title: Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2).
abstract: Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O₂ activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC₅₀ = ∼1 μm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O₂ activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O₂ activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells.
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Mahmoud M
authors.initials: MM
authors.lastname: Elguindy
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Electronic address: eikoo@mail.med.upenn.edu.
authors.forename: Eiko
authors.initials: E
authors.lastname: Nakamaru-Ogiso
mesh_terms: D051033:Apoptosis Inducing Factor / Q000235:genetics / Q000378:metabolism*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D042967:Electron Transport Complex I / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D015870:Gene Expression; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D007527:Isoenzymes / Q000172:deficiency / Q000235:genetics; D007700:Kinetics; D008565:Membrane Proteins / Q000172:deficiency / Q000235:genetics*; D051336:Mitochondrial Membranes; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C494829:AIFM1 protein, human; C462504:ferroptosis suppressor protein 1, human; D051033:Apoptosis Inducing Factor; D051017:Apoptosis Regulatory Proteins; D029968:Escherichia coli Proteins; D007527:Isoenzymes; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; C540656:NuoH protein, E coli; D011994:Recombinant Proteins; D009243:NAD; D042967:Electron Transport Complex I
keywords: apoptosis; apoptosis-inducing factor; complex I; mitochondrial metabolism; mitochondrial respiratory chain complex; nicotinamide adenine dinucleotide (NADH); rotenone; ubiquinone