Role of Tyr-288 at the dioxygen reduction site of cytochrome bo studied by stable isotope labeling and resonance raman spectroscopy

Abstract

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 d4-Tyr, 1-[13C]Tyr, or 4-[13C]Tyr has been biosynthetically incorporated. Unexpectedly, the d4-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 ν(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.