. The main quantitative difference between the two sets of spectra is the approximate 50% reduction in spectral contri- bution from sextet 2 and doublet 2 (Table I). The apparent disappearance of doublet 2 at 13.5 K is probably due to the very small signal being obscured by sextets 1 and 2. Discussion The results shown here indicate that the reactions of hydrogen sulfide gas with (i) native horse spleen ferritin solution and (ii) a suspension of ferrihydrite particles at pH 8.5 generate similar (12) St.Pierre,T.G.;Richardson, D.R.;Baker,E.; Webb,J.Biochim.Eiophys. Acta 1992, 1135, 154-158. 0 1993 American Chemical Society