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.
The cells were washed twice with 20 mM NaPi 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 NaPi 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 NaPi 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 NaPi 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 NaPi at pH7.0 (stepwise gradient of 20, 50, 100, 150, 200, 300, and 400 mM; Additional file 2).
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 NaPi 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 .
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 NaPi 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 NaPi 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.
Blue-native polyacrylamide gel electrophoresis (BN-PAGE) was performed according to the method of Schägger et al. . 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 . 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).
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. . 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 . 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.
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 .