title: Functional domains of NosR, a novel transmembrane iron-sulfur flavoprotein necessary for nitrous oxide respiration.
abstract: 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.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität Karlsruhe, Karlsruhe, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Wunsch
authors.forename: Walter G
authors.initials: WG
authors.lastname: Zumft
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C075970:NosR protein, Bacteria; D009609:Nitrous Oxide
references.citation: J Bacteriol. 2001 Mar;183(5):1663-71
references.pmid: 11160097
references.citation: Eur J Biochem. 2000 Mar;267(5):1368-81
references.pmid: 10691974
references.citation: Biochem J. 2002 Jun 15;364(Pt 3):807-15
references.pmid: 12049645
references.citation: Adv Microb Physiol. 2002;46:47-110
references.pmid: 12073657
references.citation: J Bacteriol. 2002 Oct;184(19):5330-8
references.pmid: 12218019
references.citation: J Bacteriol. 2003 Feb;185(3):887-96
references.pmid: 12533464
references.citation: Microbiology. 2003 Jan;149(Pt 1):29-36
references.pmid: 12576577
references.citation: Mol Microbiol. 2003 Mar;47(5):1459-73
references.pmid: 12603748
references.citation: J Bacteriol. 2003 Mar;185(6):1895-902
references.pmid: 12618453
references.citation: BMC Microbiol. 2003 Feb 6;3:3
references.pmid: 12625841
references.citation: Nat Struct Biol. 2000 Mar;7(3):191-5
references.pmid: 10700275
references.citation: J Mol Biol. 2000 Mar 17;297(1):49-65
references.pmid: 10704306
references.citation: FEBS Lett. 2000 Jun 2;474(2-3):165-8
references.pmid: 10838078
references.citation: J Bacteriol. 2000 Oct;182(20):5683-91
references.pmid: 11004165
references.citation: Biochemistry. 2000 Oct 24;39(42):12753-6
references.pmid: 11041839
references.citation: FEBS Lett. 2001 Jan 12,;488(1-2):5-8
references.pmid: 11163785
references.citation: J Biol Chem. 1987 May 15;262(14):6515-25
references.pmid: 3032972
references.citation: J Bacteriol. 1987 Oct;169(10):4577-80
references.pmid: 2820935
references.citation: Nucleic Acids Res. 1988 Jul 11;16(13):6127-45
references.pmid: 3041370
references.citation: Eur J Biochem. 1989 Jan 2;178(3):751-62
references.pmid: 2536326
references.citation: J Bacteriol. 1989 Feb;171(2):929-39
references.pmid: 2536685
references.citation: FEMS Microbiol Lett. 1990 Jul;58(2):221-5
references.pmid: 2121587
references.citation: J Biol Chem. 1991 Feb 5;266(4):2199-202
references.pmid: 1846617
references.citation: J Biol Chem. 1991 Feb 5;266(4):2203-9
references.pmid: 1899237
references.citation: J Bacteriol. 1992 Aug;174(16):5332-9
references.pmid: 1644760
references.citation: J Bacteriol. 1992 Oct;174(20):6444-54
references.pmid: 1400197
references.citation: J Bacteriol. 1994 Jul;176(13):4052-65
references.pmid: 8021187
references.citation: Gene. 1994 Oct 11;148(1):81-6
references.pmid: 7926843
references.citation: Biochim Biophys Acta. 1995 Nov 7;1264(2):183-90
references.pmid: 7495862
references.citation: Biochim Biophys Acta. 1995 Dec 12;1232(3):97-173
references.pmid: 8534676
references.citation: J Bacteriol. 1996 Mar;178(6):1505-14
references.pmid: 8626275
references.citation: Arch Microbiol. 1996 May;165(5):297-305
references.pmid: 8661920
references.citation: Protein Sci. 1996 Aug;5(8):1704-18
references.pmid: 8844859
references.citation: Microbiol Mol Biol Rev. 1997 Dec;61(4):533-616
references.pmid: 9409151
references.citation: J Bacteriol. 1998 Aug;180(16):4044-50
references.pmid: 9696749
references.citation: J Inorg Biochem. 1998 Jul;70(3-4):155-69
references.pmid: 9720302
references.citation: J Bacteriol. 1985 Sep;163(3):918-24
references.pmid: 2993252
references.citation: J Am Chem Soc. 2003 Dec 24;125(51):15708-9
references.pmid: 14677937
references.citation: J Am Chem Soc. 2004 Mar 17;126(10):3030-1
references.pmid: 15012115
references.citation: Antonie Van Leeuwenhoek. 2004 Apr;85(3):229-35
references.pmid: 15028871
references.citation: Biochem J. 2004 Apr 1;379(Pt 1):47-55
references.pmid: 14674886
references.citation: FEBS Lett. 2004 Jul 2;569(1-3):7-12
references.pmid: 15225600
references.citation: Dalton Trans. 2004 Apr 7;(7):996-1002
references.pmid: 15252678
references.citation: J Nat Prod. 2004 Aug;67(8):1356-67
references.pmid: 15332855
references.citation: J Biochem. 1971 Jun;69(6):991-1001
references.pmid: 5091944
references.citation: J Gen Microbiol. 1975 May;88(1):11-9
references.pmid: 1151328
references.citation: Appl Environ Microbiol. 1980 Jan;39(1):81-4
references.pmid: 7356322
references.citation: Anal Biochem. 1983 Jun;131(2):373-8
references.pmid: 6614472
references.citation: Eur J Biochem. 1984 Sep 17;143(3):499-505
references.pmid: 6383823
references.citation: Gene. 1985;33(1):103-19
references.pmid: 2985470
references.citation: Eur J Biochem. 1985 Dec 16;153(3):459-67
references.pmid: 3000778
references.citation: Mol Microbiol. 1999 Oct;34(1):24-36
references.pmid: 10540283
references.citation: Biochemistry. 1999 Dec 7;38(49):16246-52
references.pmid: 10587447
references.citation: J Bacteriol. 2001 Apr;183(8):2516-26
references.pmid: 11274111