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Experimental studies of proton translocation reactions in biological systems : Electrogenic events in heme-copper oxidases

<p>Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O<sub>2</sub> to H<sub>2</sub>O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the <i>aa</i><sub>3</sub>-type oxidases, to which cytochrome <i>c</i> oxidase (Cyt<i>c</i>O) belongs, which uses energy released in the O<sub>2</sub>-reduction for proton pumping. The bacterial nitric oxide reductases (NORs) have been identified as divergent members of the HCuO-superfamily and are involved in the denitrification pathway where they catalyze the reduction of NO to NO<sub>2</sub>. Although as exergonic as O<sub>2</sub>-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the <i>cbb</i><sub>3</sub>-type oxidases are the closest relatives to the NORs and as such provide a link between the <i>aa</i><sub>3</sub> oxidases and the NORs. The <i>cbb</i><sub>3</sub> oxidases have been shown to pump protons with nearly the same efficiency as the <i>aa</i><sub>3</sub> oxidases, despite low sequence similarity.</p><p>This thesis is focused on measurements of membrane potential generating reactions during catalysis in the Cyt<i>c</i>O and the <i>cbb</i><sub>3</sub> oxidase from <i>Rhodobacter sphaeroides</i>, and the NOR from <i>Paracoccus</i> <i>denitrificans</i>, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in Cyt<i>c</i>O showed that only one proton is taken up and that no protons are pumped, at high pH. An additional kinetic phase was also detected at high pH that presumably originates to from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of Cyt<i>c</i>O. The measurements established that electrons and protons are taken up from the same side of the membrane in NOR. In addition, the directionality for proton uptake in <i>cbb</i><sub>3</sub> oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O<sub>2</sub>-reduction.</p>

Identiferoai:union.ndltd.org:UPSALLA/oai:DiVA.org:su-8147
Date January 2008
CreatorsLepp, Håkan
PublisherStockholm University, Department of Biochemistry and Biophysics, Stockholm : Institutionen för biokemi och biofysik
Source SetsDiVA Archive at Upsalla University
LanguageEnglish
Detected LanguageEnglish
TypeDoctoral thesis, comprehensive summary, text

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