Assembly of the copper centres of nitrous oxide reductase in Paracoccus denitrificans and connections to copper detoxification/trafficking

The α-proteobacterium Paracoccus denitrificans uses nitrate as an alternative electron acceptor under anaerobic conditions to survive, reducing it stepwise to nitrogen gas in the process known as denitrification. Agricultural practices have presented large amounts of anthropogenic nitrogen to soil microbes over the past century, which has led to a significant rise in the emission of nitrous oxide (N2O), a potent ozone depleting and greenhouse gas. The last step yields N2 from N2O and is catalysed by N2OR, a homo dimeric multi-copper enzyme with 2 copper cofactor centres: the CuA electron entry site, and the CuZ site, a unique 4Cu-2S cluster. Understanding how the copper metal centres of N2OR are assembled, and the effect of copper on organisms that require this metal, is an essential pre-requisite for efforts towards mitigating N2O emissions. \nHere we present an investigation of the role of two accessory genes in the nos gene cluster, nosL and nosX, in P. denitrificans. Phenotype analysis of gene deletion mutants demonstrates the two genes are necessary for whole cell N2O reduction. Spectrosopic characterisation of N2OR from each background demonstrates the role of NosX is not to assemble the Cu centres, while NosL is involved in this process. Further studies of NosL heterologously expressed in E. coli demonstrate the capacity of NosL to specifically bind Cu(I) with a high affinity, suggesting a role for NosL as a copper binding protein involved in the copper centre biogenesis of N2OR. \nAn additional study of the P. denitrificans transcriptome in the presence of Cu excess, using RNA-Sequencing, is discussed, covering the most differentially expressed transcripts from aerobic and anaeroic datasets. Importantly, there is no complete system dedicated to copper detoxification and common to prokaryotes and eukaryotes, in P. denitrificans. We have identified, however, 2 new genes involved in Cu trafficking in this bacterium.