β-Oxochlorin cobalt(II) complexes catalyze the electrochemical reduction of CO2

Inspired by the architecture of the macrocycle of heme d1, a series of synthetic mono-, di- and tri-β-oxo-substituted porphyrinoid cobalt(II) complexes were evaluated as electrocatalytic CO2 reducers, identifying complexes of...

The crystal structure of heme d1 biosynthesis-associated small c-type cytochrome NirC reveals mixed oligomeric states in crystallo

Monoheme c-type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α-helices that surround a covalently attached heme. An intriguing feature of many monoheme c-type cytochromes is their capacity to form oligomers by exchanging at least one of their α-helices, which is often referred to as 3D domain swapping. Here, we have determined the crystal structure of NirC, a c-type cytochrome co-encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa. Crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution 2.83 Å) and initial phases were obtained by Fe-SAD phasing, revealing the presence of eleven NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D-domain-swapped dimers, one showing pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between high protein concentration required for crystallization and the structural plasticity of monoheme c-type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison to similar proteins may offer new leads to unravel the unknown function of NirC.SynopsisThe crystal structure of the c-type cytochrome NirC from Pseudomonas aeruginosa has been determined and reveals the simultaneous presence of monomers and 3D-domain-swapped dimers in the same asymmetric unit.

Structure of heme d1-free cd1 nitrite reductase NirS

A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by cd1 nitrite reductase NirS in e.g. the gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d1 as an essential part of its active site. Although NirS is mechanistically and structurally well studied, the focus of previous studies has been on the active, heme d1-bound form. The heme d1-free form of NirS reported here, representing a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, movement of a loop around W498 seems to be related to a widening of the propeller, allowing easier access to the heme d1 binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.SynopsisThe crystal structure of heme d1-free cd1 nitrite reductase NirS from Pseudomonas aeruginosa has been determined and provides insight into a premature form of the enzyme.

Crystal structure of NirF: Insights into its role in heme d1 biosynthesis

Certain facultative anaerobes such as the opportunistic human pathogen Pseudomonas aeruginosa 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 P. aeruginosa 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 d1. While heme d1 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 d1, the last intermediate of heme d1 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 via both N-termini. Further, the complex with dihydroheme d1 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 ΔnirF strain of P. aeruginosa. Together, these data implicate that NirF possesses a yet unknown enzymatic activity and is not simply a binding protein of heme d1 derivatives.

NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis

Heme d1 plays an important role in denitrification as the essential cofactor of the cytochrome cd1 nitrite reductase NirS. At present, the biosynthesis of heme d1 is only partially understood. The last step of heme d1 biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d1. Instead, the NirS purified from this strain contains the heme d1 precursor dihydro-heme d1 lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d1 was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d1 and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d1.