The electron transfer complex between nitrous oxide reductase and its electron donors

Abstract

Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (N 2 OR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of N 2 OR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica N 2 OR, or an electrostatic nature, in the case of P. denitrificans N 2 OR and A. cycloclastes N 2 OR. A set of well-conserved residues on the N 2 OR surface were identified as being part of the electron transfer pathway from the redox partner to N 2 OR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N 2 OR sequence). Moreover, we built a model for Wolinella succinogenes N 2 OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N 2 OR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the N 2 OR domain is similar to that found in the other electron transfer complexes.

Direct electron transfer from pseudoazurin to nitrous oxide reductase in catalytic N2O reduction

“…S1A and S1B), suggesting that AcPAz and cytc 552 interact with their respective N 2 ORs in a closely similar manner, and consistent with the role of Cu A as the electron entry point to the enzymes. These findings broadly agree with the recent results which were performed using the same algorithm generated independently by Dell'Acqua et al [60]. On the other hand, BHcytc-AcN 2 OR has a significantly lower average global score (2.2± 0.2) than the others indicative of non-physiological interactions (Table S2).…”

“…Mattila et al proposed that His635 (corresponding to His576 in AcN 2 OR) is involved in the ET pathway based on their docking simulations between P. pantotrophus PAz (PpPAz) and PdN 2 OR which is phylogenetically related to AcN 2 OR [2,25]. In addition, Dell'Acqua et al suggested that the ET pathway involving His566 or Leu568 in MhN 2 OR (corresponding to His576 or Leu578 in AcN 2 OR, respectively) is the most plausible ET route [60] which is consistent with our predictions. It should be noted that Asp588 (533 in AcN 2 OR), proposed as a key interfacial residue in the docking of PdN 2 OR with PpPAz [25], was not assigned as a part of ET routes using the PATHWAYS algorithm with our protocol.…”

Transferable Denitrification Capability of Thermus thermophilus

“…The recent availability of crystal structures of these enzymes from Pseudomonas aeruginosa (cNor) (20) and Geobacillus stearothermophilus (qNor) (21) supports the idea that these are not proton pumping enzymes despite their sequence and structural similarity to cytochrome c and quinol oxidases (1). In several denitrificant microorganisms, N 2 O is finally reduced to N 2 by a periplasmic reductase (NosZ), for which a cytochrome c or a type I copper protein, e.g., pseudoazurin or azurin, has to act as an electron donor (22). However, N 2 is also produced by organisms that do not encode NosZ homologues, supporting the idea that a different type of N 2 O reductase might exist (23,24).…”

Protein Network of the Pseudomonas aeruginosa Denitrification Apparatus

“…The solubilized membrane proteins present in the supernatant were mixed for 1 h at 4°C with nickle-nitrilotriacetic acid (Ni-NTA) agarose previously equilibrated with NPI-10 buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole [Protino]; MachereyNagel GmbH, Düren, Germany). The column was washed with imidazole at increasing concentrations (20,30,40, and 50 mM in NPI-20, NPI-30, NPI-40, and NPI-50 buffers consecutively). Recombinantly tagged NosR, NorC, and NorB proteins, along with their potential interaction partners, were eluted from the column by addition of 300 mM imidazole in NPI-300 buffer.…”