Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus

NADH oxidase from Thermus thermophilus is a homodimer with an unknown physiological function. As is typical for an enzyme isolated from a thermophile, the catalytic rate, k cat , is low at low temperatures and increases with temperature, achieving an optimum at the physiological temperature of the organism, i.e. at ≈ 70 °C for T. thermophilus . At low temperatures, the k cat of several enzymes from thermophilic and mesophilic organisms can be increased by chaotropic agents. The catalytic rate of NADH oxidase increases in the presence of urea. At concentrations of 1.0–1.3  m urea it reaches 250% of the activity in the absence of urea, at 20 °C. At higher urea concentrations the enzyme activity is inhibited. The urea‐dependent activity changes correlate with changes in the fluorescence intensity of Trp47, which is located in the active site of the enzyme. Both fluorescence and circular dichroism measurements indicate that the activation by chaotropic agents involves local environmental changes accompanied by increased dynamics in the active site of the enzyme. This is not related to the global structure of NADH oxidase. The presence of an aromatic amino acid interacting with the flavin cofactor is common to numerous flavin‐dependent oxidases. A comparison of the crystal structure with the activation thermodynamic parameters, Δ H * and T Δ S *, obtained from the temperature dependence of k cat, suggests that Trp47 interacts with a water molecule and the isoalloxazine flavin ring. The present investigation suggests a model that explains the role of the homodimeric structure of NADH oxidase.