Resolution of the Aerobic Respiratory System of the Thermoacidophilic Archaeon, Sulfolobus sp. Strain 7:статья из журнала
Аннотация: The aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, is unusual in that it consists of only a- and b-type cytochromes but no c-type cytochromes. In previous studies, a novel cytochrome oxidase a583-aa3 subcomplex has been purified, which showed a ferrocytochrome c oxidase but no caldariellaquinol oxidase activity (Wakagi, T., Yamauchi, T., Oshima, T., Müller, M., Azzi, A., and Sone, N.(1989) Biochem. Biophys. Res. Commun. 165, 1110-1114). We show here that the cytochrome subcomplex could be copurified with a non-CO-reactive cytochrome b562 as a novel terminal oxidase "supercomplex," which also contained a Rieske-type FeS cluster at gy = 1.89. It contained one copper and all four heme centers detectable in the archaeal membranes by the low temperature spectrophotometry and the potentiometric titration: cytochromes b562 (+146 mV), a583 (+270 mV), and aa3 (+117 and +325 mV). The presence of one copper atom indicates that it contains the conventional heme a3-Cub binuclear center for reducing molecular oxygen. In conjunction with the presence of a Rieske-type FeS center, inhibitor studies suggest that the terminal oxidase segment of the respiratory chain of Sulfolobus sp. strain 7 is a functional fusion of respiratory complexes III and IV, where cytochrome b562 and the Rieske-type FeS center probably play a central role in the oxidation of caldariellaquinol. This archaeal terminal oxidase supercomplex reconstitutes the in vitro succinate oxidase respiratory chain for the first time together with caldariellaquinone and the purified cognate succinate:caldariellaquinone oxidoreductase complex. The reconstitution system requires caldariellaquinone for the activity, and is highly sensitive to cyanide and 2-heptyl-4-hydroxy-quinoline-N-oxide. These results are also discussed in terms of the evolutionary considerations. The aerobic respiratory system of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, is unusual in that it consists of only a- and b-type cytochromes but no c-type cytochromes. In previous studies, a novel cytochrome oxidase a583-aa3 subcomplex has been purified, which showed a ferrocytochrome c oxidase but no caldariellaquinol oxidase activity (Wakagi, T., Yamauchi, T., Oshima, T., Müller, M., Azzi, A., and Sone, N.(1989) Biochem. Biophys. Res. Commun. 165, 1110-1114). We show here that the cytochrome subcomplex could be copurified with a non-CO-reactive cytochrome b562 as a novel terminal oxidase "supercomplex," which also contained a Rieske-type FeS cluster at gy = 1.89. It contained one copper and all four heme centers detectable in the archaeal membranes by the low temperature spectrophotometry and the potentiometric titration: cytochromes b562 (+146 mV), a583 (+270 mV), and aa3 (+117 and +325 mV). The presence of one copper atom indicates that it contains the conventional heme a3-Cub binuclear center for reducing molecular oxygen. In conjunction with the presence of a Rieske-type FeS center, inhibitor studies suggest that the terminal oxidase segment of the respiratory chain of Sulfolobus sp. strain 7 is a functional fusion of respiratory complexes III and IV, where cytochrome b562 and the Rieske-type FeS center probably play a central role in the oxidation of caldariellaquinol. This archaeal terminal oxidase supercomplex reconstitutes the in vitro succinate oxidase respiratory chain for the first time together with caldariellaquinone and the purified cognate succinate:caldariellaquinone oxidoreductase complex. The reconstitution system requires caldariellaquinone for the activity, and is highly sensitive to cyanide and 2-heptyl-4-hydroxy-quinoline-N-oxide. These results are also discussed in terms of the evolutionary considerations. INTRODUCTIONIt has been accepted that, regardless of the species, the major aerobic respiratory electron transport chain generally consists of one or more primary dehydrogenase (such as NADH and succinate dehydrogenases), a variety of quinone, and one or more respiratory terminal oxidase system(s)(1.Ludwig B. FEMS Microbiol. Rev. 1987; 46: 41-56Crossref Google Scholar, 2.Anraku Y. Annu. Rev. Biochem. 