Purification and biochemical properties of a cytochrome bc complex from the aerobic hyperthermophilic archaeon Aeropyrum pernix

Isolation of a membrane bound c-type cytochrome from A. pernix

Spectroscopic properties of cytochromes in A. pernix

The redox difference spectrum of membranes showed α-band peaks with maxima at 554 and 610 nm (Figure 2a), derived from c- and a-type cytochromes, respectively. The isolated cytochrome c₅₅₃ in the reduced state showed an absorption peak at 553 nm (Figure 2b, dotted line). The pyridine ferro-hemochrome spectrum showed 2 α-band peaks with maxima at 551 and 557 nm, indicating the presence of heme C and heme B (Figure 2b, solid line) [18]. The redox spectrum of the cytochrome oa₃ oxidase showed α-band peaks with maxima at 555 and 610 nm (Figure 2c, dotted line) and the pyridine ferro-hemochrome spectrum did α-band peaks with maxima at 553 and 588 nm (Figure 2c, solid line), indicating the presence of heme O and heme A [18, 19]. To determine the heme species of the oxidase in more detail, total heme was extracted from the partially purified oxidase preparation and analyzed by mass spectrometry. We observed 3 peaks at molecular masses of 630.44, 888.94, and 920.98 (Figure 3). The molecular mass of 888.94 matches that of heme O_p1, which was identified in Sulfolobus and other species [20], while the molecular mass of 920.98 matches that of heme A_s. The molecular mass of 630.44 matches that of heme B, which is probably contamination from other cytochromes, because the peak height is lower than those of hemes O_p1 and A_s, and this oxidase does not contain b-type heme (Figure 2c). The difference spectrum of the CO-bound, reduced form minus the reduced form showed a peak and a trough at 595 nm and 611 nm, respectively, in the α region (Figure 2d) and those at 432 nm and 444 nm in the γ region (data not shown), indicating that CO was bound to an a-type heme (Figure 2d), and thus the oxidase was designated a cytochrome oa₃-type.

Polypeptide composition and enzyme activities

SDS-PAGE showed mainly 3 polypeptide bands for cytochrome c₅₅₃ with apparent molecular masses of 40, 30, and 25 kDa (Figure 4a, panel 1). The 25-kDa band was visualized with heme staining (Figure 4a, panel 2). We performed mass analysis for the 3 bands at 40, 30, and 25 kDa using a MALDI-TOF/MS spectrometer. The 40- and 30-kDa polypeptides could not be identified. The 25-kDa polypeptide, which was positive for heme staining, had a molecular mass of 21,344 (Figure 5). The theoretical mass of the APE_1719.1 gene, which encodes the hypothetical cytochrome c subunit of the bc complex, was 20,813. The calculated mass of the APE_1719.1 gene product, which is the hypothetical cytochrome c polypeptide of the bc complex, is 21,429. On a BN-PAGE gel, cytochrome c₅₅₃ migrated at 80 kDa as a single band (Figure 4a, panel 3). The entire panel was excised and processed by two-dimensional SDS-PAGE. The 80-kDa band consisted of 3 main polypeptides as shown by SDS-PAGE (Figure 4a, panel 1 and panel 3) indicating that these 3 polypeptides form a complex. For partially purified cytochrome oa₃ oxidase, SDS-PAGE showed 3 polypeptide bands with apparent molecular masses of 74, 40, and 25 kDa (Figure 4b, panel 1). The 25-kDa band was visualized by heme staining, suggesting this band was derived from cytochrome c₅₅₃ (Figure 4b, panel 2). BN-PAGE showed a band at 140 kDa, which had TMPD oxidase activity, suggesting that the band contain a cytochrome c oxidase (Figure 4b, panel 3). The 140-kDa band was separated by SDS-PAGE and found to consist of 3 main polypeptides as shown by SDS-PAGE (Figure 4b, panel 1 and panel 3).

