Functional Domains of NosR, a Novel Transmembrane Iron-Sulfur Flavoprotein Necessary for Nitrous Oxide Respiration
Open Access
- 15 March 2005
- journal article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 187 (6), 1992-2001
- https://doi.org/10.1128/jb.187.6.1992-2001.2005
Abstract
Bacterial nitrous oxide (N 2 O) respiration depends on the polytopic membrane protein NosR for the expression of N 2 O 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 CX 3 CP 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, MK418 nosR ::Tn 5 . NosR's function was studied by its effects on N 2 O respiration, NosZ synthesis, and the properties of purified NosZ proteins. Although all recombinant NosR proteins allowed the synthesis of NosZ, a loss of N 2 O 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 N 2 O respiration. We also discuss its putative functions in electron donation and redox activation.Keywords
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