Role of Tyr-288 at the Dioxygen Reduction Site of Cytochrome bo Studied by Stable Isotope Labeling and Resonance Raman Spectroscopy
2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês
10.1074/jbc.m409719200
ISSN1083-351X
AutoresTakeshi Uchida, Tatsushi Mogi, Hiro Nakamura, Teizo Kitagawa,
Tópico(s)Hemoglobin structure and function
ResumoTo explore the role of a cross-link between side chains of Tyr-288 and His-284 at the heme-copper binuclear center, we prepared cytochrome bo where d4-Tyr, 1-[13C]Tyr, or 4-[13C]Tyr has been biosynthetically incorporated. Unexpectedly, the d4-Tyr-labeled enzyme showed a large decrease in the ubiquinol-1 oxidase and CO binding activities. Optical absorption and resonance Raman spectra identified the defect in the distal side of the heme-copper binuclear center. In the CO-bound d4-Tyr-labeled enzyme, a large fraction of the ν(Fe-C) mode was shifted from the normal 520-cm-1 band to a broad band centered around 491 cm-1, as found for the Y288F mutant. Our results suggested that the substitution of ring hydrogens of Tyr-288 with deuteriums slows down the formation of the His-Tyr cross-link essential for dioxygen reduction at the binuclear center. To explore the role of a cross-link between side chains of Tyr-288 and His-284 at the heme-copper binuclear center, we prepared cytochrome bo where d4-Tyr, 1-[13C]Tyr, or 4-[13C]Tyr has been biosynthetically incorporated. Unexpectedly, the d4-Tyr-labeled enzyme showed a large decrease in the ubiquinol-1 oxidase and CO binding activities. Optical absorption and resonance Raman spectra identified the defect in the distal side of the heme-copper binuclear center. In the CO-bound d4-Tyr-labeled enzyme, a large fraction of the ν(Fe-C) mode was shifted from the normal 520-cm-1 band to a broad band centered around 491 cm-1, as found for the Y288F mutant. Our results suggested that the substitution of ring hydrogens of Tyr-288 with deuteriums slows down the formation of the His-Tyr cross-link essential for dioxygen reduction at the binuclear center. Cytochrome bo is one of two terminal ubiquinol oxidases in the aerobic respiratory chain of Escherichia coli (1Mogi T. Tsubaki M. Hori H. Miyoshi H. Nakamura H. Anraku Y. J. Biochem. Mol. Biol. Biophys. 1998; 2: 79-110Google Scholar, 2Ingledew W.J. Poole R.K. Microbiol. Rev. 1984; 48: 222-271Crossref PubMed Google Scholar). It catalyzes the two-electron oxidation of ubiquinol-8 and the four-electron reduction of dioxygen to water, which are coupled with proton pumping across the cytoplasmic membrane. The redox center of cytochrome bo consists of a bound ubiquinone-8 (QH) (3Sato-Watanabe M. Mogi T. Ogura T. Kitagawa T. Miyoshi H. Iwamura H. Anraku Y. J. Biol. Chem. 1994; 269: 28908-28912Abstract Full Text PDF PubMed Google Scholar, 4Sato-Watanabe M. Mogi T. Miyoshi H. Anraku Y. Biochemistry. 1998; 37: 5356-5361Crossref PubMed Scopus (38) Google Scholar, 5Mogi T. Sato-Watanabe M. Miyoshi H. Orii Y. FEBS Lett. 1999; 457: 61-64Crossref PubMed Scopus (17) Google Scholar), two heme groups, namely, heme b and heme o, and a copper ion (CuB) (1Mogi T. Tsubaki M. Hori H. Miyoshi H. Nakamura H. Anraku Y. J. Biochem. Mol. Biol. Biophys. 1998; 2: 79-110Google Scholar, 6Hosler J.P. Ferguson-Miller S. Calhoun M.W. Thomas J.W. Hill J. Lemieux L. Ma J. Georgiou C. Fetter J. Shapleigh J. Tecklenburg M.M.J. Babcock G.T. Gennis R.B. J. Bioenerg. Biomembr. 