IR changes caused by photolysis of CO from the mixed valence form of bovine cytochrome c oxidase have been investigated over the pH/pD range 6-9.8. Band assignments were based on effects of H2O/D2O exchange and by comparisons with published IR data and crystallographic data. Changes arise both from CO photolysis and from subsequent reversed electron transfer from heme a3 to heme a. This reversed electron transfer is known to have pH-independent and, above pH 8, pH-dependent components. The pH-independent component is associated with a trough around the 1742 cm(-1) band attributable to one or more protonated carboxylic acids. Its peak position, but not extent, is pH-dependent, indicative of a titratable group with a pK of 8.2 whose acid form causes increased hydrogen bonding to the IR-detectable carboxylic group. A different protonatable group with pK above 9 controls the extent of the pH-dependent component. This phase is associated with perturbation of an arginine guanidinium that is most clearly observed as a trough at 1592 cm(-1) after H/D exchange. It is suggested that this group, probably Arg-438 that is in close contact with propionate groups of both hemes and already proposed to be of functional significance, lowers the energy of the transient charge-uncompensated electron-transfer intermediate by changing the charge distribution in response to heme-heme electron transfer. No other IR signature of a titratable group that controls the extent of the pH-dependent phase is present, and it most likely arises from a nonphysiological deprotonation of the proximal water ligand of ferric heme a3 at high pH that has been reported to exhibit a similar pK.