Covalently bound flavin in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus.
Na(+)-translocating NADH-quinone reductase (NQR) from the marine bacterium Vibrio alginolyticus is composed of six subunits (NqrA to NqrF). On SDS-PAGE of the purified complex, NqrB and NqrC subunits were found to give yellow-green fluorescent bands under UV illumination. Both the NqrB and NqrC, electroeluted from the gel, had an absorption maximum at 448 nm, and the fluorescence excitation maxima at 365 and 448 nm and the emission maximum at 514 nm. The electroeluted NqrB and NqrC, respectively, were identified from their N-terminal amino acid sequences. These results clearly indicated that the NqrB and NqrC subunits have covalently bound flavins. The two subunits were digested by protease and then the fluorescent peptide fragments were separated by a reversed-phase high performance liquid chromatography. N-Terminal amino acid sequence analyses of the fluorescent peptides revealed that the flavin is linked to Thr-235 in the NqrB and Thr-223 in the NqrC subunits. This is the first example that the flavin is linked to a threonine residue. The amino acid sequence around the flavin-linked threonine was well conserved between NqrB and NqrC. Identification of the flavin group is in progress.
Nakayama
Y
Y
Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, 263-8522, Chiba, Japan. nakayama@p.chiba-u.ac.jp
Yasui
M
M
Sugahara
K
K
Hayashi
M
M
Unemoto
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D017124:Conserved Sequence / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011808:Quinone Reductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013912:Threonine / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology*
D001426:Bacterial Proteins; D005415:Flavins; D010446:Peptide Fragments; D005182:Flavin-Adenine Dinucleotide; D013912:Threonine; D005486:Flavin Mononucleotide; C084142:sodium-translocating NADH-quinone reductase; D011808:Quinone Reductases; D010450:Endopeptidases