메뉴 건너뛰기




Volumn 1827, Issue 11-12, 2013, Pages 1309-1319

Design and use of photoactive ruthenium complexes to study electron transfer within cytochrome bc1 and from cytochrome bc1 to cytochrome c

Author keywords

Cytochrome bc1; Cytochrome c; Electron transfer; Ruthenium

Indexed keywords

CYTOCHROME B; CYTOCHROME C; CYTOCHROME C1; FERRIC ION; FERROUS ION; IRON SULFUR PROTEIN; MYXOTHIAZOL; RUTHENIUM COMPLEX; STIGMATELLIN; UBIQUINOL CYTOCHROME C REDUCTASE;

EID: 84884671725     PISSN: 00052728     EISSN: 18792650     Source Type: Journal    
DOI: 10.1016/j.bbabio.2012.09.002     Document Type: Review
Times cited : (17)

References (112)
  • 1
    • 0028290824 scopus 로고
    • Energy transduction by cytochrome complexes in mitochondrial and bacterial respiration: The enzymology of coupling electron transfer reactions to transmembrane proton translocation
    • B.L. Trumpower, and R.B. Gennis Energy transduction by cytochrome complexes in mitochondrial and bacterial respiration: the enzymology of coupling electron transfer reactions to transmembrane proton translocation Annu. Rev. Biochem. 63 1994 675 716 (Pubitemid 24218646)
    • (1994) Annual Review of Biochemistry , vol.63 , pp. 675-716
    • Trumpower, B.L.1    Gennis, R.B.2
  • 3
    • 0016754421 scopus 로고
    • The protonmotive Q cycle: A general formulation
    • P. Mitchell The protonmotive Q cycle: a general formulation FEBS Lett. 59 1975 137 139
    • (1975) FEBS Lett. , vol.59 , pp. 137-139
    • Mitchell, P.1
  • 4
    • 1942447877 scopus 로고    scopus 로고
    • 1 complex: Function in the context of structure
    • DOI 10.1146/annurev.physiol.66.032102.150251
    • 1 complex: function in the context of structure Annu. Rev. Physiol. 66 2004 689 733 (Pubitemid 40614460)
    • (2004) Annual Review of Physiology , vol.66 , pp. 689-733
    • Crofts, A.R.1
  • 9
    • 0034660152 scopus 로고    scopus 로고
    • 1 complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment
    • DOI 10.1016/S0969-2126(00)00152-0
    • 1 complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment Struct. Fold. Des. 8 2000 669 684 (Pubitemid 30409318)
    • (2000) Structure , vol.8 , Issue.6 , pp. 669-684
    • Hunte, C.1    Koepke, J.2    Lange, C.3    Rossmanith, T.4    Michel, H.5
  • 15
    • 0034624079 scopus 로고    scopus 로고
    • 1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein
    • DOI 10.1074/jbc.M007444200
    • 1 complex by the formation of an intersubunit disulfide bond between cytochrome b and the iron-sulfur protein J. Biol. Chem. 275 2000 38597 38604 (Pubitemid 32009188)
    • (2000) Journal of Biological Chemistry , vol.275 , Issue.49 , pp. 38597-38604
    • Xia, K.1    Yu, L.2    Yu, C.-A.3
  • 26
    • 1442325507 scopus 로고    scopus 로고
    • o Site Occupants and the Head Domain Position of the Fe-S Protein Subunit
    • DOI 10.1021/bi035938u
    • 1 is mediated by both the Qo site occupants and the head domain position of the Fe-S protein subunit Biochemistry 43 2004 2217 2227 (Pubitemid 38279967)
    • (2004) Biochemistry , vol.43 , Issue.8 , pp. 2217-2227
    • Cooley, J.W.1    Roberts, A.G.2    Bowman, M.K.3    Kramer, D.M.4    Daldal, F.5
  • 30
    • 0024293251 scopus 로고
    • Preparation and characterization of singly labeled ruthenium polypyridine cytochrome c derivatives
    • L.P. Pan, B. Durham, J. Wolinska, and F. Millett Preparation and characterization of singly labeled ruthenium polypyridine cytochrome c derivatives Biochemistry 27 1988 7180 7184
