Redox-induced Protein Structural Changes in Cytochrome bo Revealed by Fourier Transform Infrared Spectroscopy and [13C]Tyr Labeling
2005; Elsevier BV; Volume: 280; Issue: 38 Linguagem: Inglês
10.1074/jbc.m502072200
ISSN1083-351X
AutoresHideki Kandori, Hiro Nakamura, Yoichi Yamazaki, Tatsushi Mogi,
Tópico(s)Mitochondrial Function and Pathology
ResumoCytochrome bo is a heme-copper terminal ubiquinol oxidase of Escherichia coli under highly aerated growth conditions. Tyr-288 present at the end of the K-channel forms a Cϵ–Nϵ covalent bond with one of the CuB ligand histidines and has been proposed to be an acid-base catalyst essential for the O–O bond cleavage at the Oxy-to-P transition of the dioxygen reduction cycle (Uchida, T., Mogi, T., and Kitagawa, T. (2000) Biochemistry 39, 6669–6678). To probe structural changes at tyrosine residues, we examined redox difference Fourier transform infrared difference spectra of the wild-type enzyme in which either l-[1-13C]Tyr or l-[4-13C]Tyr has been biosynthetically incorporated in the tyrosine auxotroph. Spectral comparison between [1-13C]Tyr-labeled and unlabeled proteins indicated that substitution of the main chain carbonyl of a Tyr residue(s) significantly affected changes in the amide-I (∼1620–1680 cm–1) and -II (∼1540–1560 cm–1) regions. In contrast, spectral comparison between [4-13C]Tyr-labeled and unlabeled proteins showed only negligible changes, which was the case for both the pulsed and the resting forms. Thus, protonation of an OH group of tyrosines including Tyr-288 in the vicinity of the heme o-CuB binuclear center was not detected at pH 7.4 upon full reduction of cytochrome bo. Redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal centers and may be related to switching of the K-channel operative at the reductive phase to D-channel at the oxidative phase of the dioxygen reduction cycle via conformational changes in the middle of helix VI. Cytochrome bo is a heme-copper terminal ubiquinol oxidase of Escherichia coli under highly aerated growth conditions. Tyr-288 present at the end of the K-channel forms a Cϵ–Nϵ covalent bond with one of the CuB ligand histidines and has been proposed to be an acid-base catalyst essential for the O–O bond cleavage at the Oxy-to-P transition of the dioxygen reduction cycle (Uchida, T., Mogi, T., and Kitagawa, T. (2000) Biochemistry 39, 6669–6678). To probe structural changes at tyrosine residues, we examined redox difference Fourier transform infrared difference spectra of the wild-type enzyme in which either l-[1-13C]Tyr or l-[4-13C]Tyr has been biosynthetically incorporated in the tyrosine auxotroph. Spectral comparison between [1-13C]Tyr-labeled and unlabeled proteins indicated that substitution of the main chain carbonyl of a Tyr residue(s) significantly affected changes in the amide-I (∼1620–1680 cm–1) and -II (∼1540–1560 cm–1) regions. In contrast, spectral comparison between [4-13C]Tyr-labeled and unlabeled proteins showed only negligible changes, which was the case for both the pulsed and the resting forms. Thus, protonation of an OH group of tyrosines including Tyr-288 in the vicinity of the heme o-CuB binuclear center was not detected at pH 7.4 upon full reduction of cytochrome bo. Redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal centers and may be related to switching of the K-channel operative at the reductive phase to D-channel at the oxidative phase of the dioxygen reduction cycle via conformational changes in the middle of helix VI. Cytochrome bo is a four-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and belongs to the heme-copper terminal oxidase superfamily (1Trumpower B.L. Gennis R.B. Annu. Rev. Biochem. 