A novel copper A containing menaquinol NO reductase from Bacillus azotoformans

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 t he cytoplasmic membrane of the gram-positive bacterium Bacillus azotoforman s. The purified enzyme consists of two subunits with apparent molecular mas ses of 16 and 40 kDa based on SDS-PAGE. Analytical and spectroscopic determ inations revealed the presence of one non-heme iron, two copper atoms and o f 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 a t an angle of 60-70 degrees. The second heme b is high-spin binding CO in t he reduced state. The high-spin heme center and the non-hems iron are EPR s ilent. They are proposed to form a binuclear center where reduction of NO o ccurs. 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 ni trous oxide reductases. Second, the enzyme uses menaquinol as electron dono r, 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 prost hetic groups the B. azotoformans NO reductase is a true hybrid between copp er A containing cytochrome oxidases and NO reductases present in gram-negat ive bacteria. It may represent the most ancient "omnipotent" progenitor of the family of heme-copper oxidases.