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Journal of Bacteriology, March 2005, p. 1992-2001, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.1992-2001.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration

Patrick Wunsch and Walter G. Zumft^*

Lehrstuhl für Mikrobiologie, Universität Karlsruhe, Karlsruhe, Germany

Received 24 October 2004/ Accepted 9 December 2004

Bacterial nitrous oxide (N₂O) respiration depends on the polytopic membrane protein NosR for the expression of N₂O reductase from the nosZ gene. We constructed His-tagged NosR and purified it from detergent-solubilized membranes of Pseudomonas stutzeri ATCC 14405. NosR is an iron-sulfur flavoprotein with redox centers positioned at opposite sides of the cytoplasmic membrane. The flavin cofactor is presumably bound covalently to an invariant threonine residue of the periplasmic domain. NosR also features conserved CX₃CP motifs, located C-terminally of the transmembrane helices TM4 and TM6. We genetically manipulated nosR with respect to these different domains and putative functional centers and expressed recombinant derivatives in a nosR null mutant, MK418nosR::Tn5. NosR's function was studied by its effects on N₂O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N₂O respiration was observed upon deletion of most of the periplasmic domain or of the C-terminal parts beyond TM2 or upon modification of the cysteine residues in a highly conserved motif, CGWLCP, following TM4. Nonetheless, NosZ purified from the recombinant NosR background exhibited in vitro catalytic activity. Certain NosR derivatives caused an increase in NosZ of the spectral contribution from a modified catalytic Cu site. In addition to its role in nosZ expression, NosR supports in vivo N₂O respiration. We also discuss its putative functions in electron donation and redox activation.

Present address: InfectoPharm, D-64646 Heppenheim, Germany.

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