Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

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<p>Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O₂ to H₂O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the <i>aa</i>₃-type oxidases, to which cytochrome <i>c</i> oxidase (Cyt<i>c</i>O) belongs, which uses energy released in the O₂-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 NO₂. Although as exergonic as O₂-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the <i>cbb</i>₃-type oxidases are the closest relatives to the NORs and as such provide a link between the <i>aa</i>₃ oxidases and the NORs. The <i>cbb</i>₃ oxidases have been shown to pump protons with nearly the same efficiency as the <i>aa</i>₃ 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>₃ 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>₃ oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O₂-reduction.</p>

Terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain - the reduction of O2 to H2O, coupled to energy conservation by generation of an electrochemical proton gradient. The most extensively investigated of the HCuOs are the aa3-type oxidases, to which cytochrome c oxidase (CytcO) belongs, which uses energy released in the O2-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 NO2. Although as exergonic as O2-reduction, this reaction is completely non-electrogenic. Among the traditional HCuOs, the cbb3-type oxidases are the closest relatives to the NORs and as such provide a link between the aa3 oxidases and the NORs. The cbb3 oxidases have been shown to pump protons with nearly the same efficiency as the aa3 oxidases, despite low sequence similarity. This thesis is focused on measurements of membrane potential generating reactions during catalysis in the CytcO and the cbb3 oxidase from Rhodobacter sphaeroides, and the NOR from Paracoccus denitrificans, using a time resolved electrometric technique. The pH dependence of the membrane potential generation in CytcO 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 CytcO. 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 cbb3 oxidase appeared to be dependent on the choice of substrate while proton pumping was indicated to occur only during O2-reduction.