Protein complex formation during denitrification by Pseudomonas aeruginosa

Summary The most efficient means of generating cellular energy is through aerobic respiration. Under anaerobic conditions, several prokaryotes can replace oxygen by nitrate as final electron acceptor. During denitrification, nitrate is reduced via nitrite, NO and N2O to molecular nitrogen (N2) by four membrane‐localized reductases with the simultaneous formation of an ion gradient for ATP synthesis. These four multisubunit enzyme complexes are coupled in four electron transport chains to electron donating primary dehydrogenases and intermediate electron transfer proteins. Many components require membrane transport and insertion, complex assembly and cofactor incorporation. All these processes are mediated by fine‐tuned stable and transient protein–protein interactions. Recently, an interactomic approach was used to determine the exact protein–protein interactions involved in the assembly of the denitrification apparatus of Pseudomonas aeruginosa. Both subunits of the NO reductase NorBC, combined with the flavoprotein NosR, serve as a membrane‐localized assembly platform for the attachment of the nitrate reductase NarGHI, the periplasmic nitrite reductase NirS via its maturation factor NirF and the N2O reductase NosZ through NosR. A nitrate transporter (NarK2), the corresponding regulatory system NarXL, various nitrite (NirEJMNQ) and N2O reductase (NosFL) maturation proteins are also part of the complex. Primary dehydrogenases, ATP synthase, most enzymes of the TCA cycle, and the SEC protein export system, as well as a number of other proteins, were found to interact with the denitrification complex. Finally, a protein complex composed of the flagella protein FliC, nitrite reductase NirS and the chaperone DnaK required for flagella formation was found in the periplasm of P. aeruginosa. This work demonstrated that the interactomic approach allows for the identification and characterization of stable and transient protein–protein complexes and interactions involved in the assembly and function of multi‐enzyme complexes. The protein‐protein interactions occuring during the anaerobic process of denitrification in Pseudomonas aeruginosa were elucidated to reach a better understanding of underlying molecular mechanims.