Reconstitution of b-type cytochrome oxidase from Rhodopseudomonas capsulata in liposomes and turnover studies of proton translocation |
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Authors: | Hendrik Hüdig Gerhart Drews |
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Affiliation: | Institut für Biologie II, Mikrobiologie, Albert-Ludwigs-Universität, Schänzlestr. 1, D-7800 Freiburg F.R.G. |
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Abstract: | Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 with uptake of 1 from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K+ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c2-oxidoreductase showed a pumping activity of 0.9 , which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio. |
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Keywords: | Cytochrome oxidase reconstitution Proton translocation Liposome Electron transfer (Rps. capsulata) CCCP DCCD DEAE Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid TMPD |
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