Bacterial denitrification is a part of the global nitrogen cycle and comprises the stepwise reduction of nitrate to molecular nitrogen, which is released to the atmosphere. Cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans catalyzes the reduction of nitric oxide to nitrous oxide and water. This is a key step of the denitrification chain as it involves reformation of the N-N bond that was split in nitrogen fixation processes. In addition, nitric oxide is cytotoxic and nitrous oxide is a potent greenhouse gas. cNOR is an integral, two-subunit membrane protein, which contains several redox-active metal cofactors essential for function. In P. denitrificans the enzyme is expressed from an operon norCBQDEF, of which only norCB are the structural genes for the cNOR protein. The assembly process of cNOR, including cofactor insertion, as well as the detailed catalytic function of the enzyme are largely unknown, which motivated this study.

Our results showed that cNOR can be expressed from only the norCB genes and that norQDEF are not essential for folding, complex formation and heme cofactor assembly of the protein. However, we found that non-heme iron (Fe_B) cofactor insertion into cNOR was dependent on the NorQ and NorD proteins, which were expressed from the nor operon. These proteins were purified as a complex and our results indicate that they act as a molecular chaperone. We present the cryo-electron microscopy structure of NorQ, which formed hexameric ring-shaped oligomers and was shown to have ATPase activity. Our data further suggest that NorD functions as an adaptor protein in order to link NorQ to a specific binding site at the cytoplasmic surface of cNOR. Based on our experimental data we present a model for Fe_B cofactor insertion into cNOR.

Without co-expression of the NorQ and NorD proteins, the produced cNOR was inactive. It lacked Fe_B at the catalytic center but was otherwise structurally intact. Therefore we used this protein to investigate the role of Fe_B in the mechanism of nitric oxide and oxygen reduction of cNOR and compared our results to computational studies of the enzyme published recently.

In vitro studies of membrane proteins, such as cNOR, are challenging because their function often depends on the interaction with a biological membrane and specific phospholipids. We used two different membrane mimetic systems, lipid nanodiscs and proteoliposomes, to study the effect of a membrane environment on the function of detergent-solubilized cNOR. Our results indicate that the membrane bilayer of lipid nanodiscs and proteoliposomes, even when assembled using the same lipids, has different properties with measurable effects on cNOR function.