1993; 25: 121-136Crossref PubMed Scopus (242) Google Scholar, 7Abramson J. Riistama S. Larsson G. Jasaitis A. Svensson-Ek M. Laakkonen L. Puustinen A. Iwata S. Wikstrom M. Nature Struct. Biol. 2000; 7: 910-917Crossref PubMed Scopus (366) Google Scholar), which are all present in subunit I. In cytochrome c oxidase (CcO), 1The abbreviations used are: CcO, cytochrome c oxidase; HPLC, high-performance liquid chromatography; RR, resonance Raman; WT, wild type; P, peroxy; O, oxy. two heme a molecules (heme a and a3) and CuB are bound to subunit I, and a binuclear copper center (CuA) serves as the electron input site. Because they lead to understanding how the oxygen chemistry is coupled to the proton pumping, studies on the O2 reduction mechanism catalyzed by these oxidases have attracted considerable interests. Time-resolved spectroscopic studies have identified a number of intermediates in the reaction of the O2 reduction by bovine CcO (8Ferguson-Miller S. Babcock G.T. Chem. Rev. 1996; 96: 2889-2907Crossref PubMed Scopus (1062) Google Scholar, 9Kitagawa T. Ogura T. Progr. Inorg. Chem. 1997; 45: 431-479Google Scholar, 10Ogura T. Kitagawa T. Biochim. Biophys. Acta. 2004; 1655: 290-297Crossref PubMed Scopus (42) Google Scholar). The reaction begins with binding of O2 to the reduced heme a3. The resulting “oxy” (O) intermediate is rapidly transformed to the so-called “peroxy” (P) species, which has an oxidation state of the heme-copper moiety higher than the Fe3+/Cu2+ state by two oxidative equivalent. Although the peroxy intermediate had been expected to have an intact O-O bond (11Vygodina T.V. Konstantinov A.A. Ann. N. Y. Acad. Sci. 1988; 550: 124-138Crossref PubMed Scopus (78) Google Scholar, 12Wikström M. Morgan J.E. J. Biol. Chem. 1992; 267: 10266-10273Abstract Full Text PDF PubMed Google Scholar), our previous studies have clearly shown the definite Fe=O stretching Raman band at ∼800 cm-1 for the P intermediate, suggesting that the O-O bond is already cleaved at this step (13Proshlyakov D.A. Ogura T. Shinzawa-Itoh K. Yoshikawa S. Appelman E.H. Kitagawa T. J. Biol. Chem. 1994; 269: 29385-29388Abstract Full Text PDF PubMed Google Scholar, 14Proshlyakov D.A. Ogura T. Shinzawa-Itoh K. Yoshikawa S. Kitagawa T. Biochemistry. 1996; 35: 76-82Crossref PubMed Scopus (81) Google Scholar, 15Ogura T. Hirota S. Proshlyakov D.A. ShinzawaItoh K. Yoshikawa S. Kitagawa T. J. Am. Chem. Soc. 1996; 118: 5443-5449Crossref Scopus (85) Google Scholar, 16Hirota S. Mogi T. Ogura T. Hirano T. Anraku Y. Kitagawa T. FEBS Lett. 1994; 352: 67-70Crossref PubMed Scopus (35) Google Scholar). Other chemical experiments also support this assignment (17Fabian M. Wong W.W. Gennis R.B. Palmer G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13114-13117Crossref PubMed Scopus (108) Google Scholar, 18Fabian M. Palmer G. Biochemistry. 1999; 38: 6270-6275Crossref PubMed Scopus (31) Google Scholar). The subsequent one-electron reduction converts the P species to the next intermediate, “ferryl” (F) species with ν(Fe=O) at ∼785 cm-1 (15Ogura T. Hirota S. Proshlyakov D.A. ShinzawaItoh K. Yoshikawa S. Kitagawa T. J. Am. Chem. Soc. 1996; 118: 5443-5449Crossref Scopus (85) Google Scholar, 19Han S. Ching Y.C. Rousseau D.L. Nature. 1990; 348: 89-90Crossref PubMed Scopus (183) Google Scholar, 20Varotsis C. Babcock G.T. J. Am. Chem. Soc. 1995; 117: 11260-11269Crossref Scopus (31) Google Scholar). In cytochrome bo, two types of the F species were recently identified by time-resolved freeze-quench electron paramagnetic resonance spectroscopy (21Matsuura K. Yoshioka S. Takahashi S. Ishimori K. Mogi T. Hori H. Morishima I. Biochemistry. 2004; 43: 2288-2296Crossref PubMed Scopus (3) Google Scholar). The O-O bond scission in the O-to-P transition raised the question about the source of an additional oxidative equivalent. Cleavage of the iron-bound O-O bond requires four electrons. Three of them are provided by the binuclear center [FeII/CuI → FeIV/CuII], but the source of the fourth electron remains to be solved. Because of the formation of a porphyrin π-cation radical, oxidation of the heme macrocycle can provide one electron, as in horseradish peroxidase compound I. However, resonance Raman and optical absorption spectra eliminate this possibility (14Proshlyakov D.A. Ogura T. Shinzawa-Itoh K. Yoshikawa S. Kitagawa T. Biochemistry. 