The respiratory chain of the thermophilic archaeon Sulfolobus metallicus: studies on the type-II NADH dehydrogenase.
The membranes of the thermoacidophilic archaeon Sulfolobus metallicus exhibit an oxygen consumption activity of 0.5 nmol O(2) min(-1) mg(-1), which is insensitive to rotenone, suggesting the presence of a type-II NADH dehydrogenase. Following this observation, the enzyme was purified from solubilised membranes and characterised. The pure protein is a monomer with an apparent molecular mass of 49 kDa, having a high N-terminal amino acid sequence similarity towards other prokaryotic enzymes of the same type. It contains a covalently attached flavin, which was identified as being FMN by 31P-NMR spectroscopy, a novelty among type-II NADH dehydrogenases. Metal analysis showed the absence of iron, indicating that no FeS clusters are present in the protein. The average reduction potential of the FMN group was determined to be +160 mV, at 25 degrees C and pH 6.5, by redox titrations monitored by visible spectroscopy. Catalytically, the enzyme is a NADH:quinone oxidoreductase, as it is capable of transferring electrons from NADH to several quinones, including ubiquinone-1, ubiquinone-2 and caldariella quinone. Maximal turnover rates of 195 micromol NADH oxidized min(-1) mg(-1) at 60 degrees C were obtained using ubiquinone-2 as electron acceptor, after enzyme dilution and incubation with phospholipids.
Bandeiras
Tiago M
TM
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, 2780-156 Oeiras, Portugal.
Salgueiro
Carlos A
CA
Huber
Harald
H
Gomes
Cláudio M
CM
Teixeira
Miguel
M
D000595:Amino Acid Sequence; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000302:isolation & purification; D013053:Spectrophotometry; D016944:Sulfolobus / Q000737:chemistry / Q000201:enzymology*
D005486:Flavin Mononucleotide; D009245:NADH Dehydrogenase