Full recovery of the NADH:ubiquinone activity of complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica by the addition of phospholipids.
NADH:ubiquinone oxidoreductase (complex I) is the largest multiprotein complex of the mitochondrial respiratory chain. His-tagged complex I purified from the strictly aerobic yeast Yarrowia lipolytica exhibited electron transfer rates from NADH to n-decylubiquinone of less than 2% when compared to turnover numbers calculated for native mitochondrial membranes from this organism. Reactivation was observed upon addition of asolectin, purified phospholipids and different phospholipid mixtures. Maximal activities of 6-7 micromol NADH min(-1) mg(-1) were observed following incubation with a mixture of 76% phosphatidylcholine, 19% phosphatidylethanolamine and 5% cardiolipin. For full reactivation, 400-500 phospholipid molecules per complex I were needed. This demonstrated that the inactivation of complex I from Y. lipolytica by general delipidation could be fully reversed simply by returning the phospholipids that had been removed during the purification procedure. Thus, our homogeneous and highly pure complex I preparation had retained its full catalytic potential and no specific, functionally essential component had been lost. As the purified enzyme was also found to contain only substoichiometric amounts of ubiquinone-9 (0.2-0.4 mol/mol), a functional requirement of this endogeneous ubiquinone could also be excluded.
D042967:Electron Transport Complex I; D004789:Enzyme Activation / Q000187:drug effects; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids / Q000032:analysis*; D014451:Ubiquinone / Q000032:analysis; D025062:Yarrowia / Q000201:enzymology*
D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D014451:Ubiquinone; C024990:asolectin; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I