    • (1988) Biochemistry , vol.27 , pp. 7180-7184
    • Pan, L.P.1    Durham, B.2    Wolinska, J.3    Millett, F.4
  • 31
    • 0024431817 scopus 로고
    • Photoinduced electron-transfer kinetics of singly labeled ruthenium bis(bipyridine) dicarboxybipyridine cytochrome c derivatives
    • B. Durham, L.P. Pan, J. Long, and F. Millett Photoinduced electron-transfer kinetics of singly labeled ruthenium bis-(bipyridine) dicarboxybipyridine cytochrome c derivatives Biochemistry 28 1989 8659 8665 (Pubitemid 19269062)
    • (1989) Biochemistry , vol.28 , Issue.21 , pp. 8659-8665
    • Durham, B.1    Pan, L.P.2    Long, J.E.3    Millet, F.4
  • 32
    • 0028832237 scopus 로고
    • Design of ruthenium-cytochrome c derivatives to measure electron transfer to cytochrome c peroxidase
    • R.-Q. Liu, L. Geren, P. Anderson, J.L. Fairris, N. Peffer, A. McKee, B. Durham, and F. Millett Design of ruthenium-cytochrome c derivatives to measure electron transfer to cytochrome c peroxidase Biochimie 77 1995 549 561
    • (1995) Biochimie , vol.77 , pp. 549-561
    • Liu, R.-Q.1    Geren, L.2    Anderson, P.3    Fairris, J.L.4    Peffer, N.5    McKee, A.6    Durham, B.7    Millett, F.8
  • 33
    • 0041001475 scopus 로고
    • Electron transfer in biology and the solid state
    • M.K. Johnson, American Chemical Society Washington, D. C.
    • B. Durham, L.P. Pan, S. Hahm, J. Long, and F. Millett Electron transfer in biology and the solid state M.K. Johnson, ACS Advances in Chemistry Series 1990 American Chemical Society Washington, D. C. 181 193
    • (1990) ACS Advances in Chemistry Series , pp. 181-193
    • Durham, B.1    Pan, L.P.2    Hahm, S.3    Long, J.4    Millett, F.5
  • 34
    • 0026074926 scopus 로고
    • Photoinduced electron transfer between cytochrome c peroxidase and yeast cytochrome c labeled at Cys 102 with (4-bromomethyl-4′-methylbipyridine) [bis(bipyridine)]ruthenium
    • L. Geren, S. Hahm, B. Durham, and F. Millett Photoinduced electron transfer between cytochrome c peroxidase and yeast cytochrome c labeled at Cys 102 with (4-bromomethyl-4′-methylbipyridine)[bis(bipyridine)]ruthenium Biochemistry 30 1991 9450 9457
    • (1991) Biochemistry , vol.30 , pp. 9450-9457
    • Geren, L.1    Hahm, S.2    Durham, B.3    Millett, F.4
  • 36
    • 0028819233 scopus 로고
    • Design of a ruthenium-cytochrome c derivative to measure the electron transfer to the initial acceptor in cytochrome c oxidase
    • L.M. Geren, J.R. Beasley, B.R. Fines, A.J. Saunders, S. Hibdon, G.J. Pielak, B. Durham, and F. Millett Design of a ruthenium-cytochrome c derivative to measure the electron transfer to the initial acceptor in cytochrome c oxidase J. Biol. Chem. 270 1995 2466 2472
    • (1995) J. Biol. Chem. , vol.270 , pp. 2466-2472
    • Geren, L.M.1    Beasley, J.R.2    Fines, B.R.3    Saunders, A.J.4    Hibdon, S.5    Pielak, G.J.6    Durham, B.7    Millett, F.8
  • 37
    • 12644291216 scopus 로고    scopus 로고
    • Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase
    • DOI 10.1021/bi9611117
    • K. Wang, H. Mei, L. Geren, M.A. Miller, A. Saunders, X. Wang, J.L. Waldner, G.J. Pielak, B. Durham, and F. Millett Design of a ruthenium-cytochrome c derivative to measure electron transfer to the radical cation and oxyferryl heme in cytochrome c peroxidase Biochemistry 35 1996 15107 15119 (Pubitemid 26423849)