1994; 63: 675-716Crossref PubMed Scopus (471) Google Scholar, 2Mogi T. Tsubaki M. Hori H. Miyoshi H. Nakamura H. Anraku Y. J. Biochem. Mol. Biol. Biophys. 1998; 2: 79-110Google Scholar, 3Tsubaki M. Hori H. Mogi T. J. Inorg. Biochem. 2000; 82: 19-25Crossref PubMed Scopus (28) Google Scholar). Subunit I binds all four redox centers, the high affinity ubiquinone binding site (QH1), low spin heme b, high spin heme o, and CuB; the latter two centers form the heme o-CuB binuclear center. Quinols are oxidized at the low affinity quinol oxidation site (QL) in subunit II, and electrons are sequentially transferred through QH and heme b to the heme o-CuB binuclear metal center, where dioxygen reduction takes place (4Sato-Watanabe M. Mogi T. Ogura T. Kitagawa T. Miyoshi H. Iwamura H. Anraku Y. J. Biol. Chem. 1994; 269: 20908-20912Google Scholar, 5Tsatsos P.H. Reynolds K. Nickels E.F. He D.-Y. Yu C.-A. Gennis R.B. Biochemistry. 1998; 37: 9884-9888Crossref PubMed Scopus (34) Google Scholar, 6Ma J. Puustinen A. Wikström M. Gennis R.B. Biochemistry. 1998; 37: 11806-11811Crossref PubMed Scopus (28) Google Scholar, 7Sato-Watanabe M. Mogi T. Miyoshi H. Anraku Y. Biochemistry. 1998; 37: 12744-12752Crossref PubMed Scopus (28) Google Scholar, 8Sato-Watanabe M. Mogi T. Miyoshi H. Anraku Y. Biochemistry. 1998; 37: 5356-5361Crossref PubMed Scopus (38) Google Scholar, 9Mogi T. Sato-Watanabe M. Miyoshi H. Orii Y. FEBS Lett. 1999; 457: 61-64Crossref PubMed Scopus (17) Google Scholar, 10Kobayashi K. Tagawa S. Mogi T. Biochemistry. 2000; 39: 15620-15625Crossref PubMed Scopus (29) Google Scholar). The two-electron oxidation of ubiquinol-8 at the periplasmic side of the cytoplasmic membrane is coupled to the four-electron reduction of dioxygen at the cytoplasmic side. Accordingly, four chemical protons are apparently translocated from the cytoplasm to the periplasm, generating an electrochemical proton gradient across the membrane. In addition, the enzyme can vectorially translocate four other protons per dioxygen reduction by a pump mechanism (11Puustinen A. Finel M. Virkki M. Wikström M. FEBS Lett. 1989; 249: 163-167Crossref PubMed Scopus (178) Google Scholar). Mutagenesis (12Konstantinov A. Siletsky S. Mitchell D. Kaulen A. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9085-9090Crossref PubMed Scopus (326) Google Scholar, 13Verkhovskaya M.L. Garcia-Horsman A. Puustinen A. Rigaud J.-L. Morgan J.E. Verkhovsky M.I. Wikström M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10128-10131Crossref PubMed Scopus (141) Google Scholar, 14Karpefors M. Δdelroth P. Aagaard A. Sigurdson H. Svensson-Ek M. Brzezinski P. Biochim. Biophys. Acta. 1998; 1365: 159-168Crossref PubMed Scopus (50) Google Scholar, 15Pfitzner U. Hoffmeier K. Harrenga A. Kannt A. Michel H. Bamberg E. Richter O.-M.H. Ludwig B. Biochemistry. 2000; 39: 6756-6762Crossref PubMed Scopus (115) Google Scholar, 16Lee H. Das T.K. Rousseau D.L. Mills D. Ferguson-Miller S. Gennis R.B. Biochemistry. 2000; 39: 2989-2996Crossref PubMed Scopus (100) Google Scholar) and x-ray crystallographic (17Abramson J. Riistama S. Larsson G. Jasaitis A. Svensson-Ek M. Laakkonen L. Puustinen A. Iwata S. Wikström M. Nat. Struct. Biol. 2000; 7: 910-917Crossref PubMed Scopus (361) Google Scholar, 18Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1985) Google Scholar, 19Ostermeier C. Harrenga A. Ermler U. Michel H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10547-10553Crossref PubMed Scopus (717) Google Scholar, 20Yoshikawa 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 (976) Google Scholar, 21Soulimane T. Buse