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| Home > Publications database > Infrarotspektroskopische Untersuchungen zum Mechanismus der Cytochrom-c-Oxidase |
| Home > Publications database > Infrarotspektroskopische Untersuchungen zum Mechanismus der Cytochrom-c-Oxidase |
| Dissertation / PhD Thesis/Book | PreJuSER-44314 |
2002
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/83
Report No.: Juel-3969
Abstract: Cytochrome c oxidase is a complex biological machinery, which couples the reduction of oxygen to a vectorial transport of protons across the membrane. To achieve this, the protein takes up eight protons, four electrons, and an oxygen molecule. The electrons and four of the eight protons are used for the reduction of oxygen to produce water. The remaining four protons are pumped across the membrane. In this work, Fourier-transorm infrared spectroscopy was applied to provide insight into the structural and functional relationships of the complex catalytic cycle. To accomplish this, different strategies were applied: The results obtained with the $\textit{fully reduced}$, CO-bound cytochrome c oxidase from bovine heart showed for the first time the possibility of performing difference spectroscopy on this enzyme with a time-resolution of 5 $\mu$s in the range of 2200 to 950 cm$^{-1}$. The evaluation of the data revealed a dynamic link between the transient binding of CO to Cu$_{B}$ and the movement of an amino acid side chain (E286) approximately 12 $\mathring{A}$ away. This amino acid is part of the D-channel and thought to play an important role in guiding the protons either into the binuclear center or through the membrane. The results presented in this work suggest a mechanism in which oxygen binding is controlled via E286. The outcome of the measurements with the $\textit{two electron-reduced}$, CO-bound cytochrome c oxidase from $\textit{Rhodobacter sphaeroides}$ indicate the deprotonation of E286 during electron transfer from heme a$_{3}$ to heme a. This result contradicts the current model of the catalytic-cycle because this deprotonation suggests an uptake of just one proton rather than two through the K-channel. Redox difference spectra of wild type and mutant cytochrome c oxidase obtained with the attenuated total reflection technique pointed out the precise assignment of a band feature. This signal was clearly attributed to a conformational change of the side chain of amino acid E286. For the first time it was possible to record a difference spectrum in the IR of the oxidative part of the catalytic cycle. The F $\textit{minus}$ O spectrum showed an involvement of a tyrosine which undergoes deprotonation.
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