The isolated cytochrome oa₃ oxidase had TMPD and yeast cytochrome c oxidation activity, with values of 132 and 0.68 μmol min^-1 mg^-1, respectively, while the cytochrome c₅₅₃ complex did not show any oxidase activity. On the other hand, cytochrome c₅₅₃ oxidized menaquinol and reduced yeast cytochrome c (3.7 μmol min^-1 mg^-1), i.e. showed activity similar to that of quinone: cytochrome c oxidoreductase, while isolated cytochrome oa₃ did not oxidize menaquinol. Interestingly, after adding the fractions containing cytochrome c₅₅₃ to cytochrome oa₃ oxidase, TMPD oxidase activity increased ~ 5.0-fold (132 μmol min^-1 mg^-1 vs 664 μmol min^-1 mg^-1).

Bacterial strain and growth conditions

A. pernix K1 cells were kindly provided by Dr. Yosuke Koga, University of Occupational and Environmental Health, Japan. A. pernix was aerobically grown in 5 × T medium [2.8% (w/v) NaCl, 0.067% (w/v) KCl, 0.55% (w/v) MgCl₂·6H₂O, 0.69% (w/v) MgSO₄·7H₂O, 0.15% (w/v) CaCl₂, 0.1% (w/v) Na₂O₃S·5H₂O, 0.5% (w/v) Trypticase Peptone, 0.1% (w/v) Yeast Extract, pH 7.0] at 90°C. The preculture was carried out for 48 h in a Sakaguchi-flask containing 50-ml of medium, and a 50-ml aliquot was inoculated into a 1-L culture in a 3-L baffled flask. Cultures were incubated for about 48 h with vigorous shaking (150 rpm) until they attained the early stationary phase of growth. The cells were collected by centrifugation at 5,000 × g for 20 min.

Membrane preparation

The cells were washed twice with 20 mM NaP_i buffer at pH 7.0 and re-suspended in the same buffer. The cells were disrupted by sonication with an Ultrasonic Disrupter UD-201 (TOMY, Tokyo) using a 50% duty cycle at output 3 for 20 sec 3 times. The broken cells were precipitated by centrifugation at 16,000 × g for 20 min at 4°C. The precipitate was resuspended in 10 mM Tris-HCl buffer at pH 8.0, which contained a final concentration of 10 mM MgCl₂ and 10 μg ml^-1 DNase, and incubated at 37°C for 30 min. To remove unbroken cells, the suspension was centrifuged at 1,000 × g for 5 min at 4°C. The supernatant was then centrifuged at 100,000 × g for 20 min at 4°C. The precipitate was resuspended in 20 mM NaP_i at pH 7.0; this suspension was designated as the membrane fraction.

Solubilization and separation of cytochromes

The membranes were suspended in buffer containing 1 M LiCl and 20 mM NaP_i at pH 7.0, and then collected by centrifugation. The membrane proteins were solubilized at 10 mg protein ml^-1 in 1% (w/v) n-dodecyl-β-D-maltoside (DDM) in the presence of 0.3 M NaCl, 20 mM NaP_i at pH 7.0, and several protease inhibitors [1 mM ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5 mM benzamidine at final concentrations]. The mixture was centrifuged at 100,000 × g for 30 min, and the supernatant was dialyzed against 10 mM Tris-HCl at pH 7.0.

Cytochromes were separated into 2 components using 3 consecutive chromatography columns: DEAE-Toyopearl, Q-Sepharose, and hydroxyapatite. In brief, the solubilized protein was applied to a DEAE-Toyopearl column after dialysis. The adsorbed proteins were eluted with 3 column volumes of buffer containing 0.1% DDM, 10 mM Tris-HCl at pH 7.0, and an increasing concentration of NaCl (stepwise gradient of 20, 50, 100, 200, 300, and 500 mM). The peak fractions were dialyzed against 10 mM Tris-HCl at pH 7.0 and were applied to a Q-Sepharose column. The proteins were eluted with 15 column volumes of buffer containing 0.1% DDM, 10 mM Tris-HCl at pH 7.0, and an increasing concentration of NaCl (linear gradient of 0-300 mM; Additional file 1). The peak fractions were applied to a hydroxyapatite column for separation. The proteins were eluted with 3 column volumes of buffer containing 0.1% DDM and an increasing concentration of NaP_i at pH7.0 (stepwise gradient of 20, 50, 100, 150, 200, 300, and 400 mM; Additional file 2).