G. Bourenkov G.P. Bartunik H.D. Huber R. Than M.E. EMBO J. 2000; 19: 1766-1776Crossref PubMed Scopus (412) Google Scholar, 22Svensson-Ek M. Abramson J. Larsson G. Törnroth S. Brzezinski P. Iwata S. J. Mol. Biol. 2002; 321: 329-339Crossref PubMed Scopus (479) Google Scholar) studies on the heme-copper terminal oxidases suggest that the D- and K-channels in subunit I are operative during redox-coupled proton pumping (Fig. 1A). Iwata et al. (18Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1985) Google Scholar) identified two putative proton channels in cytochrome c oxidase from Paracoccus denitrificans, the D-channel characterized by a conserved Asp residue at the entry site and the K-channel characterized by a conserved Lys residue in the middle of the channel. They proposed that the D-channel translocates four pumped protons, whereas the K-channel delivers four chemical protons to the binuclear center (18Iwata S. Ostermeier C. Ludwig B. Michel H. Nature. 1995; 376: 660-669Crossref PubMed Scopus (1985) Google Scholar). However, it is now assumed that the K-channel delivers one or two chemical protons to the binuclear center at the initial reductive phase of dioxygen reduction and that the D-channel translocates all other chemical and pumped protons (12Konstantinov A. Siletsky S. Mitchell D. Kaulen A. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9085-9090Crossref PubMed Scopus (326) Google Scholar, 25Michel H. Biochemistry. 1998; 38: 15129-15140Crossref Scopus (243) Google Scholar, 26Gennis R.B. Biochim. Biophys. Acta. 1998; 1365: 241-248Crossref Scopus (190) Google Scholar, 27Ruitenberg M. Kannt A. Bamberg E. Ludwig B. Michel H. Fendler K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4632-4636Crossref PubMed Scopus (89) Google Scholar, 28Wikström M. Jasaitis A. Backgren C. Puustinen A. Verkovsky M.I. Biochim. Biophys. Acta. 2000; 1459: 514-520Crossref PubMed Scopus (134) Google Scholar). In the vicinity of the binuclear center, Tyr-288 (the E. coli cytochrome bo numbering: Tyr-280 in the soil bacterium P. denitrificans and Tyr-244 in bovine cytochrome c oxidase) is highly conserved in the SoxM-type terminal oxidase and has been proposed to be involved in dioxygen reduction (25Michel H. Biochemistry. 1998; 38: 15129-15140Crossref Scopus (243) Google Scholar, 26Gennis R.B. Biochim. Biophys. Acta. 1998; 1365: 241-248Crossref Scopus (190) Google Scholar, 29Wang 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, 30Proshlyakov D.A. Pressler M.A. Babcock G.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8020-8025Crossref PubMed Scopus (299) Google Scholar, 31Proshlyakov D.A. Pressler M.A. DeMaso C. Leykam J.F. DeWitt D.L. Babcock G.T. Science. 2000; 290: 1588-1591Crossref PubMed Scopus (275) Google Scholar, 32Blomberg M.R.A. Siegbahn P.E.M. Babcock G.T. Wikström M. J. Inorg. Biochem. 2000; 80: 261-269Crossref PubMed Scopus (80) Google Scholar). Upon two-electron reduction of the enzyme, two chemical protons are taken up from the cytoplasm to compensate for an increased negative charge at the binuclear center (12Konstantinov A. Siletsky S. Mitchell D. Kaulen A. Gennis R.