    • (1996) Biochemistry , vol.35 , Issue.47 , pp. 15107-15119
    • Wang, K.1    Mei, H.2    Geren, L.3    Miller, M.A.4    Saunders, A.5    Wang, X.6    Waldner, J.L.7    Pielak, G.J.8    Durham, B.9    Millett, F.10
  • 38
    • 0032552970 scopus 로고    scopus 로고
    • Single electron reduction of cytochrome c oxidase compound F: Resolution of partial steps by transient spectroscopy
    • DOI 10.1021/bi981490z
    • D. Zaslavsky, R.C. Sadoski, K. Wang, B. Durham, R.B. Gennis, and F. Millett Single electron reduction of cytochrome c oxidase compound F: resolution of partial steps by transient spectroscopy Biochemistry 37 1998 14910 14916 (Pubitemid 28487600)
    • (1998) Biochemistry , vol.37 , Issue.42 , pp. 14910-14916
    • Zaslavsky, D.1    Sadoski, R.C.2    Wang, K.3    Durham, B.4    Gennis, R.B.5    Millett, F.6
  • 39
    • 37249001770 scopus 로고    scopus 로고
    • H state of detergent-solubilized cytochrome oxidase
    • DOI 10.1021/bi701424d
    • S.E. Brand, S. Rajagukguk, K. Ganesan, L. Geren, M. Fabian, D. Han, R.B. Gennis, B. Durham, and F. Millett A new ruthenium complex to study single-electron reduction of the pulsed OH state of detergent-solubilized cytochrome oxidase Biochemistry 46 2007 14610 14618 (Pubitemid 350276366)
    • (2007) Biochemistry , vol.46 , Issue.50 , pp. 14610-14618
    • Brand, S.E.1    Rajagukguk, S.2    Ganesan, K.3    Geren, L.4    Fabian, M.5    Han, D.6    Gennis, R.B.7    Durham, B.8    Millett, F.9
  • 41
    • 0001574930 scopus 로고
    • Electron transfer across polypeptides. Intramolecular electron transfer from ruthenium (II) to iron (III) in histidine-33 modified horse heart cytochrome c
    • S.S. Isied, G. Worosila, and S.J. Atherton Electron transfer across polypeptides. Intramolecular electron transfer from ruthenium (II) to iron (III) in histidine-33 modified horse heart cytochrome c J. Am. Chem. Soc. 104 1982 7659 7661
    • (1982) J. Am. Chem. Soc. , vol.104 , pp. 7659-7661
    • Isied, S.S.1    Worosila, G.2    Atherton, S.J.3
  • 43
    • 13444265067 scopus 로고    scopus 로고
    • Free-energy dependence of electron transfer in cytochrome c labeled with ruthenium(II)- Polypyridine complexes
    • J.L. Fairris, K. Wang, L. Geren, G.J. Pielak, B. Durham, and F. Millett Intramolecular electron transfer in yeast cytochrome c covalently bonded to ruthenium(II) polypyridine complexes at Cys39 ACS Advances in Chemistry Series 254 1998 Photochemistry and Radiation Chemistry, ACS Washington, DC 99 110 (Pubitemid 128788776)
    • (1998) Advances in Chemistry Series , vol.254 , pp. 99-110
    • Wright, J.L.1    Wang, K.2    Geren, L.3    Saunders, A.J.4
  • 44
    • 0037167615 scopus 로고    scopus 로고
    • Design of photoactive ruthenium complexes to study interprotein electron transfer
    • F. Millett, and B. Durham Design of photoactive ruthenium complexes to study interprotein electron transfer Biochemistry 41 2002 11315 11324
    • (2002) Biochemistry , vol.41 , pp. 11315-11324
    • Millett, F.1    Durham, B.2
  • 45
    • 0000614078 scopus 로고
    • Driving-force effects on the rate of long-range electron transfer in ruthenium-modified cytochrome c
    • T.J. Meade, H.B. Gray, and J.R. Winkler Driving-force effects on the rate of long-range electron transfer in ruthenium-modified cytochrome c J. Am. Chem. Soc. 111 1989 4353 4356
    • (1989) J. Am. Chem. Soc. , vol.111 , pp. 4353-4356
