Isolation, Characterization and Electron Microscopic Single Particle Analysis of the NADH:Ubiquinone Oxidoreductase (Complex I) from the Hyperthermophilic Eubacterium Aquifex aeolicus

• Published in: Biochemistry (Easton) 2003-03, Vol.42 (10), p.3032-3039
• Main Authors: Peng, Guohong, Fritzsch, Günter, Zickermann, Volker, Schägger, Hermann, Mentele, Reinhardt, Lottspeich, Friedrich, Bostina, Mihnea, Radermacher, Michael, Huber, Robert, Stetter, Karl Otto, Michel, Hartmut
• Format: Article
• Language: English
• Subjects: Aquifex aeolicus; Bacteria - enzymology; Bacteria - ultrastructure; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - ultrastructure; Catalysis; Chromatography, Gel; Chromatography, Ion Exchange; Coloring Agents; decylubiquinone; Electron Transport Complex I; Enzyme Stability; Hot Temperature; Image Enhancement; Microscopy, Electron; Molybdenum; NADH, NADPH Oxidoreductases - chemistry; NADH, NADPH Oxidoreductases - isolation & purification; NADH, NADPH Oxidoreductases - ultrastructure; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Protein Subunits - chemistry; Protein Subunits - isolation & purification; Proton Pumps - chemistry; Proton Pumps - isolation & purification; Proton Pumps - ultrastructure; Solubility
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi026876v

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) has been purified from Aquifex aeolicus, a hyperthermophilic eubacterium of known genome sequence. The purified detergent solubilized enzyme is highly active above 50 °C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 °C. The A. aeolicus complex I is completely sensitive to rotenone and 2-n-decyl-quinazoline-4-yl-amine. SDS polyacrylamide gel electrophoresis shows that it may contain up to 14 subunits. N-terminal amino acid sequencing of the bands indicates the presence of a stable subcomplex, which is composed of subunits E, F, and G. The isolated complex is highly stable and active in a temperature range from 50 to 90 °C, with a half-life of about 10 h at 80 °C. The activity shows a linear Arrhenius plot at 50−85 °C with an activation energy at 31.92 J/mol K. Single particle electron microscopy shows that the A. aeolicus complex I has the typical L-shape. However, visual inspection of averaged images reveals many more details in the external arm of the complex than has been observed for complex I from other sources. In addition, the angle (90°) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.