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9085-9090Crossref PubMed Scopus (326) Google Scholar, 33Mitchell R. Rich P.R. Biochim. Biophys. Acta. 1994; 1186: 19-26Crossref PubMed Scopus (167) Google Scholar), and at least the first proton is delivered to the binuclear center by Tyr-288 at the end of the K-channel (27Ruitenberg M. Kannt A. Bamberg E. Ludwig B. Michel H. Fendler K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4632-4636Crossref PubMed Scopus (89) Google Scholar). If deprotonated at the oxidized (O) state (20Yoshikawa 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 (976) Google Scholar), Tyr-288 serves as one of proton acceptors (26Gennis R.B. Biochim. Biophys. Acta. 1998; 1365: 241-248Crossref Scopus (190) Google Scholar). However, calculations of electrostatic energy on the P. denitrificans cytochrome c oxidase indicate that Tyr-288 is protonated in the oxidized state when a negative ligand such as OH– is bound in the binuclear center (34Kannt A. Lancaster C.R.D. Michel H. J. Bioenerg. Biomembr. 1998; 30: 81-87Crossref PubMed Scopus (24) Google Scholar). Time-resolved spectroscopic studies on bovine cytochrome c oxidase have identified a number of intermediates in the reaction of dioxygen reduction (see Refs. 35Ferguson-Miller S. Babcock G.T. Chem. Rev. 1996; 96: 2889-2907Crossref PubMed Scopus (1045) Google Scholar and 36Ogura T. Kitagawa T. Biochim. Biophys. Acta. 2004; 1655: 290-297Crossref PubMed Scopus (41) Google Scholar for reviews). The reaction begins with binding of dioxygen to the reduced binuclear center to form the oxy intermediate (Feo2+–O2, CuB+). Tyr-288-OH would provide hydrogen to the iron-bound dioxygen, leading to the rapid cleavage of the O–O bond with the concomitant formation of a tyrosine neutral radical. In the resulting P intermediate (Feo4+ =O, CuB2+), the presence of the tyrosine neutral radical (Tyr-O·) has been demonstrated by spectroscopic methods (37Fabian M. Palmer G. Biochemistry. 1995; 34: 13802-13810Crossref PubMed Scopus (131) Google Scholar, 38MacMillan F. Kannt A. Behr J. Prisner T. Michel H. Biochemistry. 1999; 38: 9179-9184Crossref PubMed Scopus (151) Google Scholar, 39Chen Y.R. Gunther M.R. Mason R.P. J. Biol. Chem. 1999; 274: 3308-3314Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 40Uchida T. Mogi T. Kitagawa T. Biochemistry. 2000; 39: 6669-6678Crossref PubMed Scopus (38) Google Scholar, 41Iwaki M. Puustinen A. Wikström M. Rich P.R. Biochemistry. 2003; 42: 8800-8817Google Scholar, 42Nyquist R.M. Heitbrink D. Bolwien C. Gennis R.B. Heberle J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8715-8720Crossref PubMed Scopus (89) Google Scholar) and by radioactive iodide labeling followed by peptide mapping (31Proshlyakov D.A. Pressler M.A. DeMaso C. Leykam J.F. DeWitt D.L. Babcock G.T. Science. 2000; 290: 1588-1591Crossref PubMed Scopus (275) Google Scholar). Subsequent one-electron transfer to the binuclear center reduces the tyrosine neutral radical and converts the species to the ferryl (F) species (FeO4+ =O, CuB2+, Tyr-O–). The F intermediate is converted to the oxidized form by a further electron transfer. Crystallographic (19Ostermeier C. Harrenga A. Ermler U. Michel H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10547-10553Crossref PubMed Scopus (717) Google Scholar, 20Yoshikawa 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 (976) Google Scholar, 21Soulimane T. Buse G. Bourenkov G.P. Bartunik H.D. Huber R. Than M.E. EMBO J. 2000; 19: 1766-1776Crossref PubMed Scopus (412) Google Scholar) and protein sequencing (43Buse G. Soulimane T. Dewor M. Meyer H.E. Blüggel M. Protein Sci. 1999; 8: 985-990Crossref PubMed Scopus (108) Google Scholar) studies on cytochrome c oxidase revealed the presence of a peculiar Cϵ–Nϵ covalent bond between Tyr-288 and His-284, one of three histidine ligands of CuB. The OH group of Tyr-288 could form a hydrogen bond with a diatomic ligand like dioxygen, favoring the cross-linked tyrosine to participate in dioxygen reduction. Studies with model compounds showed that the His-Tyr linkage lowers a pKa of the phenol moiety by 1.1 to 1.8 (8.60 versus 10.23 (44McCauley K.M. Vrtis J.M. Dupont J. van der Donk W.A. J. Am. Chem. Soc. 2000; 122: 2403-2404Crossref Scopus (95) Google Scholar), 9.2 versus 10.3 (45Aki M. Ogura T. Naruta Y. Le T.H. Sato T. Kitagawa T. J. Phys. Chem. 2002; 106: 3436-3444Crossref Scopus (42) Google Scholar), 8.34 versus 10.2 (46Cappuccio J.A. Ayala I. Elliott G.I. Szundi I. Lewis J. Konopelski J.P. Barry B.A. Einarsdóttir Ó. J. Am. Chem. Soc. 2002; 124: 1750-1760Crossref PubMed Scopus (78) Google Scholar)). Consequently, the cross-linked Tyr-288 can structurally and chemically participate in the O–O bond cleavage at the Oxy-to-P transition. Fourier transform infrared (FTIR) 3The abbreviations used are: FTIR, Fourier transform infrared; WT, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. spectroscopy is a powerful technique to probe the molecular environment of the active center of terminal oxidases. Application of spectro-electrochemical cells (47Hellwig P. Rost B. Kaiser U. Ostermeier C. Michel H. Mäntele W. FEBS Lett. 1996; 385: 53-57Crossref PubMed Scopus (105) Google Scholar, 48Hellwig P. Behr J. Ostermeier C. Richter O.-M.H. Pfitzner U. Odenwald A. Ludwig B. Michel H. Mäntele W. Biochemistry. 1998; 37: 7390-7399Crossref PubMed Scopus (169) Google Scholar, 49Hellwig P. Soulimane T. Buse G. Mäntele W. FEBS Lett. 1999; 458: 83-86Crossref PubMed Scopus (40) Google Scholar, 50Behr J. Michel H. Mäntele W. Hellwig P. Biochemistry. 2000; 39: 1356-1363Crossref PubMed Scopus (66) Google Scholar, 51Hellwig P. Pfitzner U. Behr J. Rost B. Pesavento R.P. Donk W.V. Gennis R.B. Michel H. Ludwig B. Mäntele W. Biochemistry. 2002; 41: 9116-9125Crossref PubMed Scopus (56) Google Scholar, 52Schmidt B. Hillier W. McCracken J. Ferguson-Miller S. Biochim. Biophys. Acta. 2004; 1655: 248-255Crossref PubMed Scopus (11) Google Scholar), attenuated total reflection FTIR apparatus (41Iwaki M. Puustinen A. Wikström M. Rich P.R. Biochemistry. 2003; 42: 8800-8817Google Scholar, 42Nyquist R.M. Heitbrink D. Bolwien C. Gennis R.B. Heberle J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8715-8720Crossref PubMed Scopus (89) Google Scholar, 53Rich P.R. Breton J. Biochemistry. 2002; 41: 967-973Crossref PubMed Scopus (50) Google Scholar, 54Iwaki M. Breton J. Rich P.R. Biochim. Biophys. Acta. 2002; 1555: 116-121Crossref PubMed Scopus (34) Google Scholar, 55Iwaki M. Puustinen A. Wikström M. Rich P.R. Biochemistry. 2003; 42: 8809-8817Crossref PubMed Scopus (55) Google Scholar, 56Nyquist R.M. Heitbrink D. Bolwien C. Wells T.A. Gennis R.B. Heberle J. FEBS Lett. 2001; 505: 63-67Crossref PubMed Scopus (56) Google Scholar), and a photochemical reduction technique (57Lübben M. Gerwert K. FEBS Lett. 1996; 397: 303-307Crossref PubMed Scopus (73) Google Scholar, 58Lübben M. Prutsch A. Mamat B. Gerwert K. Biochemistry. 1999; 38: 2048-2056Crossref PubMed Scopus (85) Google Scholar, 59Yamazaki Y. Kandori H. Mogi T. J. Biochem. 1999; 126: 194-199Crossref PubMed Scopus (31) Google Scholar, 60Prutsch A. Vogtt K. Ludovici C. Lübben M. Biochim. Biophys. Acta. 2002; 1554: 22-28Crossref PubMed Scopus (13) Google Scholar) enabled us to examine protein structural changes upon reduction of the redox metal centers of respiratory terminal oxidases. Alternatively, photodissociation of CO from the reduced enzyme revealed the local structural change surrounding the heme-copper binuclear center of the E. coli cytochrome bo (61Riistama S. Hummer G. Puustinen A. Dyer R.B. Woodruff W.H. Wikström M. FEBS Lett. 1997; 414: 275-280Crossref PubMed Scopus (136) Google Scholar, 62Puustinen A. Bailey J.A. Dyer R.B. Mecklenburg S.L. Wikström M. Woodruff W.H. Biochemistry. 