25.06.09 Prof. Simon de Vries
New Techniques to Study Electron Transfer and Proton Pumping in Cytochrome c Oxidase
| What |
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|---|---|
| When |
25.06.2009 from 05:15 to 06:00 |
| Where | SR 1007, Albertstr. 21, 10. OG |
| Contact Name | Angelika Massler |
| Contact Phone | 203 6090 |
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Many enzymes – including those active in respiratory chains – have turnover times shorter than a millisecond. The complexity of their catalytic cycle implies that the formation and breakdown of transient intermediates is faster, in µs. To solve enzyme catalytic mechanisms the precise (electronic) structures of transient intermediates must be known. To this end we have developed the microsecond freeze-hyperquenching technique (MHQ) [1], a hundred-fold faster set up than the classical rapid-freeze-quench device. In this seminar I will briefly describe the scope and limitations of our current MHQ set up and its application in the study of cytochrome oxidases.
The catalytic mechanism – electron transfer coupled to proton pumping – of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa3 reacting with O2 enabling to trap intermediates within 75µs. Intermediates were analyzed by low temperature UV-Visible, X-band and Q-band EPR spectroscopy. A major finding was the detection of a transient tryptophan radical (see Figure), which we propose plays a key role in the proton pumping mechanism of cytochrome oxidases [2-4].