Resonance Raman spectra of the carbon monoxy derivative of the aay-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 (α and β) 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 a-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. On the other hand, the β-form has an open structure typical of heme proteins with histidine coordination to the iron trans to a Fe-C-O moiety where there is no interaction with a copper atom in the distal pocket. The functional significance of these two forms of the enzyme remains to be determined.
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