Crystal structure of NirF: Insights into its role in hemed₁biosynthesis

Abstract

<jats:title>Abstract</jats:title><jats:p>Certain facultative anaerobes such as the opportunistic human pathogen <jats:italic>Pseudomonas aeruginosa</jats:italic> can 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 eradicating <jats:italic>P. aeruginosa</jats:italic> infections. A central step in denitrification is the reduction of nitrite to nitrous oxide by nitrite reductase NirS, an enzyme that requires the unique cofactor heme <jats:italic>d<jats:sub>1</jats:sub></jats:italic>. While heme <jats:italic>d<jats:sub>1</jats:sub></jats:italic> biosynthesis 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 dihydroheme <jats:italic>d<jats:sub>1</jats:sub></jats:italic>, the last intermediate of heme <jats:italic>d<jats:sub>1</jats:sub></jats:italic> biosynthesis. 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 anchoring <jats:italic>via</jats:italic> both N-termini. Further, the complex with dihydroheme <jats:italic>d<jats:sub>1</jats:sub></jats:italic> allowed 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 Δ<jats:italic>nirF</jats:italic> strain of <jats:italic>P. aeruginosa</jats:italic>. Together, these data implicate that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme <jats:italic>d<jats:sub>1</jats:sub></jats:italic> derivatives.</jats:p>