Enzyme activities

Cytochrome oxidase activity was assayed at 60°C by measuring oxidation of a yeast cytochrome c (Sigma-Aldrich, St. Louis MO), which had been reduced with sodium dithionite, in a final volume 800 μL containing a suitable amount of enzyme, 20 mM NaP_i at pH 7.0, and 10 μM yeast cytochrome c. The oxidation of reduced cytochrome c was followed by measuring the decrease in absorbance at 549 nm, and activity was calculated using a millimolar absorption coefficient of 21.2 mM^-1 cm^-1 [24].

N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) oxidase activity was assayed by measuring the increase in absorbance at 562 nm using a mixture of 25 mM TMPD, 0.1 M NaCl, and 50 mM NaP_i at pH 6.5, and calculated using a millimolar absorption coefficient of 10.5 mM^-1 cm^-1. To avoid the auto-oxidation of TMPD, the assay was performed at 40°C.

Menaquinol oxidase activity was assayed at 40°C by measuring the oxidation rate of menaquinol-1, which had been reduced with sodium dithionite, in a final volume of 700 μL containing a suitable amount of enzyme, 20 mM NaP_i at pH 7.0, 0.1% (w/v) DDM, 1 mM EDTA, and 0.2 mM menaquinol-1. The oxidation of reduced menaquinone was followed by measuring the increase in absorbance at 270.7 nm, and the activity was calculated using a millimolar absorption coefficient of 8.13 mM^-1 cm^-1.

Electrophoretic analyses

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was performed according to the method of Schägger et al. [25]. Nondenaturating electrophoresis was started at 100 V until the sample was within the stacking gel and continued with the voltage and current limited to 350 V and 15 mA, respectively. For two-dimensional analysis, a slice of the BN-PAGE gel was excised and soaked in 1% sodium dodecyl sulfate (SDS) and 1% mercaptoethanol buffer for 1 h and then embedded in a separating gel containing 15% acrylamide. Two-dimensional analysis was performed at room temperature with the current limited to 20 mA. SDS-PAGE was performed according to the method of Laemmli [26]. The gel was stained for protein with CBB and for heme with o-toluidine in the presence of H₂O₂. Gels were immersed in a solution containing 1% (w/v) o-tolidine, 80% (v/v) CH₃OH and 10% (v/v) CH₃COOH for 10 min, and then H₂O₂ was added at final concentration of 1% (v/v).

Mass analysis

Matrix-assisted laser desorption ionization, time-of-flight (MALDI-TOF) mass spectrometry of proteins was performed using 2- (4-hydroxyphenylazo) benzoic acid (HABA) as the matrix as described by Ghaim et al. [27]. The cytochromes extracted from the SDS-PAGE gel were precipitated with trichloroacetic acid (TCA) and were dissolved in 99% formic acid before mixing at a 1:5 ratio with a 50% acetonitrile solution containing 1.3 mg HABA ml^-1 and 0.1% trifluoroacetic acid. The mixture was spotted onto a sample plate and analyzed using a MALDI-TOF mass spectrometer.

For heme analysis, heme was extracted from partially purified cytochrome oa₃ oxidase with acetone containing 10% concentrated HCl as described previously [28]. After centrifugation, the heme in the supernatant was extracted with ethyl acetate. The heme-containing upper phase was removed, and the ethyl acetate was evaporated under a stream of nitrogen. Heme was dissolved in 30% acetonitrile and then mixed at a 1:1 ratio with a 50% acetonitrile solution containing 10 mg α-cyano-4-hydroxy cinnamic acid ml^-1 and 0.1% trifluoroacetic acid. The mixture was spotted onto a sample plate and analyzed using a MALDI-TOF mass spectrometer.

Additional analyses

Absorption spectra were measured with a recording spectrophotometer (Beckman DU70) at room temperature. Spectra of pyridine ferro-hemochromes were measured in the presence of 10% (v/v) pyridine, 0.05 N NaOH, and 1% (w/v) SDS. For membrane preparations, samples were mixed with 5% (w/v) Triton X-100 and centrifuged at 100,000 × g for 20 min at 4°C, as a common procedure to minimize turbidity. Protein concentration was determined using a modified Lowry method [29].