Purification and characterization of NADH oxidase from the archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus

Mariorosario Masullo, Gennaro Raimo, Antonio Dello Russo, Vincenzo Bocchini, Joe V. Bannister

Research output: Contribution to journalArticlepeer-review


The enzyme NADH oxidase (EC has been isolated from the two thermoacidophilic archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus and characterized. In both organisms the enzyme oxidizes specifically β-NADH in the presence of molecular oxygen and requires the presence of a flavin cofactor, showing a high specificity for FAD. A stoicheiometric amount of hydrogen peroxide to NADH is formed as the end product of the reaction, indicating that both enzymes are two-electron donors. The purified enzymes exhibit quite different molecular properties. S. acidocaldarius NADH oxidase is a monomeric protein with an estimated molecular mass of about 27 kDa, whereas S. solfataricus NADH oxidase is a dimeric protein with a molecular mass of 35 kDa per subunit; S. solfataricus NADH oxidase is purified as an FAD-containing protein, whereas S. acidocaldarius NADH oxidase does not contain a flavin molecule. Furthermore, a comparison of the N-terminal amino acid sequence shows no similarities either between the two proteins or to any other NADH oxidases. Both enzymes are essentially thermophilic. In the temperature range 20-80°C, the energy of activation is almost the same for both activities, suggesting that similar energetic parameters are required. Also both oxidases display a great stability to heat. The half-life of heat inactivation is about 180 min at 90°C for S. acidocaldarius NADH oxidase and 77 min at 98°C for the S. solfataricus enzyme. The activity of the two enzymes is inhibited by urea and guanidine and are regulated very differently by several organic solvents.

Original languageEnglish
Pages (from-to)47-54
Number of pages8
JournalBiotechnology and Applied Biochemistry
Issue number1
Publication statusPublished - 1996

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology

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