Role of Tyr-288 at the dioxygen reduction site of cytochrome bo studied by stable isotope labeling and resonance raman spectroscopy.
To explore the role of a cross-link between side chains of Tyr-288 and His-284 at the heme-copper binuclear center, we prepared cytochrome bo where d(4)-Tyr, 1-[(13)C]Tyr, or 4-[(13)C]Tyr has been biosynthetically incorporated. Unexpectedly, the d(4)-Tyr-labeled enzyme showed a large decrease in the ubiquinol-1 oxidase and CO binding activities. Optical absorption and resonance Raman spectra identified the defect in the distal side of the heme-copper binuclear center. In the CO-bound d(4)-Tyr-labeled enzyme, a large fraction of the nu((Fe-C)) mode was shifted from the normal 520-cm(-1) band to a broad band centered around 491 cm(-1), as found for the Y288F mutant. Our results suggested that the substitution of ring hydrogens of Tyr-288 with deuteriums slows down the formation of the His-Tyr cross-link essential for dioxygen reduction at the binuclear center.
Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Myodaiji, Okazaki, Aichi 444-8787, Japan.
Takeshi
T
Uchida
Tatsushi
T
Mogi
Hiro
H
Nakamura
Teizo
T
Kitagawa
D002248:Carbon Monoxide / Q000737:chemistry; D002384:Catalysis; D003300:Copper / Q000737:chemistry*; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D007477:Ions; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D009573:Nitrites / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D011808:Quinone Reductases / Q000737:chemistry*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman / Q000379:methods; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry*
D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D007477:Ions; D008670:Metals; D009573:Nitrites; D014443:Tyrosine; D006418:Heme; D006639:Histidine; D003300:Copper; D002248:Carbon Monoxide; D006859:Hydrogen; C017168:cytochrome bo, E coli; D011808:Quinone Reductases; C030914:ubiquinol-1 oxidase; D010100:Oxygen