    • Meade, T.J.1    Gray, H.B.2    Winkler, J.R.3
  • 46
    • 13544274971 scopus 로고
    • High-driving force electron transfer in metalloproteins: Intramolecular oxidation of ferrocytochrome c by Ru(2,2′bpy)2(im)(His-33)3 +
    • I.-J. Chang, H.B. Gray, and J.R. Winkler High-driving force electron transfer in metalloproteins: intramolecular oxidation of ferrocytochrome c by Ru(2,2′bpy)2(im)(His-33)3 + J. Am. Chem. Soc. 113 1991 7056 7757
    • (1991) J. Am. Chem. Soc. , vol.113 , pp. 7056-7757
    • Chang, I.-J.1    Gray, H.B.2    Winkler, J.R.3
  • 50
    • 0010884753 scopus 로고
    • On the theory of oxidation-reduction reactions involving electron transfer
    • R.A. Marcus On the theory of oxidation-reduction reactions involving electron transfer J. Chem. Phys. 24 1956 966 978
    • (1956) J. Chem. Phys. , vol.24 , pp. 966-978
    • Marcus, R.A.1
  • 57
    • 0020173148 scopus 로고
    • A Q-cycle mechanism for the cyclic electron transfer chain of Rp. Sphaeroides
    • A.R. Crofts, and S.W. Meinhardt A Q-cycle mechanism for the cyclic electron transfer chain of Rp. sphaeroides Biochem. Soc. Trans. 10 1982 201 203
    • (1982) Biochem. Soc. Trans. , vol.10 , pp. 201-203
    • Crofts, A.R.1    Meinhardt, S.W.2
  • 62
    • 0028220770 scopus 로고
    • Two pK values of the oxidised 'Rieske' [2Fe-2S] cluster observed by CD spectroscopy
    • DOI 10.1016/0005-2728(94)90196-1
    • T.A. Link Two pK values of the oxidized Rieske [2Fe2S] cluster observed by CD spectroscopy Biochim. Biophys. Acta 1185 1994 81 84 (Pubitemid 24098471)
    • (1994) Biochimica et Biophysica Acta - Bioenergetics , vol.1185 , Issue.1 , pp. 81-84
    • Link, T.A.1
  • 63
    • 0032407459 scopus 로고    scopus 로고
    • 1 complex and in membranes
    • DOI 10.1016/S0014-5793(98)01493-8, PII S0014579398014938
    • 1 complex and in membranes FEBS Lett. 440 1998 409 413 (Pubitemid 29052324)
    • (1998) FEBS Letters , vol.440 , Issue.3 , pp. 409-413
    • Ugulava, N.B.1    Crofts, A.R.2
  • 69
    • 1342325555 scopus 로고    scopus 로고
    • Reversible redox energy coupling in electron transfer chains
    • DOI 10.1038/nature02242
    • A. Osyczka, C.C. Moser, F. Daldal, and P.L. Dutton Reversible redox energy coupling in electron transfer chains Nature 427 2004 607 612 (Pubitemid 38248474)
    • (2004) Nature , vol.427 , Issue.6975 , pp. 607-612
    • Osyczka, A.1    Moser, C.C.2    Daldal, F.3    Dutton, P.L.4
  • 72
    • 0033619733 scopus 로고    scopus 로고
    • 1 complex from mitochondria and photosynthetic bacteria
    • 1 complex from mitochondria and photosynthetic bacteria Biochemistry 38 1999 15827 15839 (Pubitemid 129520474)
    • (1999) Biochemistry , vol.38 , Issue.48 , pp. 15827-15839
    • Crofts, A.R.1    Hong, S.2    Zhang, Z.3    Berry, E.A.4
  • 73
    • 77950567917 scopus 로고    scopus 로고
    • 1 complex: Effects on the biophysical properties of the Rieske iron-sulfur protein and on the kinetics of the complex
    • 1 complex: effects on the biophysical properties of the Rieske iron-sulfur protein and on the kinetics of the complex J. Biol. Chem. 285 2010 9233 9248
    • (2010) J. Biol. Chem. , vol.285 , pp. 9233-9248
    • Lhee, S.1    Kolling, D.2    Nair, S.3    Dikanov, S.4    Crofts, A.R.5
  • 74
    • 0037452557 scopus 로고    scopus 로고
    • 1 complex
    • DOI 10.1021/bi026656h
    • 1 complex Biochemistry 42 2003 1499 1507 (Pubitemid 36205956)
