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

Traditional terminal heme-copper oxidases (HCuOs) are transmembrane proteins that catalyze the final step in the respiratory chain − the exergonic reduction of O2 to water, coupled to energy conservation by generation of an electrochemical proton gradient. This is usually achieved by taking electrons and protons from opposite sides of the membrane. 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. Sequence homology searches have identified the bacterial nitric oxide reductases (NORs) as divergent members of the HCuOsuperfamily. The NORs are involved in the denitrification pathway where they catalyze the reduction of NO to NO2. Although as exergonic as O2reduction, this reaction is completely non-electrogenic as protons and electrons are taken from the same side of the membrane and no protons are pumped. In addition, some NORs are capable of reducing O2. Among the traditional HCuOs, the cbb3-type oxidases are the closest relatives to the NORs and as such provide a link between the aa3-type oxidases and the NORs. They are the HCuOs with the highest NO-reduction capability (excluding the NORs) but have also a high affinity for O2. 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 (conventionally termed electrogenic events), which occur during the catalytic reaction, 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 potential buildup in CytcO revealed 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 a large extent from charge-transfer within the K-pathway. Possible reasons for uncoupling, and the extent of charge-transfer, were studied using structural variants of CytcO, where the D-pathway had been altered. The electrometric measurements also established that both electrons and protons are taken up from the same side of the membrane in the NOR. The directionality for proton uptake in the cbb3 oxidase appeared to be dependent on the choice of substrate and proton pumping was indicated to occur only during the reduction of O2. List of publications This thesis is based on the following publications, which will be referred to by their roman numerals: I Håkan Lepp and Peter Brzezinski, “pH dependence of voltage changes associated with intramolecular electron and proton transfer reactions in cytochrome c oxidase”, Manuscript in preparation. II Håkan Lepp, Emelie Svahn, Kristina Faxén and Peter Brzezinski, “Charge transfer in the K proton pathway linked to electron transfer to the catalytic site in cytochrome c oxidase”, Biochemistry, 2008. 47(17): 4929−4935. III Håkan Lepp, Lina Salomonsson, Jia-Peng Zhu, Robert B. Gennis and Peter Brzezinski, “Impaired proton pumping in cytochrome c oxidase upon structural alteration of the D pathway”, Biochim. Biophys. Acta − Bioenergetics, 2008. 1777(7-8): 897−903. IV Andreas Namslauer, Håkan Lepp, Magnus Brändén, Audrius Jasaitis, Michael I. Verkhovsky and Peter Brzezinski, “Plasticity of proton pathway structure and water coordination in cytochrome c oxidase”, J. Biol. Chem., 2007. 282(20): 15148−15158. V Joachim Reimann, Ulrika Flock, Håkan Lepp, Alf Honigmann and Pia Ädelroth, “A pathway for protons in nitric oxide reductase from Paracoccus denitrificans”, Biochim. Biophys. Acta − Bioenergetics, 2007. 1767(5): 362−373. VI Yafei Huang, Joachim Reimann, Håkan Lepp, Nadjia Drici and Pia Ädelroth, “Substrate control of vectorial proton transfer in a hemecopper oxidase”, Submitted to Proc. Natl. Acad. Sci. USA.