Bacterial nitrous oxide (N₂O) respiration depends on the polytopic membrane protein NosR for the expression of N₂O reductase from the nosZ gene. We have constructed His-tagged NosR and purified it from detergent-solubilized membranes of Pseudomonas stutzeri ATCC14405. NosR is an iron-sulfur flavoprotein with the redox centers positioned at opposite sites of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR features also conserved CX₃CP motifs, located C-terminally of transmembrane helices TM4 and TM6, respectively. We have genetically manipulated nosR with respect to these different domains and putative functional centers and have expressed recombinant derivatives in the nosR null mutant, MK418nosR::Tn5. NosR function was studied by its effect on N₂O respiration, NosZ synthesis, and properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, loss of N₂O respiration was observed on deleting most of the periplasmic domain, the C-terminal parts beyond TM2, or by modifying the cysteine residues in the highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused in NosZ an increase of the spectral contribution from a modified catalytic Cu site. In addition to its role for nosZ expression, NosR supports in vivo N₂O respiration. We discuss putative functions of electron donation or redox activation.