title: Engineering the respiratory complex I to energy-converting NADPH:ubiquinone oxidoreductase.
abstract: The respiratory complex I couples the electron transfer from NADH to ubiquinone with a translocation of protons across the membrane. Its nucleotide-binding site is made up of a unique Rossmann fold to accommodate the binding of the substrate NADH and of the primary electron acceptor flavin mononucleotide. Binding of NADH includes interactions of the hydroxyl groups of the adenosine ribose with a conserved glutamic acid residue. Structural analysis revealed that due to steric hindrance and electrostatic repulsion, this residue most likely prevents the binding of NADPH, which is a poor substrate of the complex. We produced several variants with mutations at this position exhibiting up to 200-fold enhanced catalytic efficiency with NADPH. The reaction of the variants with NAD(P)H is coupled with proton translocation in an inhibitor-sensitive manner. Thus, we have created an energy-converting NADPH:ubiquinone oxidoreductase, an activity so far not found in nature. Remarkably, the oxidation of NAD(P)H by the variants leads to an enhanced production of reactive oxygen species.
authors.affiliation: Institut für Organische Chemie und Biochemie, Albert-Ludwigs-Universität, 79104 Freiburg, Germany.
authors.forename: Klaudia
authors.initials: K
authors.lastname: Morina
authors.forename: Marius
authors.initials: M
authors.lastname: Schulte
authors.forename: Florian
authors.initials: F
authors.lastname: Hubrich
authors.forename: Katerina
authors.initials: K
authors.lastname: Dörner
authors.forename: Stefan
authors.initials: S
authors.lastname: Steimle
authors.forename: Stefan
authors.initials: S
authors.lastname: Stolpe
authors.forename: Thorsten
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D001665:Binding Sites; D042967:Electron Transport Complex I / Q000737:chemistry*; D004583:Electrons; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000737:chemistry; D009249:NADP / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*; D011487:Protein Conformation; D015202:Protein Engineering; D011522:Protons; D017382:Reactive Oxygen Species
chemical_list: D011522:Protons; D017382:Reactive Oxygen Species; D009243:NAD; D009249:NADP; D010088:Oxidoreductases; D042967:Electron Transport Complex I