Mass spectrometric analysis of the ubiquinol-binding site in cytochrome bd from Escherichia coli.
Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu(278)-Lys(283), where a photoproduct of azido-Q(2) was linked to the carboxylic side chain of I-Glu(280). This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu(280).
Matsumoto
Yushi
Y
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan.
Murai
Masatoshi
M
Fujita
Daisuke
D
Sakamoto
Kimitoshi
K
Miyoshi
Hideto
H
Yoshida
Masasuke
M
Mogi
Tatsushi
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D045222:Electron Transport Chain Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D004583:Electrons; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry; D008024:Ligands; D008027:Light; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008666:Metalloendopeptidases / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014451:Ubiquinone / Q000031:analogs & derivatives* / Q000737:chemistry; D014466:Ultraviolet Rays
D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D010455:Peptides; D014451:Ubiquinone; D018698:Glutamic Acid; D010088:Oxidoreductases; C020094:duroquinol oxidase; C432586:ubiquinol oxidase; C040077:cytochrome bd terminal oxidase complex, E coli; D010450:Endopeptidases; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N; D008239:Lysine; C003741:ubiquinol