Semiquinone and Cluster N6 Signals in His-tagged Proton-translocating NADH:Ubiquinone Oxidoreductase (Complex I) from Escherichia coli

• Published in: The Journal of biological chemistry Vol. 288; no. 20; pp. 14310 - 14319
• Main Authors: Narayanan, Madhavan, Gabrieli, David J., Leung, Steven A., Elguindy, Mahmoud M., Glaser, Carl A., Saju, Nitha, Sinha, Subhash C., Nakamaru-Ogiso, Eiko
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-05-2013; American Society for Biochemistry and Molecular Biology
• Subjects: Bioenergetics; Cluster Analysis; Complex I; Crystallography, X-Ray; Electron Paramagnetic Resonance (EPR); Electron Spin Resonance Spectroscopy; Electron Transport Complex I - chemistry; Electron Transport Complex I - genetics; Escherichia coli - enzymology; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Flavin; Flavins - chemistry; Histidine - chemistry; Hydrogen-Ion Concentration; Iron-Sulfur Protein; Iron-Sulfur Proteins - chemistry; Mutation; NADH Dehydrogenase; Oxidation-Reduction; Proton Pumps; Proton Pumps - chemistry; Quinones; Quinones - chemistry; Semiquinone; Quinones; Flavin; Electron Paramagnetic Resonance (EPR); NADH Dehydrogenase; Iron-Sulfur Protein; Complex I; Proton Pumps; Semiquinone
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.467803

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 P½ (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.
Background: Complex I is the largest proton pump in mitochondria, yet its mechanism is unknown.
Results: For the first time, inhibitor-sensitive semiquinone and cluster N6 signals were detected in affinity-purified E. coli complex I.
Conclusion: The semiquinone species is involved in the redox-driven proton-pumping mechanism.
Significance: Our highly pure complex I will help advance the mechanistic study of the protein.