title: Effective pumping proton collection facilitated by a copper site (CuB) of bovine heart cytochrome c oxidase, revealed by a newly developed time-resolved infrared system.
abstract: X-ray structural and mutational analyses have shown that bovine heart cytochrome c oxidase (CcO) pumps protons electrostatically through a hydrogen bond network using net positive charges created upon oxidation of a heme iron (located near the hydrogen bond network) for O2 reduction. Pumping protons are transferred by mobile water molecules from the negative side of the mitochondrial inner membrane through a water channel into the hydrogen bond network. For blockage of spontaneous proton back-leak, the water channel is closed upon O2 binding to the second heme (heme a3) after complete collection of the pumping protons in the hydrogen bond network. For elucidation of the structural bases for the mechanism of the proton collection and timely closure of the water channel, conformational dynamics after photolysis of CO (an O2 analog)-bound CcO was examined using a newly developed time-resolved infrared system feasible for accurate detection of a single C=O stretch band of α-helices of CcO in H2O medium. The present results indicate that migration of CO from heme a3 to CuB in the O2 reduction site induces an intermediate state in which a bulge conformation at Ser-382 in a transmembrane helix is eliminated to open the water channel. The structural changes suggest that, using a conformational relay system, including CuB, O2, heme a3, and two helix turns extending to Ser-382, CuB induces the conformational changes of the water channel that stimulate the proton collection, and senses complete proton loading into the hydrogen bond network to trigger the timely channel closure by O2 transfer from CuB to heme a3.
authors.affiliation: From the Picobiology Institute,; PRESTO and.
authors.forename: Minoru
authors.initials: M
authors.lastname: Kubo
authors.affiliation: From the Picobiology Institute.
authors.forename: Satoru
authors.initials: S
authors.lastname: Nakashima
authors.affiliation: Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamighori, Akoh, Hyogo 678-1297 and.
authors.forename: Satoru
authors.initials: S
authors.lastname: Yamaguchi
authors.affiliation: From the Picobiology Institute,; Department of Life Science, Graduate School of Life Science, University of Hyogo, 3-2-1 Kouto, Kamighori, Akoh, Hyogo 678-1297 and; CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ogura
authors.affiliation: From the Picobiology Institute.
authors.forename: Masao
authors.initials: M
authors.lastname: Mochizuki
authors.affiliation: From the Picobiology Institute.
authors.forename: Jiyoung
authors.initials: J
authors.lastname: Kang
authors.affiliation: From the Picobiology Institute,; CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tateno
authors.affiliation: From the Picobiology Institute.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Shinzawa-Itoh
authors.affiliation: From the Picobiology Institute.
authors.forename: Koji
authors.initials: K
authors.lastname: Kato
authors.affiliation: From the Picobiology Institute,; CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: yoshi@sci.u-hyogo.ac.jp.
authors.forename: Shinya
authors.initials: S
authors.lastname: Yoshikawa
mesh_terms: D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D017433:Protein Structure, Secondary; D017494:Proton Pumps / Q000737:chemistry / Q000378:metabolism; D013055:Spectrophotometry, Infrared
chemical_list: D009124:Muscle Proteins; D017494:Proton Pumps; D006418:Heme; D003300:Copper; D003576:Electron Transport Complex IV
keywords: Bioenergetics; Biophysics; Infrared Spectroscopy; Membrane Proteins; Respiratory Chain