1996; 35: 76-82Crossref PubMed Scopus (81) Google Scholar, 22Watmough N.J. Cheesman M.R. Greenwood C. Thomson A.J. Biochem. J. 1994; 300: 469-475Crossref PubMed Scopus (53) Google Scholar, 23Fabian M. Palmer G. Biochemistry. 1995; 34: 13802-13810Crossref PubMed Scopus (132) Google Scholar). Another possibility is oxidation of an amino acid in the vicinity of the active center to form a so-called compound ES observed in cytochrome c peroxidase and prostaglandin H synthase (24Yonetani T. Schleyer H. J. Biol. Chem. 1966; 241: 3240-3243Abstract Full Text PDF PubMed Google Scholar, 25Wittenberg B.A. Kampa L. Wittenberg J.B. Blumberg W.E. Peisach J. J. Biol. Chem. 1968; 243: 1863-1870Abstract Full Text PDF PubMed Google Scholar, 26Hoffman B.M. Roberts J.E. Brown T.G. Kang C.H. Margoliash E. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 6132-6136Crossref PubMed Scopus (64) Google Scholar). Tyr-288 (if not stated otherwise, we adopt here the residue numbering of cytochrome bo) is highly conserved in the SoxM-type oxidases and is a potential candidate for such protein-based electron donors at the distal side of heme a3 (or o). Tyr-288 is also involved in the uptake of protons through the K channel during the initial reduction of the binuclear center. Crystallographic (27Ostermeier C. Harrenga A. Ermler U. Michel H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10547-10553Crossref PubMed Scopus (719) Google Scholar, 28Yoshikawa S. Shinzawa-Itoh K. Nakashima R. Yaono R. Yamashita E. Inoue N. Yao M. Fei M.J. Libeu C.P. Mizushima T. Yamaguchi H. Tomizaki T. Tsukihara T. Science. 1998; 280: 1723-1729Crossref PubMed Scopus (986) Google Scholar) and protein sequencing (29Buse G. Soulimane T. Dewor M. Meyer H.E. Bluggel M. Protein Sci. 1999; 8: 985-990Crossref PubMed Scopus (111) Google Scholar) studies on CcOs revealed the presence of a peculiar Cϵ-Nϵ covalent bond between Tyr-288 and His-284, one of three histidine ligands of CuB (Fig. 1). The OH group of Tyr-288 could form a hydrogen bond with a diatomic ligand like O2 (28Yoshikawa S. Shinzawa-Itoh K. Nakashima R. Yaono R. Yamashita E. Inoue N. Yao M. Fei M.J. Libeu C.P. Mizushima T. Yamaguchi H. Tomizaki T. Tsukihara T. Science. 1998; 280: 1723-1729Crossref PubMed Scopus (986) Google Scholar), favoring the cross-linking to His-284 to participate in O2 reduction (30Wang J. Takahashi S. Hosler J.P. Mitchell D.M. Ferguson-Miller S. Gennis R.B. Rousseau D.L. Biochemistry. 1995; 34: 9819-9825Crossref PubMed Scopus (61) Google Scholar, 31Sucheta A. Szundi I. Einarsdottir O. Biochemistry. 1998; 37: 17905-17914Crossref PubMed Scopus (87) Google Scholar, 32Gennis R.B. Biochim. Biophys. Acta. 1998; 1365: 241-248Crossref Scopus (192) Google Scholar, 33Proshlyakov D.A. Pressler M.A. Babcock G.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8020-8025Crossref PubMed Scopus (303) Google Scholar, 34Proshlyakov D.A. Pressler M.A. DeMaso C. Leykam J.F. DeWitt D.L. Babcock G.T. Science. 2000; 290: 1588-1591Crossref PubMed Scopus (278) Google Scholar, 35Blomberg M.R.A. Siegbahn P.E. Babcock G.T. Wikstrom M. J. Inorg. Biochem. 