Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex I

• Published in: Molecular microbiology 2015-10, Vol.98 (1), p.151-161
• Main Authors: Steimle, Stefan, Schnick, Christian, Burger, Eva‐Maria, Nuber, Franziska, Krämer, Dorothée, Dawitz, Hannah, Brander, Sofia, Matlosz, Bartlomiej, Schäfer, Jacob, Maurer, Katharina, Glessner, Udo, Friedrich, Thorsten
• Format: Article
• Language: English
• Subjects: Amino acids; Arm; Cysteine; E coli; Electron Spin Resonance Spectroscopy; Electron transfer; Electron Transport; Electron transport chain; Electron Transport Complex I - chemistry; Electron Transport Complex I - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Flexibility; Fluorescence; Fluorescent indicators; Labeling; Membranes; Models, Molecular; Mutation; NAD - metabolism; NADH; NADH Dehydrogenase - chemistry; NADH Dehydrogenase - metabolism; NADH-ubiquinone oxidoreductase; Nicotinamide adenine dinucleotide; Oxidation-Reduction; Protons; Redox reactions; Scanning; Translocation; Ubiquinone; Ubiquinone - metabolism; Ubiquinone oxidoreductase
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.13112

Summary The NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the redox reaction and a membrane arm catalyzing proton translocation. The membrane arm is almost completely aligned by a 110 Å unique horizontal helix that is discussed to transmit conformational changes induced by the redox reaction in a piston‐like movement to the membrane arm driving proton translocation. Here, we analyzed such a proposed movement by cysteine‐scanning of the helix of the Escherichia coli complex I. The accessibility of engineered cysteine residues and the flexibility of individual positions were determined by labeling the preparations with a fluorescent marker and a spin‐probe, respectively, in the oxidized and reduced states. The differences in fluorescence labeling and the rotational flexibility of the spin probe between both redox states indicate only slight conformational changes at distinct positions of the helix but not a large movement. The proposed movement of the horizontal helix of respiratory complex I was directly investigated by labeling distinct positions of the helix. There was not significant change in TMR‐labeling upon reduction of the oxidized complex. The mobility of an EPR spin‐label attached to these positions was very similar in the oxidized and the reduced state of the complex indicating small local conformational changes within the helix in both redox states.