    • (2003) Biochemistry , vol.42 , Issue.6 , pp. 1499-1507
    • Darrouzet, E.1    Daldal, F.2
  • 75
    • 3542991515 scopus 로고    scopus 로고
    • The quinone chemistry of bc complexes
    • DOI 10.1016/j.bbabio.2004.04.021, PII S000527280400146X
    • P.R. Rich The quinone chemistry of bc complexes Biochim. Biophys. Acta 1658 2004 165 171 (Pubitemid 39013247)
    • (2004) Biochimica et Biophysica Acta - Bioenergetics , vol.1658 , Issue.1-2 , pp. 165-171
    • Rich, P.R.1
  • 78
    • 23244438766 scopus 로고    scopus 로고
    • 1 complex: Reaction mechanism and prevention of short-circuiting
    • DOI 10.1016/j.bbabio.2005.03.009, PII S0005272805000927
    • 1 complex: reaction mechanism and prevention of short-circuiting Biochim. Biophys. Acta 1709 2005 5 34 (Pubitemid 41096088)
    • (2005) Biochimica et Biophysica Acta - Bioenergetics , vol.1709 , Issue.1 , pp. 5-34
    • Mulkidjanian, A.Y.1
  • 85
    • 73649089642 scopus 로고    scopus 로고
    • 1 complex: Enzyme with one inactive monomer is fully active but unable to activate the second ubiquinol oxidation site in response to ligand binding at the ubiquinone reduction site
    • 1 complex: enzyme with one inactive monomer is fully active but unable to activate the second ubiquinol oxidation site in response to ligand binding at the ubiquinone reduction site J. Biol. Chem. 285 2010 502 510
    • (2010) J. Biol. Chem. , vol.285 , pp. 502-510
    • Castellani, M.1    Covian, R.2    Kleinschroth, T.3    Anderka, O.4    Ludwig, B.5    Trumpower, B.6
  • 87
    • 33846029501 scopus 로고    scopus 로고
    • 1 complexes of phototrophic bacteria: Introducing the activated Q-cycle
    • DOI 10.1039/b517522d
    • 1 complexes of phototrophic bacteria: introducing the activated Q-cycle Photochem. Photobiol. Sci. 6 2007 19 34 (Pubitemid 46046002)
    • (2007) Photochemical and Photobiological Sciences , vol.6 , Issue.1 , pp. 19-34
    • Mulkidjanian, A.Y.1
  • 89
    • 79952393685 scopus 로고    scopus 로고
    • Intermonomer electron transfer between the low-potential b hemes of cytochrome bca
    • P. Lanciano, D.W. Lee, H. Yang, E. Darrouzet, and F. Daldal Intermonomer electron transfer between the low-potential b hemes of cytochrome bca Biochemistry 50 2011 1651 1663
    • (2011) Biochemistry , vol.50 , pp. 1651-1663
    • Lanciano, P.1    Lee, D.W.2    Yang, H.3    Darrouzet, E.4    Daldal, F.5
  • 91
    • 84856431524 scopus 로고    scopus 로고
    • Enzymatic activities of isolated cytochrome bca-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains
    • M. Czapla, A. Borek, M. Sarewicz, and A. Osyczka Enzymatic activities of isolated cytochrome bca -like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains Biochemistry 51 2012 829 835
    • (2012) Biochemistry , vol.51 , pp. 829-835
    • Czapla, M.1    Borek, A.2    Sarewicz, M.3    Osyczka, A.4
  • 93
    • 0018173851 scopus 로고
    • Effect of specific lysine modification on the reduction of cytochrome c by succinate-cytochrome c reductase
    • A.J. Ahmed, H.T. Smith, M.B. Smith, and F. Millett Effect of specific lysine modification on the reduction of cytochrome c by succinate-cytochrome c reductase Biochemistry 17 1978 2479 2483 (Pubitemid 8363354)
    • (1978) Biochemistry , vol.17 , Issue.13 , pp. 2479-2483
    • Ahmed, A.J.1    Smith, H.T.2    Smith, M.B.3    Millett, F.S.4
  • 95
    • 0018400889 scopus 로고