1988; 57 (1–132): 10Crossref Scopus (170) Google Scholar, 3.Poole K. Anthony C. Bacterial Energy Transduction. Academic Press, London1988: 231-291Google Scholar, 4.Sone N. Krulwich T.A. The Bacteria. XII. Academic Press, New York1990: 1-32Google Scholar, 5.Gennis R.B. Biochim. Biophys. Acta. 1991; 1058: 21-24Crossref PubMed Scopus (34) Google Scholar). Unlike the mitochondrial system, the organizations of the prokaryotic respiratory systems are far more complex and versatile to changes in the environmental conditions (for recent reviews, see (6.Saraste M. Q. Rev. 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Gennis R.B. J. Bacteriol. 1994; 176: 5587-5600Crossref PubMed Scopus (393) Google Scholar).For the aerobic archaea, several aa3-type respiratory heme-copper oxidases have been purified from two different species of Sulfolobales(19.An emüller S. Schäfer G. Eur. J. Biochem. 1990; 191: 297-305Crossref PubMed Scopus (80) Google Scholar, 20.Wakagi T. Yamauchi T. Oshima T. Müller M. Azzi A. Sone N. Biochem. Biophys. Res. Commun. 1989; 165: 1110-1114Crossref PubMed Scopus (21) Google Scholar, 21.Lübben M. Kolmerer B. Saraste M. EMBO J. 1992; 11: 805-812Crossref PubMed Scopus (116) Google Scholar, 22.Lübben M. Warne A. Albracht S.P.J. Saraste M. Mol. Microbiol. 1994; 13: 327-335Crossref PubMed Scopus (55) Google Scholar, 23.Lübben M. Arnaud S. Castresana J. Warne A. Albracht S.P.J. Saraste M. Eur. J. Biochem. 1994; 224: 151-159Crossref PubMed Scopus (84) Google Scholar), Desulfurolobus ambiva lens(24.Anemüller S. Schmidt C.L. Pacheco I. Schäfer G. Teixeira M. FEMS Microbiol. 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FEBS Lett. 1991; 292: 331-335Crossref Scopus (21) Google Scholar, 31.Fujiwara T. Fukumori Y. Yamanaka T. J. Biochem. (Tokyo). 1993; 113: 48-54Crossref PubMed Scopus (13) Google Scholar). On the basis of the absence of any c-type cytochrome, the insensitivity of a respiratory activity toward several complex III-specific respiratory inhibitors such as antimycin A(27.Anemüller S. Lübben M. Schäfer G. FEBS Lett. 1985; 193: 83-87Crossref Scopus (55) Google Scholar, 28.Wakagi T. Oshima T. Syst. Appl. Microbiol. 1986; 7: 295-304Crossref Scopus (31) Google Scholar, 32.von Jagow G. Link T.A. Methods Enzymol. 1986; 126: 253-271Crossref PubMed Scopus (312) Google Scholar), and a purification of a "single subunit" cytochrome aa3 with a quinol oxidase activity(19.An emüller S. Schäfer G. Eur. J. Biochem. 1990; 191: 297-305Crossref PubMed Scopus (80) Google Scholar, 33.Anemüller S. Schäfer G. FEBS Lett. 1989; 244: 451-455Crossref Scopus (38) Google Scholar), earlier studies have postulated that the archaeal aerobic respiratory chain might be very simple such that an aa3-type cytochrome oxidase functions as a terminal quinol oxidase(34.Schäfer G. Anemüller S. Moll R. Meyer W. Lübben M. FEMS Microbiol. Rev. 1990; 75: 335-348Crossref Google Scholar). Neither "a1-like" cytochrome (a586 in Sulfolobus acidocaldarius strain DSM 639 (27.Anemüller S. Lübben M. Schäfer G. FEBS Lett. 1985; 193: 83-87Crossref Scopus (55) Google Scholar, 30.Becker M. Schäfer G. FEBS Lett. 1991; 292: 331-335Crossref Scopus (21) Google Scholar) and a583 in Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius strain 7)( 1The organism had been isolated from Beppu hot springs, Japan, originally named as Sulfolobus acidocaldarius strain 7, but was recently re-designated tentatively as "Sulfolobus sp. strain 7" due to the small difference in 16 S rRNA base sequences of strain 7 and S. acidocaldarius type strain DSM 639. The preliminary 16 S rRNA sequence analysis suggests that the isolate is a novel species belonging to the genus Sulfolobus.) (28.Wakagi T. Oshima T. Syst. Appl. Microbiol. 1986; 7: 295-304Crossref Scopus (31) Google Scholar), respectively) predominant in the Sulfolobus membranes, nor non-CO-reactive b-type cytochrome were involved.Quite recently, the molecular genetic and biochemical evidence suggest that the archaeal respiratory terminal oxidase systems are in fact more complex than previously speculated. Lübben et al.