Properties of a Dissimilatory Nitrate Reductase from the Halophilic Archaeon Haloferax volcanii

Abstract

Grown under anaerobic conditions in presence of nitrate, Haloferax volcanii shows nitrate reduction and accumulation of nitrite in the culture medium. We found a membrane-associated nitrate reductase, which could easily be solubilized by gently stirring of isolated membranes. Surprisingly, this nitrate reductase requires no NaCl for its activity. A medium pH of 7.5 and high temperatures up to 80 °C are necessary for optimum activity. Kinetic studies showed that the apparent KM was 0.36 mmol/1 for nitrate and 80 µmol/1 for dithionite-reduced methyl viologen. The respiratory chain inhibitor cyanide effects nitrate reduction noncompetitively with respect to nitrate with a Kx of 0.3 mmol/1. Azide was a strong inhibitor: The concentration required for half maximal inhibition was 60 µmol/1, whereas thiocyanate and chlorate were much weaker inhibitors. The isolated enzyme was partially purified by fractionated precipitation using polyethylene glycol. SDS gel electrophoresis resulted in three putative subunits of the nitrate reductase of molecular masses of about 100, 61, and 31 kDa. © 1995 Verlag der Zeitschrift fur Naturforschung. All rights reserved.