nosX is essential for whole-cell N(2)O reduction in Paracoccus denitrificans but not for assembly of copper centres of nitrous oxide reductase

Nitrous oxide (N(2)O) is a potent greenhouse gas that is produced naturally as an intermediate during the process of denitrification carried out by some soil bacteria. It is consumed by nitrous oxide reductase (N(2)OR), the terminal enzyme of the denitrification pathway, which catalyses a reduction reaction to generate dinitrogen. N(2)OR contains two important copper cofactors (Cu(A) and Cu(Z) centres) that are essential for activity, and in copper-limited environments, N(2)OR fails to function, contributing to rising levels of atmospheric N(2)O and a major environmental challenge. Here we report studies of nosX, one of eight genes in the nos cluster of the soil dwelling α-proteobaterium Paraccocus denitrificans. A P. denitrificans ΔnosX deletion mutant failed to reduce N(2)O under both copper-sufficient and copper-limited conditions, demonstrating that NosX plays an essential role in N(2)OR activity. N(2)OR isolated from nosX-deficient cells was found to be unaffected in terms of the assembly of its copper cofactors, and to be active in in vitro assays, indicating that NosX is not required for the maturation of the enzyme; in particular, it plays no part in the assembly of either of the Cu(A) and Cu(Z) centres. Furthermore, quantitative Reverse Transcription PCR (qRT-PCR) studies showed that NosX does not significantly affect the expression of the N(2)OR-encoding nosZ gene. NosX is a homologue of the FAD-binding protein ApbE from Pseudomonas stutzeri , which functions in the flavinylation of another N(2)OR accessory protein, NosR. Thus, it is likely that NosX is a system-specific maturation factor of NosR, and so is indirectly involved in maintaining the reaction cycle of N(2)OR and cellular N(2)O reduction.