Observation of the Fe–O₂ 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 O-20 ps following photolysis of the enzyme-CO adduct in the presence of 0,, we observed the Fe&& stretching Raman band at 568 cm-' which shifted to 535 cm-' with the "0, derivative. These frequencies are remarkably close to those of other oxyhemoproteins including dioxygen-bound hemoglobin and aa,-type cytochrome c oxidase. In the later time range (20-40 ps), other oxygen-isotope-sensitive Raman bands were observed at 788 and 361 cm-'. Since the 781 cm-' band exhibited a downshift by 37 cm-' upon '*O, substitution, we assigned it to the Fe'"=0 stretching mode. This band is considered to arise from the ferry1 intermediate, but its appearance was much earlier than the corresponding intermediate of bovine cytochrome c oxidase (>I00 ps). The 361 cn-' band showed the '60/'s0 isotopic frequency shift of I4 cm-' similar to the case of bovine cytochrome c oxidase reaction.

Role of Tyr-288 at the Dioxygen Reduction Site of Cytochrome bo Studied by Stable Isotope Labeling and Resonance Raman Spectroscopy

“…The resulting "oxy" (O) intermediate is rapidly transformed to the so-called "peroxy" (P) species, which has an oxidation state of the hemecopper moiety higher than the Fe 3ϩ /Cu 2ϩ state by two oxidative equivalent. Although the peroxy intermediate had been expected to have an intact O-O bond (11,12), our previous studies have clearly shown the definite FeϭO stretching Raman band at ϳ800 cm Ϫ1 for the P intermediate, suggesting that the O-O bond is already cleaved at this step (13)(14)(15)(16). Other chemical experiments also support this assignment (17,18).…”

Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen

“…Time‐resolved resonance Raman studies showed the formation of the oxoferryl intermediate with a rate constant of about 2×10 4 s −1 [20,21] which is comparable to about 5×10 4 s −1 for the fast phase observed by visible spectroscopy [13,16,19,22]. The final product of the fast reaction with peaks at 557 and about 420 nm could be the oxoferryl intermediate [19] which then decays to the oxidized state within 1 s [13,16,21,22].…”

Infrared and EPR Studies on Cyanide Binding to the Heme-Copper Binuclear Center of Cytochrome bo-type Ubiquinol Oxidase from Escherichia coli

“…Although the dioxygen reduction mechanism at the hemecopper binuclear center is thought to be identical in both enzymes (12)(13)(14), these differences raise the questions whether the electron transfer reactions from the substrates to dioxygen and the proton pumping mechanism coupled to these redox reactions are alike or distinct. It is proposed that the last two steps of the four-electron transfer reactions to dioxygen are linked to proton pumping by cytochrome c oxidase (15)(16)(17).…”