NO Reductase from Bacillus azotoformans Is a Bifunctional Enzyme Accepting Electrons from Menaquinol and a Specific Endogenous Membrane-Bound Cytochrome c551.

Bacillus azotofonnans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique men aquinol-dependent nitric oxide reductase (qCuANOR) with a CuA center in its small subunit. The qCuANOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the Em for all is close to 150 mV. Only one of these c cytochromes, which exhibits an a-band maximum at 551 nm, acts as a direct electron donor to qCuANOR. Further investigation demonstrated that this cytochrome c551 possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c551 is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCuANOR. The ability of qCUANOR to accept electrons from both menaquinol and cytochrome c551 might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus den itnflcans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c551, is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b6f complex serving the bioenergetic needs of the organism. [ABSTRACT FROM AUTHOR]