PURIFICATION AND CHARACTERIZATION OF NADH OXIDASE FROM THE ARCHAEA SULFOLOBUS-ACIDOCALDARIUS AND SULFOLOBUS-SOLFATARICUS

The enzyme NADH oxidase (EC 1.6.99.3) has been isolated from the two t hermoacidophilic archaea Sulfolobus acidocaldarius and Sulfolobus solf ataricus and characterized. In both organisms the enzyme oxidizes spec ifically beta-NADH in the presence of molecular oxygen and requires th e 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-elec tron donors. The purified enzymes exhibit quite different molecular pr operties, S. acidocaldarius NADH oxidase is a monomeric protein with a n estimated molecular mass of about 27 kDa, whereas S. solfataricus NA DH oxidase is a dimeric protein with a molecular mass of 35 kDa per su bunit; S. solfataricus NADH oxidase is purified as an FAD-containing p rotein, whereas S. acidocaldarius NADH oxidase does not contain a flav in molecule. Furthermore, a comparison of the N-terminal amino acid se quence shows no similarities either between the two proteins or to any other NADH oxidases. Both enzymes are essentially thermophilic. In th e temperature range 20-80 degrees C, the energy of activation is almos t the same for both activities, suggesting that similar energetic para meters are required. Also both oxidases display a great stability to h eat. The half-life of heat inactivation is about 180 min at 90 degrees C for S. acidocaldarius NADH oxidase and 77 min at 98 degrees C for t he S. solfataricus enzyme. The activity of the two enzymes is inhibite d by urea and guanidine and are regulated very differently by several organic solvents.