Time-resolved FT-IR studies on the CO adduct of Paracoccus denitrificans cytochrome c oxidase: Comparison of the fully reduced and the mixed valence form

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans i n the fully reduced and in the half-reduced (mixed valence) form as a funct ion of temperature was investigated using time-resolved rapid-scan FT-IR sp ectroscopy in the mid-IR (1200-2100 cm(-1)). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction. At 84 K, nonreversible transfer of CO from heme a(3) to Cu-B was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR d ifference spectra which show similarities in the amide I, amide II, and hem e modes. Both processes, however, differ in spectral features characteristi c for amino acid side chain modes and may thus be indicative for the motion al constraint of CO at low temperature. Rebinding of photodissociated CO fo r the mixed-valence enzyme at 268 K is also biphasic, but much slower as co mpared to the fully reduced enzyme. FT-IR difference spectra show band feat ures similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes in duced by electron and coupled proton transfer. These signals disappear when the temperature is lowered to 84 K. At 268 K, a difference signal at 1746 cm(-1) is observed which is shifted by 6 cm(-1) to 1740 cm(-1) in (H2O)-H-2 . The absence of this signal for the mutant Glu 278 Gin allows assignment t o the COOH stretching mode of Glu 278, and indicates changes of the conform ation, proton position, or protonation of this residue upon electron transf er.