Observation of the FeO 2 and Fe IVO stretching Raman bands for dioxygen reduction intermediates of cytochrome bo isolated from Escherichia coli

Abstract

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 μs following photolysis of the enzyme—CO adduct in the presence of O 2, we observed the FeO 2 stretching Raman band at 568 cm −1 which shifted to 535 cm −1 with the 18O 2 derivative. These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen-bound hemoglobin and aa 3-type cytochrome c oxidase. In the later time range (20–40 μs), 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 18O 2 substitution, we assigned it to the Fe IV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 μs). 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.