Transient binding of CO to Cu(B) in cytochrome c oxidase is dynamically linked to structural changes around a carboxyl group: a time-resolved step-scan Fourier transform infrared investigation.
The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the mid-infrared range (2300-950 cm(-1)). Difference spectra of the bovine complex were obtained at -20 degrees C with 5 micros time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu(B) and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK(a) of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme.
Heitbrink
Dirk
D
Forschungszentrum Jülich, IBI-2: Structural Biology, 52425 Jülich, Germany.
Sigurdson
Håkan
H
Bolwien
Carsten
C
Brzezinski
Peter
P
Heberle
Joachim
J
D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry*; D002264:Carboxylic Acids / Q000737:chemistry; D002417:Cattle; D003300:Copper / Q000737:chemistry*; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008929:Mitochondria, Heart / Q000201:enzymology; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014867:Water
D002264:Carboxylic Acids; D014867:Water; D003300:Copper; D002248:Carbon Monoxide; D003576:Electron Transport Complex IV