1997; 36: 13195-13200Crossref PubMed Scopus (113) Google Scholar). Previous studies using site-directed mutants revealed that a COOH group of Glu-286 at the end of the D-channel undergoes hydrogen bond changes upon full reduction of the metal centers (48Hellwig P. Behr J. Ostermeier C. Richter O.-M.H. Pfitzner U. Odenwald A. Ludwig B. Michel H. Mäntele W. Biochemistry. 1998; 37: 7390-7399Crossref PubMed Scopus (169) Google Scholar, 50Behr J. Michel H. Mäntele W. Hellwig P. Biochemistry. 2000; 39: 1356-1363Crossref PubMed Scopus (66) Google Scholar, 59Yamazaki Y. Kandori H. Mogi T. J. Biochem. 1999; 126: 194-199Crossref PubMed Scopus (31) Google Scholar, 60Prutsch A. Vogtt K. Ludovici C. Lübben M. Biochim. Biophys. Acta. 2002; 1554: 22-28Crossref PubMed Scopus (13) Google Scholar) or CO photolysis of the reduced, CO-bound enzyme (61Riistama S. Hummer G. Puustinen A. Dyer R.B. Woodruff W.H. Wikström M. FEBS Lett. 1997; 414: 275-280Crossref PubMed Scopus (136) Google Scholar, 62Puustinen A. Bailey J.A. Dyer R.B. Mecklenburg S.L. Wikström M. Woodruff W.H. Biochemistry. 1997; 36: 13195-13200Crossref PubMed Scopus (113) Google Scholar). In contrast, a lack of CuB and the substitution of high spin heme o for heme b in the Tyr-288 mutants (63Kawasaki M. Mogi T. Anraku Y. J. Biochem. 1997; 122: 422-429Crossref PubMed Scopus (25) Google Scholar, 64Mogi T. Minagawa 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) obscured protein structural changes attributable to Tyr-288 at the end of the K-channel (59Yamazaki Y. Kandori H. Mogi T. J. Biochem. 1999; 126: 194-199Crossref PubMed Scopus (31) Google Scholar). Recently, it has been reported that Glu-286 deprotonates upon one-electron reduction of the fully oxidized enzyme (42Nyquist R.M. Heitbrink D. Bolwien C. Gennis R.B. Heberle J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8715-8720Crossref PubMed Scopus (89) Google Scholar) or reverse electron transfer in the mixed valence enzyme (65McMahon B.H. Fabian M. Tomson F. Causgrove T.P. Bailey J.A. Rein F.N. Dyer R.B. Palmer G. Gennis R.B. Woodruff W.H. Biochim. Biophys. Acta. 2004; 1655: 321-331Crossref PubMed Scopus (41) Google Scholar). In the present study, to probe redox-induced structural changes at tyrosines, we prepared the wild-type cytochrome bo into which either l-[1-13C]Tyr or l-[4-13 C]Tyr (Fig. 1B) had been biosynthetically incorporated and measured their photo-reduced minus oxidized redox difference FTIR spectra. We found that redox-induced main chain changes at a Tyr residue(s) are associated with structural changes at Glu-286 near the binuclear metal center. In addition, protonation of an OH group of tyrosines was not detected at pH 7.4 and 8.5 under steady-state conditions. On the basis of present findings, we will discuss redox-induced protein structural changes in subunit I. Preparation of Isotope-labeled Enzymes—Unlabeled wild-type enzyme (12C-WT) and [13C]Tyr-labeled enzymes were isolated from E. coli GO103/pHN3795-1 (66Hirota S. Mogi T. Ogura T. Hirano T. Anraku Y. Kitagawa T. FEBS Lett. 1994; 352: 67-70Crossref PubMed Scopus (35) Google Scholar) and GO103Y (cyo+ Δcyd::Kmr ΔtyrA16::Tn10)/pHN3795-1 (67Uchida T. Mogi T. Kitagawa T. J. Biol. Chem. 2004; 279: 53613-53620Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) as described previously. [13C]Tyrosines used are l-[1-13C]Tyr (99 atom %, MassTrace) and l-[4-13C]Tyr (99 atom %, ICON), and final yields of the [1-13C]Tyr- and [4-13C]Tyr-labeled enzymes were 0.39 and 0.35 μmol, respectively, from the 10-liter culture. Purified enzymes in 50 mm Tris-HCl (pH 7.4) containing 0.1% sucrose monolaurate (Mitsubishi-Kagaku Foods Co., Tokyo) were stored at –80 °C. The concentration of the enzyme was calculated from the heme content determined by the pyridine ferrohemochromogen method, as described previously (68Tsubaki M. Mogi T. Anraku Y. Hori H. Biochemistry. 