    • Definition of cytochrome c binding domains by chemical modification: Kinetics of reaction with beef mitochondrial reductase
    • S.H. Speck, S. Ferguson-Miller, N. Osheroff, and E. Margoliash Definition of cytochrome c binding domains by chemical modification: kinetics of reaction with beef mitochondrial reductase Proc. Natl. Acad. Sci. U. S. A. 76 1979 155 160
    • (1979) Proc. Natl. Acad. Sci. U. S. A. , vol.76 , pp. 155-160
    • Speck, S.H.1    Ferguson-Miller, S.2    Osheroff, N.3    Margoliash, E.4
  • 98
    • 0021103057 scopus 로고
    • Interaction between cytochrome c and ubiquinone-cytochrome c oxidoreductase: A study with water-soluble carbodiimides
    • H.E. Gutweniger, C. Grassi, and C. Bisson Interaction between cytochrome c and ubiquinone-cytochrome c oxidoreductase: a study with water-soluble carbodiimides Biochem. Biophys. Res. Commun. 116 1983 272 283
    • (1983) Biochem. Biophys. Res. Commun. , vol.116 , pp. 272-283
    • Gutweniger, H.E.1    Grassi, C.2    Bisson, C.3
  • 100
    • 47749126543 scopus 로고    scopus 로고
    • Structure of complex III with bound cytochrome c in reduced state and definition of a minimal core interface for electron transfer
    • S.R.N. Solmaz, and C. Hunte Structure of complex III with bound cytochrome c in reduced state and definition of a minimal core interface for electron transfer J. Biol. Chem. 283 2008 17542 17549
    • (2008) J. Biol. Chem. , vol.283 , pp. 17542-17549
    • Solmaz, S.R.N.1    Hunte, C.2
  • 104
    • 79953801818 scopus 로고    scopus 로고
    • 2 upon interaction with oppositely charged macromolecules probed by SR EPR: Implications for the role of dipole moment to facilitate collisions in proper configuration for electron transfer
    • 2 upon interaction with oppositely charged macromolecules probed by SR EPR: implications for the role of dipole moment to facilitate collisions in proper configuration for electron transfer Metallomics 3 2011 404 409
    • (2011) Metallomics , vol.3 , pp. 404-409
    • Sarewicz, M.1    Pietras, R.2    Froncisz, W.3    Osyczka, A.4
  • 105
    • 0023645713 scopus 로고
    • 1 complex. Nucleotide sequence and homologies between bacterial and mitochondrial subunits
    • 1 complex. Nucleotide sequence and homologies between bacterial and mitochondrial subunits J. Biol. Chem. 262 1987 13805 13811
    • (1987) J. Biol. Chem. , vol.262 , pp. 13805-13811
    • Kurowski, B.1    Ludwig, B.2
  • 106
    • 0023655323 scopus 로고
    • 1 and c. Equilibrium constants and kinetic probes
    • 1 and c. Equilibrium constants and kinetic probes J. Biol. Chem. 262 1987 8103 8108
    • (1987) J. Biol. Chem. , vol.262 , pp. 8103-8108
    • Kim, C.H.1    Balny, C.2    King, T.E.3
  • 108
    • 34447643582 scopus 로고    scopus 로고
    • A Novel Approach to Analyze Membrane Proteins by Laser Mass Spectrometry: From Protein Subunits to the Integral Complex
    • DOI 10.1016/j.jasms.2007.04.013, PII S1044030507003741
    • N. Morgner, T. Kleinschroth, H.D. Barth, B. Ludwig, and B. Brutschy A novel approach to analyze membrane proteins by laser mass spectrometry: from protein subunits to the integral complex J. Am. Soc. Mass Spectrom. 18 2007 1429 1438 (Pubitemid 47088995)
    • (2007) Journal of the American Society for Mass Spectrometry , vol.18 , Issue.8 , pp. 1429-1438
    • Morgner, N.1    Kleinschroth, T.2    Barth, H.-D.3    Ludwig, B.4    Brutschy, B.5


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.