2000; 80: 261-269Crossref PubMed Scopus (81) Google Scholar). Studies with model compounds showed that the His-Tyr linkage lowers the pKa of the phenol moiety by 1.1 to 1.8 (36McCauley K.M. Vrtis J.M. Dupont J. van der Donk W.A. J. Am. Chem. Soc. 2000; 122: 2403-2404Crossref Scopus (97) Google Scholar, 37Aki M. Ogura T. Naruta Y. Le T.H. Sato T. Kitagawa T. J. Phys. Chem. A. 2002; 106: 3436-3444Crossref Scopus (43) Google Scholar, 38Cappuccio J.A. Ayala I. Elliott G.I. Szundi I. Lewis J. Konopelski J.P. Barry B.A. Einarsdottir O. J. Am. Chem. Soc. 2002; 124: 1750-1760Crossref PubMed Scopus (79) Google Scholar). Consequently, Tyr-288 could provide hydrogen to the iron-bound O-O, which leads to rapid cleavage of the bond with the concomitant formation of a tyrosine neutral radical. Electron paramagnetic resonance studies on the reaction of H2O2 with the oxidized enzyme revealed an electron paramagnetic resonance signal with partially resolved hyperfine structure ascribable to a tyrosine radical (17Fabian M. Wong W.W. Gennis R.B. Palmer G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13114-13117Crossref PubMed Scopus (108) Google Scholar, 39MacMillan F. Kannt A. Behr J. Prisner T. Michel H. Biochemistry. 1999; 38: 9179-9184Crossref PubMed Scopus (151) Google Scholar, 40Chen Y.R. Gunther M.R. Mason R.P. J. Biol. Chem. 1999; 274: 3308-3314Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Using radioactive iodide labeling followed by peptide mapping, Proshlyakov et al. (34Proshlyakov D.A. Pressler M.A. DeMaso C. Leykam J.F. DeWitt D.L. Babcock G.T. Science. 2000; 290: 1588-1591Crossref PubMed Scopus (278) Google Scholar) demonstrated the presence of the tyrosine neutral radical at the cross-linked His-284-Tyr-288 during P formation. In model studies, a C-O stretching vibration of the tyrosine radical was assigned to 1516 (38Cappuccio J.A. Ayala I. Elliott G.I. Szundi I. Lewis J. Konopelski J.P. Barry B.A. Einarsdottir O. J. Am. Chem. Soc. 2002; 124: 1750-1760Crossref PubMed Scopus (79) Google Scholar) or 1530 (37Aki M. Ogura T. Naruta Y. Le T.H. Sato T. Kitagawa T. J. Phys. Chem. A. 2002; 106: 3436-3444Crossref Scopus (43) Google Scholar) cm-1, which is higher than the 1489 cm-1 found in the reaction of H2O2 with the oxidized cytochrome bo (41Uchida T. Mogi T. Kitagawa T. Biochemistry. 2000; 39: 6669-6678Crossref PubMed Scopus (39) Google Scholar). Recent attenuated total reflection-Fourier transform infrared study on bovine CcO revealed strong negative bands at 1313 and 1547 cm-1 and a positive band at ∼1519 cm-1 in the PM intermediate (42Iwaki M. Puustinen A. Wikstrom M. Rich P.R. Biochemistry. 2003; 42: 8809-8817Crossref PubMed Scopus (56) Google Scholar). These results indicate that the cross-linked Tyr facilitates the cleavage of the O-O bond during P formation. To explore the role of the Tyr-His covalent bond, we prepared three stable isotope-labeled enzymes. We constructed a Tyr auxotrophic strain harboring an overexpression vector for cytochrome bo and grew it in a synthetic medium supplemented with l-d4-Tyr, l-1-[13C]Tyr, or l-4-[13C]Tyr as a sole l-Tyr source. Unexpectedly, the d4-Tyr-labeled enzyme showed a reduction in the ubiquinol-1 oxidase and CO binding activities. Optical absorption and resonance Raman (RR) spectra identified the defect in the distal side of the heme-copper binuclear center. Our results suggest that the substitution of ring hydrogens of Tyr-288 with deuteriums slows down the formation of the His-Tyr cross-link essential for dioxygen reduction at the binuclear center. A possible self-catalyzed mechanism for the formation of the His-Tyr bond is discussed. Materials—l-d4- and l-4-13C-labeled Tyr (98 and 99 atom%, respectively) were purchased from ICON Services Inc. (Summit, NJ), and l-1-[13C]Tyr (99 atom%) was from MassTrace Inc. (Woburn, MA). Bacterial Strains—E. coli strains GO103/pHN3795–1 (cyo+ Δcyd/cyo+), ST4533/pHN3795-H333A (Δcyo cyd+/cyo-) (43Mogi T. Hirano T. Nakamura H. Anraku Y. Orii Y. FEBS Lett. 1995; 370: 259-263Crossref PubMed Scopus (37) Google Scholar), and ST4676/pMFO9-Y288F (Δcyo cyd+/cyo-) (44Kawasaki M. Mogi T. Anraku Y. J. Biochem. 