The hyperthermophilic archaeon Acidianus ambivalens expresses a membrane-bound aa(3)-type quinol oxidase, when grown aerobically, that we have studied by resonance Raman spectroscopy. The purified aa(3) oxidase, which does not contain bound quinol, undergoes a reversible slow conformational change at heme a(3) upon reduction, as indicated by a change in the frequency of its heme formyl stretching mode, from 1,660 cm(-1) to 1,667 cm(-1). In contrast, upon reduction of the integral membrane enzyme or the purified enzyme preincubated with decylubiquinol, this mode appears at 1,667 cm(-1) much more rapidly, suggesting a role of the bound quinol in controlling the redox-linked conformational changes. The shift of the formyl mode to higher frequency is attributed to a loss of hydrogen bonding that is associated with a group having a pKa of approximately 3.8. Based on these observations, a crucial element for proton translocation involving a redox-linked conformational change near the heme a(3) formyl group is postulated.