(21.Lübben M. Kolmerer B. Saraste M. EMBO J. 1992; 11: 805-812Crossref PubMed Scopus (116) Google Scholar, 23.Lübben M. Arnaud S. Castresana J. Warne A. Albracht S.P.J. Saraste M. Eur. J. Biochem. 1994; 224: 151-159Crossref PubMed Scopus (84) Google Scholar) isolated one terminal oxidase operon (soxABCD) and other isolated genes encoding the fused subunit I + III of the alternative oxidase (soxM) of S. acidocaldarius strain DSM 639, and showed that the partially purified SoxABCD and SoxM oxidases contain the catalytic core subunits of mitochondrial respiratory complex III (SoxC) and complex IV (SoxAB and SoxM) by immunological cross-reactivities. In addition, it appears that they contain unusual hemes such as "heme AS," which has a hydroxyethylgeranylgeranyl side chain at position 2 of the iron porphyrin instead of the hydroxyethylfarnesyl group in heme A, beside a conventional protoheme IX(22.Lübben M. Warne A. Albracht S.P.J. Saraste M. Mol. Microbiol. 1994; 13: 327-335Crossref PubMed Scopus (55) Google Scholar, 35.Lübben M. Morand K. J. Biol. Chem. 1994; 269: 21473-21479Abstract Full Text PDF PubMed Google Scholar). These findings suggest that the archaeal respiratory and heme biosynthetic systems may represent unique evolutionary events. However, the Sulfolobus oxidase complex preparations (20.Wakagi T. Yamauchi T. Oshima T. Müller M. Azzi A. Sone N. Biochem. Biophys. Res. Commun. 1989; 165: 1110-1114Crossref PubMed Scopus (21) Google Scholar, 21.Lübben M. Kolmerer B. Saraste M. EMBO J. 1992; 11: 805-812Crossref PubMed Scopus (116) Google Scholar, 22.Lübben M. Warne A. Albracht S.P.J. Saraste M. Mol. Microbiol. 1994; 13: 327-335Crossref PubMed Scopus (55) Google Scholar, 23.Lübben M. Arnaud S. Castresana J. Warne A. Albracht S.P.J. Saraste M. Eur. J. Biochem. 1994; 224: 151-159Crossref PubMed Scopus (84) Google Scholar) have be en poorly characterized at the protein level, and leave some uncertainties for the functional assignment of the individual redox metal centers. In particular, we note that the reduced-minus-oxidized difference spectrum of the SoxABCD oxidase from S. acidocaldarius strain DSM 639 (22.Lübben M. Warne A. Albracht S.P.J. Saraste M. Mol. Microbiol. 1994; 13: 327-335Crossref PubMed Scopus (55) Google Scholar) is similar to that of a novel three-subunit cytochrome oxidase a583-aa3 subcomplex from Sulfolobus sp. strain 7, which did not show any caldariellaquinol but a ferrocytochrome c oxidase activity (20.Wakagi T. Yamauchi T. Oshima T. Müller M. Azzi A. Sone N. Biochem. Biophys. Res. Commun. 1989; 165: 1110-1114Crossref PubMed Scopus (21) Google Scholar, 36.Iwasaki T. Wakagi T. Isogai Y. Iizuka T. Oshima T. J. Biol. Chem. 1995; 270: 30893-30901Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). More recently, we found that the b-type cytochrome is in the upstream of the a-type cytochromes of the archaeal respiratory chain, and its partially-denatured form to be copurified with the cytochrome a583-aa3 subcomplex, which, however, did not retain any caldariellaquinol oxidase activity. ( 2T. Iwasaki, T. Wakagi, and T. Oshima, manuscript in preparation.) In addition, the complexity of the archaeal aerobic respiratory chain is further empathized by the recent findings that the Sulfolobus membranes in fact contain the respiratory Rieske-type FeS clusters(37.Anemüller S. Schmidt C.L. Schäfer G. Teixeira M. FEBS Lett. 1993; 318: 61-64Crossref PubMed Scopus (27) Google Scholar, 38.Iwasaki T. Isogai T. Iizuka T. Oshima T. J. Bacteriol. 1995; 177: 2576-2582Crossref PubMed Google Scholar).In a series of the present studies, we have carried out the purification, characterization(36.Iwasaki T. Wakagi T. Isogai Y. Iizuka T. Oshima T. J. Biol. Chem. 1995; 270: 30893-30901Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 39.Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), and reconstitution in vitro of the functionally active respiratory complexes of the chemoheterotrophically grown thermoacidophilic archaeon, Sulfolobus sp. strain 7, in order to reveal the overview of the archaeal aerobic respiratory system and to discuss them in terms of the unique evolutionary and phylogenetic status of thermoacidophilic archaea. In this paper, we show that the active respiratory terminal oxidase segment of Sulfolobus sp. strain 7 contains one non-CO-reactive b-type cytochrome (b562) and two different a-type cytochromes (a583 and aa3), in addition to one copper and a Rieske-type FeS cluster(38.Iwasaki T. Isogai T. Iizuka T. Oshima T. J. Bacteriol. 1995; 177: 2576-2582Crossref PubMed Google Scholar), which, as a whole, function as an active caldariellaquinol oxidase supercomplex. In addition, evidence is presented that cytochrome b562, which recently appeared to be in the upstream of and tightly associated with the a-type cytochromes,2 is probably functionally equivalent to cytochrome bII of the respiratory complex III (cytochrome bc1-b6f complex), while cytochrome a583(36.Iwasaki T. Wakagi T. Isogai Y. Iizuka T. Oshima T. J. Biol. Chem. 1995; 270: 30893-30901Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) may correspond to the c-type cytochrome of the c-aa3 or c-bb3 type heme-copper oxidase(8.García-Horsman J.A. Barquera B. Rumbley J. Ma J. Gennis R.B. J. Bacteriol. 1994; 176: 5587-5600Crossref PubMed Scopus (393) Google Scholar, 40.Berry E.A. Trumpower B.L. J. Biol. Chem. 1985; 260: 2458-2467Abstract Full Text PDF PubMed Google Scholar, 41.Sone N. Sekimachi M. Kutoh E. J. Biol. Chem. 1987; 262: 15386-15391Abstract Full Text PDF PubMed Google Scholar). Furthermore, the purified terminal oxidase supercomplex was reconstituted in vitro for the first time together with caldariellaquinone and the cognate respiratory complex II(39.Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar).EXPERIMENTAL PROCEDURESMaterialsAntimycin A, HOQNO, ( 3The abbreviations used are: HOQNO2-heptyl-4-hydroxy-quinoline-N-oxideMEGA-9nanoyl-N-methylglucamideMEGA-10decanoyl-N-methylglucamidePAGEpolyacrylamide gel electrophoresisSM-1200sucrose monolaurate.) and myxothiazol are from Sigma, and stigmatellin was purchased from Fluka. MEGA-9 and MEGA-10 were purchased from Dohjin (Kumamoto, Japan), sodium cholate from Sigma, sucrose monolaurate (SM-1200) from Mitsubishi Kasei Co. (Japan), and lauryl maltoside (n-dodecyl β-D-maltoside) from Boehringer Mannheim, respectively. Hydroxylapatite (Bio-Gel HTP) was from Bio-Rad. Heme A, protoheme, heme O, and heme AS were prepared by HCl/acetone extraction from bovine heart cytochrome aa3, hemoglobin (Sigma), Escherichia coli membranes(14.Puustinen A. Wikström M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6122-6126Crossref PubMed Scopus (144) Google Scholar), and Thermus thermophilus membranes(35.Lübben M. Morand K. J. Biol. Chem. 1994; 269: 21473-21479Abstract Full Text PDF PubMed Google Scholar, 42.Hon-nami K. Oshima T. Biochemistry. 1984; 23: 454-460Crossref Scopus (43) Google Scholar), respectively. Caldariellaquinone was isolated from the membrane of Sulfolobus sp. strain 7 as described previously by De Rosa et al.(43.De Rosa M. De Rosa S. Gambacorta A. Minale L. Thomson R.H. Worthington R.D. J. Chem. Soc. Perkin. Trans. 1977; 1: 653-657Crossref Scopus (75) Google Scholar). Horse heart cytochrome c was purchased from Sigma. Water was purified by the Milli-Q purification system (Millipore). Other chemicals mentioned in this study were of analytical grade.Organism, Cell Culture, and Membrane PreparationSulfolobus sp. strain 7 was cultivated aerobically and chemoheterotrophically at pH 2.5-3 and 75-80°C as described previously(28.Wakagi T. Oshima T. Syst. Appl. Microbiol. 1986; 7: 295-304Crossref Scopus (31) Google Scholar, 44.Wakao H. Wakagi T. Oshima T. J. Biochem. (Tokyo). 