Resonance Raman spectra of the carbon monoxy derivative of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides show two distinct Fe-CO stretching modes (519 and 493 cm-1) at room temperature. The frequency of the mode at 519 cm-1 coincides with that of other terminal oxidases at neutral pH. Two C-O stretching modes, one at 1966 cm-1 and one at 1955 cm-1, are also found. The splitting of the C-O stretching mode is consistent with the FTIR spectra of cytochrome c oxidases at cryogenic temperatures in which two different conformations (alpha and beta) of the catalytic site of the enzyme are present. The splitting of both the Fe-CO and C-O stretching modes under our conditions indicates that these two forms of the enzyme are also present at room temperature, and with the additional information on the Fe-CO modes provided here, a structural origin for the two forms may be postulated. The alpha-form has the same general structure of the active site as mammalian oxidase, a structure in which the copper atom that is the part of the Fe-CuB binuclear site interacts strongly with the bound CO. We postulate that the copper atom exerts a strong polar or steric effect on the heme-bound CO, resulting in either compression of the Fe-CO bond or distortion of the Fe-CO moiety.(ABSTRACT TRUNCATED AT 250 WORDS)