1997; 122: 422-429Crossref PubMed Scopus (25) Google Scholar) were used for isolation of the wild type cytochrome bo and H333A and Y288F mutant oxidases, respectively. The tyrosine auxotroph, GO103Y, was constructed as follows. The ΔtyrA16::Tn10 locus was transduced into GO103 by P1 phage grown on strain N3087 (CGSC 6662). A transductant was verified by Tetr and Tyr- phenotypes and transformed with pHN3795–1 to give GO103Y/pHN3795–1. Purification of Unlabeled Cytochrome bo—E. coli cells were grown in 1.5× minimal medium A (45Davis B.D. Mingioloi E.S. J. Bacteriol. 1959; 60: 17-28Crossref Google Scholar) supplemented with 1% Bacto tryptone, 0.5% Bacto casamino acid, 0.5% Bacto yeast extract, 1% glycerol, 100 μg/ml sodium ampicillin, 50 μg/ml l-Trp, 50 μg/ml FeSO4·7H2O, and 25 μg/ml CuSO4·4H2O (IM-metal-glycerol medium). Cytochrome bo was solubilized from isolated cytoplasmic membranes with sucrose monolaurate (Mitsubishi-Kagaku Foods Co., Tokyo) and purified by anion-exchange HPLC as described previously (46Tsubaki M. Mogi T. Anraku Y. Hori H. Biochemistry. 1993; 32: 6065-6072Crossref PubMed Scopus (83) Google Scholar). Purified enzymes were stored in 50 mm Tris-HCl (pH 7.4) containing 0.1% sucrose monolaurate at -80 °C until use. The concentration of the enzymes was determined by the pyridine ferrohemochromogen method using a millimolar extinction coefficient of 20.7 for heme B, assuming that the enzyme has two hemes. Reduced forms were prepared by adding a slight excess of freshly prepared anaerobic dithionite solution under argon atmosphere into the degassed enzyme solution. CO-bound forms were prepared by introducing gaseous CO into the reduced samples. Isotopic Labeling of Wild Type Enzyme—To incorporate specifically isotope-labeled Tyr into cytochrome bo, GO103Y/pHN3795–1 was grown in a modified minimal medium A supplemented with 20 amino acids. The 10-liter medium contained1gof isotope-labeled l-Tyr and 19 unlabeled l-amino acids (5.6 g of Ala, 6.6 g of Arg, 1.4 g of Asn, 12 g of Asp, 5.2 g of Cys-HCl, 2.4 g of Gln, 40 g of Glu, 3.4 g of Gly, 5.4 g of His, 10.0 g of Ile, 17.8 g of Leu, 18.4 g of Lys-HCl, 5.4 g of Met, 9.0 g of Phe, 20.6 g of Pro, 10.0 g of Ser, 7.4 g of Thr, 4.0 g of Trp, 12.4 g of Val) (47Kainosho M. Tsuji T. Biochemistry. 1982; 21: 6273-6279Crossref PubMed Scopus (173) Google Scholar), as well as 5.0 g of adenine, 2.0 g of cytosine, 6.5 g of guanosine, 2.0 g of thymine, 5.0 g of uracil, 15.0 g of sodium acetate, 15.0 g of sodium succinate, 104 g of K2HPO4,30gofKH2PO4, 7.5 g of sodium citrate, and 25.0 g of (NH4)2SO4. After autoclaving, the following solutions were added after filter sterilization: 400 ml of 50% glucose, 100 ml of vitamin mixture containing thiamine-HCl (0.50 g), niacin (0.50 g), biotin (0.01 g) (48Muchmore D.C. McIntosh L.P. Russell C.B. Anderson D.E. Dahlquist F.W. Methods Enzymol. 1989; 177: 44-73Crossref PubMed Scopus (478) Google Scholar), and sodium ampicillin (1.0 g), 10 ml of metal mixture A containing FeCl3·6H2O (0.25 g) and CaCl2·2H2O (0.04 g), and 100 ml of metal mixture B containing MgSO4·7H2O (10 g), CuSO4·5H2O (0.125 g), ZnSO4·7H2O (0.04 g), and MnSO4·5H2O (0.04 g). During cell growth, 10 N NaOH was added to maintain the pH between 7.2 and 7.5. Isotopically labeled enzymes were purified by anion-exchange HPLC (46Tsubaki M. Mogi T. Anraku Y. Hori H. Biochemistry. 