1987; 102: 255-262Crossref PubMed Scopus (48) Google Scholar), and was harvested in the early to middle exponential phase of growth and stored at −80°C until use. The cells were suspended in 200 ml of 100 mM Tris-Cl buffer, pH 7.5, containing 0.2 mM PMSF and 10 mM EDTA (∼4 ml/g of cells (wet weight)), and disrupted with a French press (Otake Works, Tokyo) at 1500 kg/cm2 twice. The membranes pelleted by ultracentrifugation with a Beckman 45Ti rotor at 130,000 × g for 120 min at 15°C were composed of two layers. The upper soft layer was carefully collected and resuspended in 50 mM Tris-Cl buffer, pH 7.5, containing 0.2 mM PMSF and 10 mM EDTA, then pelleted by ultracentrifugation at 130,000 × g for 90 min at 20°C. The membrane fraction was washed once more by resuspending in the same buffer and collected by ultracentrifugation; the pellet thus obtained was suspended in 15 mM Tris-Cl buffer, pH 7.3, containing 20% (v/v) glycerol and 0.2 mM PMSF at ∼20 mg of protein/ml, and used for the subsequent purification of the archaeal respiratory complexes (see below). Alternatively, the membranes were suspended in 40 mM potassium phosphate buffer, pH 6.8, containing 0.2 mM PMSF at ∼20 mg of protein/ml, then stored at −80°C until use. The pH value of 6.8 was preferably used in the present studies, because it is the optimal value for the succinate-dependent oxygen uptake and succinate:ubiquinone-1 oxidoreductase activities of the archaeal membrane fraction.Solubilization and Purification of the Sulfolobus Membrane-bound Cytochromes as a SupercomplexAll purification steps were carried out at 4°C, except for Step 2 at room temperature, and following the absorption at 280 and 423 nm of each fraction at different purification steps.Step 1: Cholate/MEGA-9 ExtractionTo the washed membrane suspension was added 50 mM potassium phosphate buffer, pH 6.8, containing 5% (w/v) sodium cholate, 1% (w/v) MEGA-9, 10% (v/v) glycerol, and 0.6 mM PMSF. The concentration of MEGA-9 to the amount of protein was critical for avoiding destruction of interactions among cytochromes to the minimal extent. The detergent/protein mixture was gently stirred for 12 h at 4°C, and was ultracentrifuged at 130,000 × g for 90 min at 4°C.Step 2: Hydroxylapatite Column ChromatographyThe solubilized material was directly applied to a hydroxylapatite column (2.0 × 31 cm) which had been equilibrated with 0.5% cholate, 0.1% MEGA-9, 10 mM succinate, 1 mM PMSF, and 10 mM potassium phosphate buffer, pH 6.8. The column was washed with 100 ml of the same detergent buffer, followed by 500 ml of the same buffer containing a linear gradient of 10-200 mM potassium phosphate, pH 6.8. It was then washed with 100 ml of 0.5% cholate, 0.1% MEGA-9, 10 mM succinate, 1 mM PMSF, and 200 mM potassium phosphate buffer, pH 6.8. Finally, the Sulfolobus cytochromes of both a- and b-types were concomitantly eluted with the same buffer containing 500 mM potassium phosphate.Step 3: Sucrose Density GradientThe pooled fractions after Step 2 were subsequently loaded on a sucrose density gradient (20-70%, w/v) containing 0.5% cholate, 0.6% MEGA-9, 25 mM potassium phosphate buffer, pH 6.8, and the tubes of a Beckman SW 28Ti rotor were spun at 28,000 rpm for 30 h at 4°C. The greenish band near the bottom of a tube (at ∼55-60% sucrose, w/v) was collected by a tubing, and was checked for purity by SDS-PAGE. When required, this step was repeated for a higher purity. The pooled fractions were combined and stored as the purified cytochrome supercomplex at −80°C until use.Analytical MethodsAbsorption spectra were recorded with either a Shimadzu MPS-2000 or UV-3000 spectrophotometer, or a Hitachi U-3210 spectrophotometer equipped with a thermoelectric cell holder. EPR measurements were carried out using a JEOL JEX-RE1X spectrometer equipped with an Air Products model LTR-3 Heli-Tran cryostat system, in which temperature was monitored with a Scientific Instruments series 5500 temperature indicator/controller.The oxidation and reduction potentials of the Sulfolobus membrane-bound cytochromes were measured in a Thunberg-type cell similar to that described by Dutton (45.Dutton P.L. Methods Enzymol. 