Properties of a thermostable nitrate reductase from the hyperthermophilic archaeon Pyrobaculum aerophilum

The nitrate reductase of the hyperthermophilic archaeon Pyrobaculum aerophi lum was purified 137-fold from the cytoplasmic membrane. Based on sodium do decyl sulfate-polyacrylamide gel electrophoresis analysis, the enzyme compl ex consists of three subunits with apparent molecular weights of 130,000, 5 2,000, and 32,000. The enzyme contained molybdenum (0.8-mol/mol complex), i ron (15.4-mol/mol complex) and cytochrome b (0.49-mol/mol complex) as cofac tors. The P. aerophilum nitrate reductase distinguishes itself from nitrate reductases of mesophilic bacteria and archaea by its very high specific ac tivity using reduced benzyl viologen as the electron donor (V-max with nitr ate, 1,162 s(-1) (326 U/mg); V-max with chlorate, 1,348 s(-1) (378 U/mg) [a ssayed at 75 degreesC]). The K-m values for nitrate and chlorate were 58 an d 140 muM, respectively. Azide was a competitive inhibitor and cyanide was a noncompetitive inhibitor of the nitrate reductase activity. The temperatu re optimum for activity was > 95 degreesC. When incubated at 100 degreesC, the purified nitrate reductase had a half-life of 1.5 h. This study constit utes the first description of a nitrate reductase from a hyperthermophilic archaeon.