Isolation, characterization and electron microscopic single particle analysis of the NADH:ubiquinone oxidoreductase (complex I) from the hyperthermophilic eubacterium Aquifex aeolicus.
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 degrees C. The specific activity for electron transfer from NADH to decylubiquinone is 29 U/mg at 80 degrees 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 degrees C, with a half-life of about 10 h at 80 degrees C. The activity shows a linear Arrhenius plot at 50-85 degrees 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 degrees ) between the cytoplasmic peripheral arm and the membrane intrinsic arm of the complex appears to be invariant.
Peng
Guohong
G
Max-Planck-Institut für Biophysik, Frankfurt a. M., Germany.
Fritzsch
Günter
G
Zickermann
Volker
V
Schägger
Hermann
H
Mentele
Reinhardt
R
Lottspeich
Friedrich
F
Bostina
Mihnea
M
Radermacher
Michael
M
Huber
Robert
R
Stetter
Karl Otto
KO
Michel
Hartmut
H
D001419:Bacteria / Q000201:enzymology* / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004396:Coloring Agents; D042967:Electron Transport Complex I; D004795:Enzyme Stability; D006358:Hot Temperature; D007089:Image Enhancement; D008854:Microscopy, Electron; D008982:Molybdenum; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D017494:Proton Pumps / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure; D012995:Solubility
D001426:Bacterial Proteins; D004396:Coloring Agents; D010446:Peptide Fragments; D021122:Protein Subunits; D017494:Proton Pumps; D008982:Molybdenum; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I; C022175:ammonium molybdate