In Yarrowia Lipolityca Mitochondria the Association of NADH Dehydrogenase Type II with the Cytochrome Complexes Depends on the Growth Phase

Abstract

In Yarrowia lipolytica mitochondria, the electron transport from NADH to O2 is branched by alternative respiratory components. One external NADH dehydrogenase (NDH2e) and an alternative oxidase (AOX). Both enzymes are peripheral single-subunit oxido-reductases not implicated in proton gradient formation. Thus, if electrons pass through those two enzymes, the oxidation of NADH is not able to conserve energy. During exponential growth, this is undesirable; however, during the stationary phase this process may help to maintain a high rate of oxygen consumption. To prevent the electron flux between alternative components, either AOX may be interacting with the complex I or NDH2e with complexes III and IV. We have evaluated the participation of the alternative components on electron transport and on supramolecular structures of mitochondria from wild type and a αnubm mutant, where complex I is inactive and NDH2 was redirected to the matrix side (NDH2i)4. In order to determine whether there are specific interactions between NDH2e and other respiratory complexes, we measured oxygen consumption rates with different respiratory substrates and inhibitors. We suggested an interaction of NDH2e (but not NDH2i) with cytochrome complexes, indicating that the interaction sites are located in the intermembrane face of the cytochromic complexes. Furthermore, we identified by native PAGE, in-gel activity and mass spectrometry this interaction between NDH2e with complexes III and IV in the wild type. Also, larger supercomplexes5 and a complex V dimer were found. This association pattern seems to vary during the stationary phase as NDH2e is overproduced saturating its binding site in Cyt IV and thus appearing as the free enzyme.