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 in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy 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 a3 to CuB was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes. Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature. Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme. FT-IR difference spectra show band features similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes induced 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 2H2O. The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.
Rost
B
B
Institut für Biophysik der Universität Frankfurt, Germany.
Behr
J
J
Hellwig
P
P
Richter
O M
OM
Ludwig
B
B
Michel
H
H
Mäntele
W
W
D002248:Carbon Monoxide / Q000737:chemistry*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*; D010782:Photolysis; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors
D011522:Protons; D005973:Glutamine; D018698:Glutamic Acid; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV