Proton pumping by complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica reconstituted into proteoliposomes
- 作者：Stefan Drö ; se ; Alexander Galkin and Ulrich Brandt
- 关键词：Mitochondria ; Complex I ; Reconstitution ; H+-pumping ; Yarrowia lipolytica
- 刊名：Biochimica et Biophysica Acta (BBA)/Bioenergetics
- 出版年：2005
- 期刊代码：13_00052728
- 类别：bio
- 出版时间：20 December 2005
- 卷：1710
- 期：2-3
- 页码：87-95
- 文件大小：446 K
摘要
The mechanism of energy converting NADH:ubiquinone oxidoreductase (complex I) is still unknown. A current controversy centers around the question whether electron transport of complex I is always linked to vectorial proton translocation or whether in some organisms the enzyme pumps sodium ions instead. To develop better experimental tools to elucidate its mechanism, we have reconstituted the affinity purified enzyme into proteoliposomes and monitored the generation of ΔpH and Δψ. We tested several detergents to solubilize the asolectin used for liposome formation. Tightly coupled proteoliposomes containing highly active complex I were obtained by detergent removal with BioBeads after total solubilization of the phospholipids with n-octyl-β-d-glucopyranoside. We have used dyes to monitor the formation of the two components of the proton motive force,ΔpH and Δψ, across the liposomal membrane, and analyzed the effects of inhibitors, uncouplers and ionophores on this process. We show that electron transfer of complex I of the lower eukaryote Y. lipolytica is clearly linked to proton translocation. While this study was not specifically designed to demonstrate possible additional sodium translocating properties of complex I, we did not find indications for primary or secondary Na+ translocation by Y. lipolytica complex I.