1993; 32: 6065-6072Crossref PubMed Scopus (83) Google Scholar), and their yields were ∼0.4 μmol from the 10-liter culture. Resonance Raman Measurements—RR spectra were obtained with a single polychromator (DG-1000; Ritsu Oyo Kogaku) equipped with a liquid N2-cooled CCD detector (CCD-1340/400-EB; Princeton Instruments). The excitation wavelength used was 413.1 nm from a Kr ion laser (BeamLok 2060; Spectra Physics) for the air-oxidized, reduced, and CO-bound forms. The laser power at the sample point was adjusted to 1.2 mW for the reduced form and to 0.05–0.1 mW for the air-oxidized and CO-bound forms to prevent photoreduction and photodissociation, respectively. Raman shifts were calibrated with indene, CCl4, and aqueous solution of ferrocyanide, and the accuracy of the peak positions of the well defined Raman bands was ± 1 cm-1. All measurements were performed at room temperature with a spinning cell. The enzyme concentration for RR experiments was 50 μm heme in the 50 mm Tris-HCl (pH 7.4) with 0.1% sucrose monolaurate. To measure absorption and RR spectra of the fully oxidized Y288F, the air-oxidized form, where hemes are partly reduced (49Das T.K. Tomson F.L. Gennis R.B. Gordon M. Rousseau D.L. Biophys. J. 2001; 80: 2039-2045Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 50Pinakoulaki E. Pfitzner U. Ludwig B. Varotsis C. J. Biol. Chem. 2002; 277: 13563-13568Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), was treated with ferricyanide and passed through a Sephadex G-200 column to remove the excess ferricyanide. Metal Content Analysis—The purified oxidase solutions (0.2–1 mm) were diluted to 0.5–1 μm by Nanopure water (18.3 mΩ purity; Barnstead), and copper and iron contents were determined by inductively coupled plasma atomic emission spectroscopy at Shimadzu Analysis Center. Each analysis was complemented by appropriate control experiments. Miscellaneous—Optical absorption spectra of purified oxidases (∼5 μm heme) were recorded on a Hitachi UV-3200 UV/Vis spectrophotometer at room temperature. The samples were sealed inside a 1-cm path length quartz cuvette. Heme contents of the oxidases were analyzed by reverse-phase HPLC after extraction with acid acetone as described previously (51Saiki K. Mogi T. Anraku Y. Biochem. Biophys. Res. Commun. 1992; 189: 1491-1497Crossref PubMed Scopus (85) Google Scholar). Ubiquinol-1 oxidase activity was measured spectrophotometrically with a millimolar extinction coefficient of 13.25 at 275 nm at 0.5 mm ubiquinol-1 (3Sato-Watanabe M. Mogi T. Ogura T. Kitagawa T. Miyoshi H. Iwamura H. Anraku Y. J. Biol. Chem. 1994; 269: 28908-28912Abstract Full Text PDF PubMed Google Scholar). Effects of Isotope Labeling of Tyrosines on Quinol Oxidase Activity and Metal Contents—Three different isotope-labeled tyrosines were biosynthetically incorporated into the wild type cytochrome bo in the Tyr auxotroph: 1) l-1-[13C]Tyr with a 13C atom at the main chain carbonyl, 2) l-4-[13C]Tyr with a 13C atom bound to the phenolic oxygen, and 3) l-d4-Tyr where all ring hydrogens are deuterated (Fig. 1). In the enzymes, all tyrosines are isotopically labeled. Ubiquinol-1 oxidase activity and metal contents are summarized in Table I. Those of 1-[13C]Tyr and 4-[13C]Tyr-labeled wild type enzyme (1-[13C]Tyr-WT and 4-[13C]Tyr-WT, respectively) were comparable with those of unlabeled wild type enzyme (12C WT). The elimination of one of the three His ligands of CuB in the H333A mutant and removal of a phenolic hydroxyl group at position 288 in the Y288F and Y288L mutants resulted in a complete loss of oxidase activity (Table I) as reported previously (43Mogi T. Hirano T. Nakamura H. Anraku Y. Orii Y. FEBS Lett. 1995; 370: 259-263Crossref PubMed Scopus (37) Google Scholar, 44Kawasaki M. Mogi T. Anraku Y. J. Biochem. 1997; 122: 422-429Crossref PubMed Scopus (25) Google Scholar, 52Tsubaki M. Mogi T. Hori H. Hirota S. Ogura T. Kitagawa T. Anraku Y. J. Biol. Chem. 1994; 269: 30861-30868Abstract Full Text PDF PubMed Google Scholar, 53Thomas J.W. Calhoun M.W. Lemieux L.J. Puustinen A. Wikstrom M. Alben J.O. Gennis R.B. Biochemistry. 1994; 33: 13013-13021Crossref PubMed Scopus (40) Google Scholar, 54Mogi T. Mina J. Hirano T. Sato-Watanabe M. Tsubaki M. Uno T. Hori H. Nakamura H. Nishimura Y. Anraku Y. Biochemistry. 