Membrane-bound denitrification in the Gram-positive bacterium Bacillus azotoformans

Gram+ bacteria are capable of complete denitrification just like Gram− (Gram-negative) bacteria. However, Gram+ (Gram-positive) bacteria have a very small periplasmic-like space. This leads to the question of whether those enzymes and electron carriers involved in denitrification, which are normally located in the periplasmic space in Gram− bacteria, are located in the periplasmic-like space in Gram+ bacteria or have been modified as membrane-bound proteins. Using Bacillus azotoformans as a Gram+ bacterial model, our study demonstrates that anaerobic denitrification is catalysed by four membrane-bound enzymes and that the electron carriers are membrane-bound c-type cytochromes and menaquinol. NADH dehydrogenase is coupled with the denitrification pathway providing menaquinol. In addition, the cytochrome b₆f complex forms part of the denitrification pathway, oxidizing menaquinol and reducing at least three different membrane-bound c-type cytochromes. We determined that the NO reductase, qCu_ANOR (where NOR stands for nitric oxide reductase), can accept electrons from two donors, a specific cytochrome c₅₅₁ and menaquinol. Similarly, nitrite reductase, a copper enzyme, and nitrous oxide reductase may be bifunctional enzymes. Regarding the bifunctionality of qCu_ANOR, we propose that the menaquinol-linked pathway is involved in the detoxification of NO.