title: Semiquinone and cluster N6 signals in His-tagged proton-translocating NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli.
abstract: NADH:ubiquinone oxidoreductase (complex I) pumps protons across the membrane using downhill redox energy. The Escherichia coli complex I consists of 13 different subunits named NuoA-N coded by the nuo operon. Due to the low abundance of the protein and some difficulty with the genetic manipulation of its large ~15-kb operon, purification of E. coli complex I has been technically challenging. Here, we generated a new strain in which a polyhistidine sequence was inserted upstream of nuoE in the operon. This allowed us to prepare large amounts of highly pure and active complex I by efficient affinity purification. The purified complex I contained 0.94 ± 0.1 mol of FMN, 29.0 ± 0.37 mol of iron, and 1.99 ± 0.07 mol of ubiquinone/1 mol of complex I. The extinction coefficient of isolated complex I was 495 mM(-1) cm(-1) at 274 nm and 50.3 mM(-1) cm(-1) at 410 nm. NADH:ferricyanide activity was 219 ± 9.7 μmol/min/mg by using HEPES-Bis-Tris propane, pH 7.5. Detailed EPR analyses revealed two additional iron-sulfur cluster signals, N6a and N6b, in addition to previously assigned signals. Furthermore, we found small but significant semiquinone signal(s), which have been reported only for bovine complex I. The line width was ~12 G, indicating its neutral semiquinone form. More than 90% of the semiquinone signal originated from the single entity with P&frac12; (half-saturation power level) = 1.85 milliwatts. The semiquinone signal(s) decreased by 60% when with asimicin, a potent complex I inhibitor. The functional role of semiquinone and the EPR assignment of clusters N6a/N6b are discussed.
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Madhavan
authors.initials: M
authors.lastname: Narayanan
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: David J
authors.initials: DJ
authors.lastname: Gabrieli
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Leung
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.
authors.forename: Carl A
authors.initials: CA
authors.lastname: Glaser
authors.affiliation: Johnson Research Foundation, Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Nitha
authors.initials: N
authors.lastname: Saju
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037.
authors.forename: Subhash C
authors.initials: SC
authors.lastname: Sinha
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: D016000:Cluster Analysis; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics*; D005415:Flavins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D009154:Mutation; D010084:Oxidation-Reduction; D017494:Proton Pumps / Q000737:chemistry; D011809:Quinones / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; D005415:Flavins; D007506:Iron-Sulfur Proteins; D017494:Proton Pumps; D011809:Quinones; C033223:polyhistidine; D006639:Histidine; C556263:NuoE protein, E coli; D042967:Electron Transport Complex I
keywords: Complex I; Electron Paramagnetic Resonance (EPR); Flavin; Iron-Sulfur Protein; NADH Dehydrogenase; Proton Pumps; Quinones; Semiquinone
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