Redox-induced protein structural changes in cytochrome bo revealed by Fourier transform infrared spectroscopy and [13C]Tyr labeling.
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 Cepsilon-Nepsilon covalent bond with one of the Cu(B) 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 (approximately 1620-1680 cm(-1)) and -II ( approximately 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-Cu(B) 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.
Kandori
Hideki
H
Department of Materials Science and Engineering, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555. kandori@nitech.ac.jp
Nakamura
Hiro
H
Yamazaki
Yoichi
Y
Mogi
Tatsushi
T
D000818:Animals; D001671:Biochemistry / Q000379:methods; D002417:Cattle; D003300:Copper / Q000737:chemistry; D003580:Cytochromes / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008027:Light; D008956:Models, Chemical; D008958:Models, Molecular; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry; D011188:Potassium / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D014443:Tyrosine / Q000737:chemistry*
D003580:Cytochromes; D008024:Ligands; D018698:Glutamic Acid; D014443:Tyrosine; D006639:Histidine; D003300:Copper; D006859:Hydrogen; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV; D011188:Potassium; D010100:Oxygen