1998; 37: 1632-1639Crossref PubMed Scopus (18) Google Scholar). In Y288F, the loss of ubiquinol oxidase activity was accompanied by the replacement of heme o with heme b at the binuclear center and a partial loss of the CO binding activity (44Kawasaki M. Mogi T. Anraku Y. J. Biochem. 1997; 122: 422-429Crossref PubMed Scopus (25) Google Scholar) as reported for Y288L (54Mogi T. Mina J. Hirano T. Sato-Watanabe M. Tsubaki M. Uno T. Hori H. Nakamura H. Nishimura Y. Anraku Y. Biochemistry. 1998; 37: 1632-1639Crossref PubMed Scopus (18) Google Scholar) and heme bb-type wild type oxidase, which has been isolated from the heme O synthase mutant (55Saiki K. Mogi T. Hori H. Tsubaki M. Anraku Y. J. Biol. Chem. 1993; 268: 26927-26934Abstract Full Text PDF PubMed Google Scholar). The CO binding activity and the copper content of H333A were higher than those reported previously (52Tsubaki M. Mogi T. Hori H. Hirota S. Ogura T. Kitagawa T. Anraku Y. J. Biol. Chem. 1994; 269: 30861-30868Abstract Full Text PDF PubMed Google Scholar), probably because of a difference in expression vectors (i.e. a multicopy vector pHN3795-H333A versus a single copy vector pMFO1-H333A) (43Mogi T. Hirano T. Nakamura H. Anraku Y. Orii Y. FEBS Lett. 1995; 370: 259-263Crossref PubMed Scopus (37) Google Scholar).Table ICharacterization of the metal centers in the unlabeled and Tyr isotope-labeled wild types and H333A and Y288F mutants of cytochrome boOxidasesQ1H2 oxidase activityCO binding activityHeme b: heme o: CuBReference%%12C WT1001000.98: 1.02: 1.1This work1-[13C]Tyr-WT1011000.98: 1.05: 1.1This work4-[13C]Tyr-WT54940.90: 1.10: 1.2This workd4-Tyr-WT19631.05: 0.95: 0.93This workH333A0.1781.25: 0.75: 0.45This workH333ANRaNR, not reportedNR1.58: 0.42: 0.05(52Tsubaki M. Mogi T. Hori H. Hirota S. Ogura T. Kitagawa T. Anraku Y. J. Biol. Chem. 1994; 269: 30861-30868Abstract Full Text PDF PubMed Google Scholar)Y288F0.3541.83: 0.17: 0.55This workY288L0.3141.87: 0.13: 0.17(54Mogi T. Mina J. Hirano T. Sato-Watanabe M. Tsubaki M. Uno T. Hori H. Nakamura H. Nishimura Y. Anraku Y. Biochemistry. 1998; 37: 1632-1639Crossref PubMed Scopus (18) Google Scholar)WT (ΔE2)0.1>182.00: 0: 1.02(55Saiki K. Mogi T. Hori H. Tsubaki M. Anraku Y. J. Biol. Chem. 1993; 268: 26927-26934Abstract Full Text PDF PubMed Google Scholar)a NR, not reported Open table in a new tab Unexpectedly, the quinol oxidase and CO binding activities of d4-Tyr-WT were reduced to 19 and 63%, respectively, of those of 12C WT. Labeling of cytochrome bo with l-d4-Tyr appears to affect the structural environment at the heme o-CuB binuclear center. Because d4-Tyr-WT normally contains all three redox metal centers as in [12C]Tyr-WT, 1-[13C]Tyr-WT, and 4-[13C]Tyr-WT (Table I), effects of the d4-Tyr labeling on the oxidase activity must be different from those of the Y288F and H333A mutations. Accordingly, effects of the d4-Tyr labeling on the binuclear center were examined by optical and resonance Raman spectroscopy. Effects of Isotope Labeling of Tyrosines on Optical Absorption Spectra of Cytochrome bo—Optical absorption maxima of 1-[13C] Tyr-WT and 4-[13C]Tyr-WT in the air-oxidized, dithionite-reduced, and reduced CO-bound states are summarized in Table II. They are similar to those of 12C WT with the Soret peak at 409, 428, and 418 nm, respectively. The Soret maxima of the CO-bound form of d4-Tyr-WT was shifted to 426 nm from 418 nm of 12C WT (Table II) because of significant contribution from the unbound form (428 nm), which results from the low CO binding activity of heme o (Table I) as found for the heme bb-type Y288F mutant. However, a peak (i.e. heme o2+-CO) and trough (heme o2+) in the Soret region of the reduced CO-bound minus reduced difference spectrum of d4-Tyr-WT are identical to those of [12C]Tyr-WT, 1-[13C]Tyr-WT, 4-[13C]Tyr-WT, and H333A as shown in Fig. 2. In contrast, peaks at 415.5, 532, and 567 nm and troughs at 430 and 551 nm of 12C WT are shifted to 419.5, 543, and 577 nm and 431.5 and 561 nm, respectively, for the Y288F mutant.Table IIAbsorption maxima of the absolute optical spectra at room temperature for unlabeled and Tyr isotope-labeled WTs and H333A and Y288F mutants of cytochrome boOxidasesAir-oxidizedReducedCO-bound12C WT409.5, 531428, 532, 562418, 532, 5641-[13C]Tyr-WT408, 529427.5, 531.5, 561.5418.5, 532, 5634-[13C]Tyr-WT408,532428, 531.5, 561418.5, 532, 563d4-Tyr-WT408, 532428, 531.5, 561.5426.5, 532, 562.5Y288F414, 532.5428, 531, 562426, 532, 562.5H333A409, 534428, 532, 561.5419, 532, 563 Open table in a new tab Effects of Isotope Labeling of Tyrosines on Resonance Raman Spectra in the High Frequency Region—To gain further insight into the molecular structure of the heme-copper binuclear center, we have employed RR spectroscopy. The 413.1-nm excited RR spectra of the air-oxidized, reduced, and CO-bound forms are shown in Fig. 3, A, B, and C, respectively. It is well known that bands in the high frequency region can be used as sensitive markers of the oxidation state (ν4) and spin and coordination states (ν2 and ν3) of the heme iron (56Spiro T.G. Li X.-Y. Spiro T.G. Biological Applications of Raman Spectroscopy. III. John Wiley & Sons, New York1988: 1-37Google Scholar). In the air-oxidized states, all the Tyr-labeled WT and H333A mutant enzymes showed almost similar RR spectra to that of the unlabeled 12C WT enzyme (Fig. 3A). The ν4 band of all the preparations was observed at 1371 cm-1 with a shoulder at 1361 cm-1. The shoulder intensity becomes relatively stronger upon increase of laser power and, therefore, is attributed to the photoreduced species (data not shown). Typically, 5-coordinate high spin, 6-coordinate high spin, and 6-coordinate low spin hemes give the ν3 band at 1490–1500, 1475–1485, and 1500–1510 cm-1, respectively (57Spiro T.G. Stong J.D. Stein P. J. Am. Chem. Soc. 1979; 101: 2648-2655Crossref Scopus (265) Google Scholar). Therefore, the ν3 bands observed at 1477 and 1505 cm-1 for 12C WT, Tyr-labeled WT, and H333A mutant are assignable to high spin heme o and low spin heme b, respectively. d4-Tyr-WT exhibited no specific features compared with 12C WT. In the heme bb-type Y288F mutant, a high spin heme b at the binuclear center was partly photoreduced and gave the ν4 and ν3 bands at 1361 and 1492 cm-1 at the expense of the 1371- and 1477-cm-1 bands. For the dithionite-reduced 12C WT, Tyr-labeled WT, and H333A mutant, the ν3 bands observed at 1472 and 1492 cm-1 were assigned to 5-coordinate high spin heme o and 6-coordinate low spin heme b, respectively (Fig. 3B). Again, there are no specific features in d4-Tyr-WT. In the reduced Y288F mutant, the 1472-cm-1 band disappeared and only the 1492-cm-1 band was observed, which coincides with the conversion of heme bo-type to heme bb-type oxidase (Table I). When CO was added to the dithionite-reduced state, a new ν4 band appeared at 1371 cm-1 for [12C]Tyr-WT, 1-[13C]Tyr-WT, and 4-[13C]Tyr-WT enzymes, suggesting that CO binds to high spin heme o (Fig. 3C). The presence of the ν3 band at 1504 cm-1 supports this assignment. For d4-Tyr-WT, on the other hand, ν4 and ν3 appeared at 1361 and 1493 cm-1, respectively, which were distinct from 12C WT but close to the H333A mutant. This would suggest the formation of an amino acid-bound 6-coordinate low spin state in heme o similar to the heme b moiety. In the case of the Y288F mutant, the whole spectrum was little affected by addition of CO, presumably because of photodissociation of the iron-bound CO even under the quite low la
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