Crystal structure of NirF: Insights into its role in hemed1biosynthesis

Abstract

AbstractCertain facultative anaerobes such as the opportunistic human pathogenPseudomonas aeruginosacan respire on nitrate, a process generally known as denitrification. This enables denitrifying bacteria to survive in anoxic environments and contributes e.g. to the formation of biofilm, hence increasing difficulties in eradicatingP. aeruginosainfections. A central step in denitrification is the reduction of nitrite to nitrous oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor hemed1. While hemed1biosynthesis is mostly understood, the role of the essential periplasmatic protein NirF in this pathway remains unclear. Here, we have determined crystal structures of NirF and its complex with dihydrohemed1, the last intermediate of hemed1biosynthesis. We found that NirF forms a bottom-to-bottom β-propeller homodimer and confirmed this by multi-angle light and small-angle X-ray scattering. The N-termini are immediately neighbored and project away from the core structure, which hints at simultaneous membrane anchoringviaboth N-termini. Further, the complex with dihydrohemed1allowed us to probe the importance of specific residues in the vicinity of the ligand binding site, revealing residues not required for binding or stability of NirF but essential for denitrification in experiments with complemented mutants of a ΔnirFstrain ofP. aeruginosa. Together, these data implicate that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of hemed1derivatives.