Materials and methods

Microorganisms, medium and conditions of cultivation

The bacterium used throughout this study was A. ferrooxidans ATCC 23270. The sulfur medium (pH 2.5) used in the cultivation of A. ferrooxidans ATCC 23270 contained elemental sulfur (1%), (NH4)2SO4 (3%) K2HPO4 (0.05%), MgSO4.7H2O (0.05%), KCl (0.01%) and Ca(NO3)2 (0.001%). Sulfur-grown ATCC 23270 cells were cultured in 300 ml of sulfur medium (pH 2.5) in 3000 ml Erlenmeyer flasks with shaking at 30 °C. Seven liters of ATCC 23270 cells grown in the sulfur medium for 3 days (exponential phase) was filtered with Toyo filter paper (no. 2) to remove the elemental sulfur particles, and centrifuged at 10,000 ×g to collect the cells.

Purification of rusticyanin, aa3-type cytochrome coxidase and cytochrome c from sulfur-grown ATCC23270 cells

The iron oxidation enzyme system from the sulfur-grown A. ferrooxidans cells, was directly extracted from the resting cells (325.0 mg protein) by using 30.0 ml of 1 M Na2SO4 in 0.1 M β-alanine-SO42-(pH 2) buffer containing2% 1-O-n-octyl-β-D-glucopyranoside(OGL). To the obtained cell extract (46.5 mg protein in 27 ml), solid ammonium sulfate was added slowly with continuous stirring to give 40 % of saturation. The resulting turbid solution was centrifuged at 10,000 ×g for 20 min. The reddish precipitate obtained contained cytochrome c. The supernatant (32.3 mg protein in 29.0 ml) with greenish color containing aa3-type cytochromec oxidase and rusticyanin was dialyzed 2 times against 10 mM β-alanine-SO42-buffer (pH 3), concentrated with polyethylene glycol 6000 and centrifuged at 10000 ×g for 20 min. The greenish precipitate was solubized with 5 ml of the same extraction buffer (pH 3) and then applied to Phenyl-650M column (2.0 × 10.0 cm).The columnwas eluted at 0.5 ml/min with a 1M-0M ammonium sulfate gradientin 10 mMβ-alanine-SO42-(pH 3) containing 0.2 % OGL. The fractions eluted by 1 M ammonium sulfate contained aa3-type cytochrome oxidase while those eluted by 0 M ammonium sulfate, contained rusticyanin. Both of the aa3-type cytochrome oxidase (5.2 mg protein in 5 ml) and rusticyanin (3.7 mg in 5ml) fractions were passed through Phenyl-650M column (Toyopearl, Tosoh) (2.0 × 10.0 cm) for two times. The aa3-type cytochromec oxidase and rusticyanin fractions from the previous step were concentrated and passed through SuperdexTM 75 column(Amersham Biosciences) (2.0 × 20.0 cm) using 1Mammonium sulfate in 10 mMβ-alanine-SO42-(pH 3) containing 0.2 % OGL as eluting solution to give finally about 0.9 mg protein of aa3-type cytochrome oxidase and about 0.7 mg protein of rusticyanin.

For purification of cytochrome c, the precipitate obtained after 40% ammonium sulfate fractionation containing cytochrome c was resolubilized with the 10 ml of the same extraction buffer with 2% OGL without ammonium sulfate (pH 3) and passed through Phenyl-650M column (Toyopearl, Tosoh) (2.0 × 10.0 cm) for three times.The columnwas eluted at 0.5 ml/min with a 1M-0M ammonium sulfate gradientin 10 mMβ-alanine-SO42-(pH 3) containing 0.2 % Octyl. The cytochrome c fractions were eluted by 0 M ammonium sulfate then concentrated (4.8 mg protein in 2 ml) and passed through SuperdexTM 75 column (Amersham biosciences) (2.0 × 20.0 cm) using 1.0 Mammonium sulfate in10 mMβ-alanine-SO42-(pH 3) containing 0.2 % OGL for elution to give finally about 1.2 mg protein of cytochrome c.

Ferrous iron oxidation activity

The activity was determined by measuring the Fe2+oxidized in the reaction mixture under aerobic conditions. The reaction mixture was composed of 0.1 M β-alanine-SO42- buffer (pH 3.0), the protein(s), and ferrous sulfate (4 μmol). The total volume was 3.0 ml. The reaction was carried out by shaking the reaction mixture at 30 °C. A sample from the reaction mixture (0.2 ml) was withdrawn and centrifuged at 12000 ×g for 2 min, and the concentration of Fe2+ in 0.1 ml of the supernatant was determined spectrophotometrically by the o-phenanthroline method (Saywell and Cunningham 1937).

Protein measurement

Protein concentration was measured by the method of Lowry (Lowry et al. 1951) with crystalline bovine serum albumin as the reference protein.

Spectrum measurement

The spectra of cytochrome c, aa3-type cytochrome oxidase and rusticyanin were measured with Shimadzu spectrophotometer UV-1700. Reduced forms of cytochromes and rusticyanin were prepared by adding small amounts of sodium hydrosulfite.

Electrophoresis

Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) was done in 12.5% polyacrylamide gel by the method of Laemmli (Laemmli 1970). The concentrated protein sample was incubated with the same volume of solution containing SDS (10%), 2-mercaptoethanol (16.7%), glycerol (16.7%) and Tris-HCl buffer (pH 6.8) (83.3 mM) at 30°C for 3 h (to ensure complete solubilization and denaturation of the proteins before loading) before it was loaded on SDS-PAGE. Lysozyme (14,3000 Mr), soybean trypsin inhibitor A (20,1000 Mr), carbonic anhydrase II (29,000 Mr), ovalbumin (45,000 Mr ), bovine serum albumin (66,400 Mr) and phosphorylase b (97,200 Mr) were used as protein size markers. SDS-PAGE was done on 12.5% polyacrylamide gel for about 2 h at the current of 20 mA. After electrophoresis, the gel was stained with 0.1% coomassie blue G-250 in 50% methanol for 2 h and then destained by 10% acetic acid in 50% methanol.

Amino acid sequencing

After electrophoresis, the protein bands on the SDS-PAGE gel weretransferred to polyvinylidene difluoride membrane (PVDF) following the technique described by Ausubel et al. (1992). The intended bands were excised, dried at room temperature and kept at -20 °C until sequencing with Applied Biosystems 491 protein sequencer.

References

1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith and K. Struhl. 1992. Current Protocols in Molecular Biology.Greene Publishing. New York, N.Y.

2. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.

3. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

4. Saywell, L. G. and B. B. Cunningham.1937. Determination of Iron: Colorimetric o-Phenanthroline Method.Ind.Eng. Chem. Anal. 9:67-69.

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