Characterization of a subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I) lacking the flavoprotein part of the N-module |
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Authors: | Volker Zickermann |
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Institution: | Johann Wolfgang Goethe-Universität, Fachbereich Medizin, Molekulare Bioenergetik, Centre of Excellence Frankfurt “Macromolecular Complexes”, D-60590 Frankfurt am Main, Germany |
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Abstract: | Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron-sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b. |
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Keywords: | DBQ n-decylubiquinone dSDS-PAGE doubled sodium dodecylsulfate polyacrylamide gel electrophoresis DQA 2-decyl-4-quinazolinyl amine EPR electron paramagnetic resonance HAR hexaammineruthenium(III) MES 2-(N-Morpholino)-ethanesulfonic acid Tris Tris(hydroxymethyl)aminomethane |
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