1993; 32: 6065-6072Crossref PubMed Scopus (81) Google Scholar). Preparation of the Pulsed Form—A pulsed form of cytochrome bo was prepared according to Moody and Rich (69Moody A.J. Rich P.R. Eur. J. Biochem. 1994; 226: 731-737Crossref PubMed Scopus (31) Google Scholar) as follows. The resting enzyme (∼0.25 mm) in 50 mm Tricine-NaOH (pH 8.5) containing 0.1% sucrose monolaurate and 0.5 mm sodium EDTA were incubated aerobically in the dark with 8 mm sodium ascorbate (pH 7.6) and 0.1 mm phenazine methosulfate (final concentrations) at 23 °C for 1 h. Then the sample was dialyzed at 4 °C against 300 volumes of 50 mm Tricine-NaOH (pH 8.5) containing 0.1% sucrose monolaurate and 0.5 mm sodium EDTA with four changes of the buffer every hour. After the dialysis, insoluble materials were removed by centrifugation at 16,000 rpm (22,640 × g) and 4 °C for 10 min. The sample solution was concentrated to a final concentration of about 0.5 mm using a Centricon 100 apparatus (Amicon) for 2 h at 4°C. The control sample (the resting form) was prepared as for the pulsed form except for the omission of the reduction with 8 mm sodium ascorbate and 0.1 mm phenazine methosulfate. The pulsed form showed the Soret peak at 407 nm and an intense g = 3.7 signal, whereas the resting form had its Soret peak at 409 nm, consistent with previous reports (69Moody A.J. Rich P.R. Eur. J. Biochem. 1994; 226: 731-737Crossref PubMed Scopus (31) Google Scholar). FTIR Spectroscopy—The samples for spectroscopic analysis were prepared essentially according to Lübben and Gerwert (57Lübben M. Gerwert K. FEBS Lett. 1996; 397: 303-307Crossref PubMed Scopus (73) Google Scholar). Five μl of the reaction mixture containing about 0.25 mm air-oxidized enzyme (resting form), 0.25 mm riboflavin, 50 mm sodium EDTA, 50 mm sodium phosphate (pH 7.4), and 0.1% sucrose monolaurate was placed on a BaF2 window and concentrated to some extent in a vacuum desiccator (59Yamazaki Y. Kandori H. Mogi T. J. Biochem. 1999; 126: 194-199Crossref PubMed Scopus (31) Google Scholar). The hydrated enzyme/detergent paste was then sandwiched by another BaF2 window and covered by an aperture of 6-mm diameter. Once the enzyme was mixed with riboflavin, all of the procedures were carried out under dim red light conditions. Oxford cryostat (DN-1704) was equipped in the FTIR spectrometer, and the sample was attached to the sample holder for the cryostat (70Kandori H. Maeda A. Biochemistry. 1995; 34: 14220-14229Crossref PubMed Scopus (78) Google Scholar). Photoreduction of the air-oxidized enzymes was conducted at 15 °C in the presence of riboflavin as photoactivatable electron donor (63Kawasaki M. Mogi T. Anraku Y. J. Biochem. 1997; 122: 422-429Crossref PubMed Scopus (25) Google Scholar). The 256 interferograms at 2 cm–1 resolution were recorded with a Bio-Rad FTS-40 spectrometer and converted to the infrared absorption spectrum by use of a reference interferogram recorded in the absence of the sample. The spectral difference before and after irradiation was compared with the base line as the difference between the two spectra without intervening irradiation, and if necessary the base line value was subtracted from the data. The spectra in Figs. 2 and 3 are the averages of 4–6 independent measurements.FIGURE 3Spectral comparison in the typical frequency region of protein backbone (1760–1525 cm–1).a, infrared difference spectra of [4-13C]Tyr-labeled (solid line) and unlabeled (dotted line) forms of cytochrome bo. b, infrared difference spectra of [1-13C]Tyr-labeled (solid line) and unlabeled (dotted line) forms of cytochrome bo.