1978; 54: 411-435Crossref PubMed Scopus (725) Google Scholar) under anaerobic conditions with continuous flow of N2 gas while st irring. The medium used was 60 mM potassium phosphate buffer, pH 6.8, with the following redox mediators: 40 μM 2,3,5,6-tetramethylphenylenediamine, 100 μM EDTA-Fe, 10 μM phenazine methosulfate, 3 μM pyocyanin, and 20 μM menadione. Ambient redox potentials (Eh) were monitored with a Pt-Ag/AgCl electrode (Type PS-165F, Toa Electronic Ltd., Tokyo, Japan). Desired potentials were attained by adding a small volume of ferricyanide or dithionite solution, and obtained absorption spectra were recorded with a Shimadzu UV-3000 spectrophotometer equipped with a Fujitsu FM 16β HD-II personal computer. All midpoint redox potentials stated in the text were calculated from titration curves using a fitting program (written at the Department of Biology, Tokyo Metropolitan University).Test for the presence of a cyanide-sensitive branched respiratory chain in Sulfolobus sp. strain 7 was carried out polarographically at 55°C, as a function of HOQNO and KCN concentrations, with a Clark-type oxygen electrode (Oxygen analyzer MP-1000, Iizima Products, Tokyo, Japan) equipped with temperature-controlled cells. The standard reaction mixture contained 480-1440 μg of membrane proteins/ml of 40 mM potassium phosphate buffer, pH 6.8, in a total volume of 2.1 ml, and the reaction was initiated by addition of 5 mM succinate.Presteady state reduction of the membrane-bound cytochromes b562 and a583 in the presence of 5 mM KCN was monitored at 562-552 and 583-573 nm, respectively, with a Shimadzu UV-3000 dual wavelength spectrophotometer at room temperature.A ferrocytochrome c oxidase activity was measured spectrophotometrically at 50°C with horse heart cytochrome c (Sigma) as an electron donor, as described previously (20.Wakagi T. Yamauchi T. Oshima T. Müller M. Azzi A. Sone N. Biochem. Biophys. Res. Commun. 1989; 165: 1110-1114Crossref PubMed Scopus (21) Google Scholar).The in vitro reconstitution experiments of the archaeal aerobic succinoxidase chain were carried out polarographically at 50°C with a Clark-type oxygen electrode (Oxygen analyzer MP-1000, Iizima Products, Tokyo, Japan) equipped with temperature-controlled cells. The standard reaction mixture contained 0.5% (w/v) cholate, 0.6% (w/v) MEGA-9, 20% (w/v) sucrose in 25 mM potassium phosphate buffer, pH 6.8, in a total volume of 2.1 ml; for the inhibitor studies, the reaction mixture also contained 5 mM succinate, 22.1-44.3 μg of the purified respiratory complex II (the preparations obtained after Step 5b were used because of a slightly superior purity(39.Iwasaki T. Wakagi T. Oshima T. J. Biol. Chem. 1995; 270: 30902-30908Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar)), 7 μM caldariellaquinone, and the purified cytochrome supercomplex. The concentration of caldariellaquinone in the mixture was kept low because of its very hydrophobic nature and limited availability.The succinate-driven reduction behavior of cytochromes in the terminal oxidase supercomplex in the in vitro reconstitution system was monitored spectrophotometrically at 20°C with a Beckman DU7400 spectrophotometer; the second-order finite derivative spectrum of the 5 mM succinate-reduced minus oxidized difference spectra of the terminal oxidase supercomplex was recorded every 30 s (the average scanning time, ∼1-2 s), in 25 mM potassium phosphate buffer, pH 6.8, containing the purified respiratory complex II, 7 μM caldariellaquinone, 0.6% (w/v) MEGA-9, and 0.5% (w/v) cholate.Protein was measured by the BCA assay (Pierce Chemical) with bovine serum albumin as a standard. Types of hemes were determined as described by Sone and Fujiwara(15.Sone N. Fujiwara Y. FEBS Lett. 1991; 288: 154-158Crossref PubMed Scopus (54) Google Scholar), using hemes A, As, and O, and protoheme IX as standards (14.Puustinen A. Wikström M. Proc. Natl.
Год издания: 1995
Авторы: Toshio Iwasaki, Katsumi Matsuura, Tairo Oshima
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Photosynthetic Processes and Mechanisms, Hemoglobin structure and function, Photoreceptor and optogenetics research
Другие ссылки: Journal of Biological Chemistry (PDF)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Journal of Biological Chemistry (HTML)
PubMed (HTML)
Открытый доступ: hybrid
Том: 270
Выпуск: 52
Страницы: 30881–30892