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 thermoacidophilic archaea Sulfolobus acidocaldarius and Sulfolobus solfataricus and characterized. In both organisms the enzyme oxidizes specifically beta-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 degrees 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 degrees C for S. acidocaldarius NADH oxidase and 77 min at 98 degrees 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.
Masullo
M
M
Department of Biochemistry and Medical Biotechnologies, University of Naples Federico II, Italy.
Raimo
G
G
Dello Russo
A
A
Bocchini
V
V
Bannister
J V
JV
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000302:isolation & purification*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D012997:Solvents; D013053:Spectrophotometry; D016944:Sulfolobus / Q000201:enzymology*; D016945:Sulfolobus acidocaldarius / Q000201:enzymology*; D014508:Urea
D006146:Guanidines; D009097:Multienzyme Complexes; D012997:Solvents; D014508:Urea; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019791:Guanidine