Resonance Raman evidence for an exchangeable protein hydrogen associated with the hemeagroup of cytochrome oxidase

Abstract

When cytochrome–c oxidase is soaked in D₂O, downshifts of the cytochrome a formyl C=O stretching mode are seen in the resonance Raman (RR) spectra (413.1 nm excitation) of both the resting and reduced forms. Other changes observed in the reduced protein RR spectra are consistent with involvement of the cytochrome a formyl group in the deuterium effect. The D₂O–induced RR changes are fully developed during 3–5 days incubation, but are incomplete after 1 h. Extraction of the heme a chromophore in deuterated solvents eliminates these changes, implying that the exchangeable proton is on a protein group in the cytochrome a pocket which H–bonds to the heme formyl. The rate of the D₂O exchange process is unaffected by enzyme turnover, thus reducing the likelihood that the cytochrome a formyl H–bond is directly involved in the redox–linked mechanism of proton pumping.