Functional domains of NosR, a novel transmembrane iron-sulfur flavoprotein necessary for nitrous oxide respiration.
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.
Wunsch
Patrick
P
Lehrstuhl für Mikrobiologie, Universität Karlsruhe, Karlsruhe, Germany.
Zumft
Walter G
WG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004734:Energy Metabolism; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009609:Nitrous Oxide / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D044223:Pseudomonas stutzeri / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C075970:NosR protein, Bacteria; D009609:Nitrous Oxide
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