Observation of the Fe-O2 and FeIV=O stretching Raman bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli.
Reaction intermediates in dioxygen reduction by the E. coli cytochrome bo-type ubiquinol oxidase were studied by time-resolved resonance Raman spectroscopy using the artificial cardiovascular system. At 0-20 microseconds following photolysis of the enzyme-CO adduct in the presence of O2, we observed the Fe-O2 stretching Raman band at 568 cm-1 which shifted to 535 cm-1 with the 18O2 derivative. These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen-bound hemoglobin and aa3-type cytochrome c oxidase. In the later time range (20-40 microseconds), other oxygen-isotope-sensitive Raman bands were observed at 788 and 361 cm-1. Since the 781 cm-1 band exhibited a downshift by 37 cm-1 upon 18O2 substitution, we assigned it to the FeIV=O stretching mode. This band is considered to arise from the ferryl intermediate, but its appearance was much earlier than the corresponding intermediate of bovine cytochrome c oxidase (> 100 microseconds). The 361 cm-1 band showed the 16O/18O isotopic frequency shift of 14 cm-1 similar to the case of bovine cytochrome c oxidase reaction.
Hirota
S
S
Graduate University for Advanced Studies, Okazaki National Research Institutes, Japan.
Mogi
T
T
Ogura
T
T
Hirano
T
T
Anraku
Y
Y
Kitagawa
T
T
D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D007501:Iron / Q000737:chemistry; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry*; D013059:Spectrum Analysis, Raman
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C066317:ferryl iron; C017168:cytochrome bo, E coli; D007501:Iron; D010100:Oxygen