The molecular biology and biochemistry of denitrification in gram-negative bacteria has been studied extensively. However, little is known about this process in gram-positive bacteria. We have purified the NO reductase from the cytoplasmic membrane of the gram-positive bacterium Bacillus azotoformans. The purified enzyme consists of two subunits with apparent molecular masses of 16 and 40 kDa based on SDS-PAGE. Analytical and spectroscopic determinations revealed the presence of one non-heme iron, two copper atoms and of two b-type hemes per enzyme complex. Heme c was absent. Using EPR and UV-visible spectroscopy, it was determined that one of the hemes is a low-spin heme b, in which the two axial histidine imidazole planes are positioned at an angle of 60-70 degrees. The second heme b is high-spin binding CO in the reduced state. The high-spin heme center and the non-heme iron are EPR silent. They are proposed to form a binuclear center where reduction of NO occurs. There are two novel features of this enzyme that distinguish it from other NO reductases. First, the enzyme contains copper in form of copper A, an electron carrier up to now only detected in cytochrome oxidases and nitrous oxide reductases. Second, the enzyme uses menaquinol as electron donor, whereas cytochrome c, which is the substrate of other NO reductases, is not used. Copper A and both hemes are reducible by menaquinol. This new NO reductase is thus a menaquinol:NO oxidoreductase. With respect to its prosthetic groups the B. azotoformans NO reductase is a true hybrid between copper A containing cytochrome oxidases and NO reductases present in gram-negative bacteria. It may represent the most ancient "omnipotent" progenitor of the family of heme-copper oxidases.