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Redox-induced Infrared Spectral Changes of Isotope-labeled Resting Oxidized Forms at pH 7.4—Fig. 2 shows photo-reduced minus air-oxidized (as prepared) infrared spectra unlabeled difference of wild-type (12 C-WT), [4-13 C]Tyr-labeled WT ([4-13C]Tyr-WT), and [1-13C]Tyr-labeled WT ([1-13C]Tyr-WT). All of the spectra showed a negative 1743-cm–1 band, assignable to the COOH group of Glu-286 in the oxidized state (42Nyquist R.M. Heitbrink D. Bolwien C. Gennis R.B. Heberle J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8715-8720Crossref PubMed Scopus (89) Google Scholar, 48Hellwig P. Behr J. Ostermeier C. Richter O.-M.H. Pfitzner U. Odenwald A. Ludwig B. Michel H. Mäntele W. Biochemistry. 1998; 37: 7390-7399Crossref PubMed Scopus (169) Google Scholar, 58Lübben M. Prutsch A. Mamat B. Gerwert K. Biochemistry. 1999; 38: 2048-2056Crossref PubMed Scopus (85) Google Scholar, 59Yamazaki Y. Kandori H. Mogi T. J. Biochem. 1999; 126: 194-199Crossref PubMed Scopus (31) Google Scholar, 60Prutsch A. Vogtt K. Ludovici C. Lübben M. Biochim. Biophys. Acta. 2002; 1554: 22-28Crossref PubMed Scopus (13) Google Scholar). A positive feature at 1720 cm–1 was not symmetrical and did not show a downshift upon substitution with Asp (59Yamazaki Y. Kandori H. Mogi T. J. Biochem. 1999; 126: 194-199Crossref PubMed Scopus (31) Google Scholar). In bovine cytochrome c oxidase, two negative bands at 1748–1749 and 1737–1738 cm–1 were identified in the fully reduced minus oxidized difference spectrum in the unligated enzyme (49Hellwig P. Soulimane T. Buse G. Mäntele W. FEBS Lett. 1999; 458: 83-86Crossref PubMed Scopus (40) Google Scholar, 52Schmidt B. Hillier W. McCracken J. Ferguson-Miller S. Biochim. Biophys. Acta. 2004; 1655: 248-255Crossref PubMed Scopus (11) Google Scholar, 54Iwaki M. Breton J. Rich P.R. Biochim. Biophys. Acta. 2002; 1555: 116-121Crossref PubMed Scopus (34) Google Scholar, 55Iwaki M. Puustinen A. Wikström M. Rich P.R. Biochemistry. 2003; 42: 8809-8817Crossref PubMed Scopus (55) Google Scholar, 56Nyquist R.M. Heitbrink D. Bolwien C. Wells T.A. Gennis R.B. Heberle J. FEBS Lett. 2001; 505: 63-67Crossref PubMed Scopus (56) Google Scholar, 71Okuno D. Iwase T. Shinzawa-Itoh K. Yoshikawa S. Kitagawa T. J. Am. Chem. Soc. 2003; 125: 7209-7218Crossref PubMed Scopus (61) Google Scholar). As postulated by crystallographic studies (20Yoshikawa 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 (976) Google Scholar), the latter signal has been tentatively assigned to the deprotonation of Asp-51 in subunit I (49Hellwig P. Soulimane T. Buse G. Mäntele W. FEBS Lett. 1999; 458: 83-86Crossref PubMed Scopus (40) Google Scholar, 71Okuno D. Iwase T. Shinzawa-Itoh K. Yoshikawa S. Kitagawa T. J. Am. Chem. Soc. 2003; 125: 7209-7218Crossref PubMed Scopus (61) Google Scholar). However, the 1588 cm–1 band assigned to a νasym(COO–) mode from Asp-51 (49Hellwig P. Soulimane T. Buse G. Mäntele W. FEBS Lett. 1999; 458: 83-86Crossref PubMed Scopus (40) Google Scholar) can be found in cytochrome c oxidase from Rhodobacter sphaeroides (52Schmidt B. Hillier W. McCracken J. Ferguson-Miller S. Biochim. Biophys. Acta. 2004; 1655: 248-255Crossref PubMed Scopus (11) Google Scholar), in which the mammalian-specific Asp-51 is not conserved. Similar spectral changes in the oxidized cyanide-bound (53Rich P.R. Breton J. Biochemistry. 2002; 41: 967-973Crossref PubMed Scopus (50) Goog
Referência(s)