Purification and characterization of an NADH oxidase from extremely thermophilic anaerobic bacterium Thermotoga hypogea

Abstract

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90 degrees C. It was found to be able to grow in the presence of micromolar molecular oxygen (O2). Activity of NADH oxidase was detected in the cell-free extract of T. hypogea, from which an NADH oxidase was purified to homogeneity. The purified enzyme was a homodimeric flavoprotein with a subunit of 50 kDa, revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It catalyzed the reduction of O2 to hydrogen peroxide (H2O2), specifically using NADH as electron donor. Its catalytic properties showed that the NADH oxidase had an apparent Vmax value of 37 micromol NADH oxidized min(-1) mg(-1) protein. Apparent Km values for NADH and O2 were determined to be 7.5 microM and 85 microM, respectively. The enzyme exhibited a pH optimum of 7.0 and temperature optimum above 85 degrees C. The NADH-dependent peroxidase activity was also present in the cell-free extract, which could reduce H2O2 produced by the NADH oxidase to H2O. It seems possible that O2 can be reduced to H2O by the oxidase and peroxidase, but further investigation is required to conclude firmly if the purified NADH oxidase is part of an enzyme system that protects anaerobic T. hypogea from accidental exposure to O2.