Molecular characterization of Desulfovibrio gigas neelaredoxin, a protein involved in oxygen detoxification in anaerobes.
Desulfovibrio gigas neelaredoxin is an iron-containing protein of 15 kDa, having a single iron site with a His(4)Cys coordination. Neelaredoxins and homologous proteins are widespread in anaerobic prokaryotes and have superoxide-scavenging activity. To further understand its role in anaerobes, its genomic organization and expression in D. gigas were studied and its ability to complement Escherichia coli superoxide dismutase deletion mutant was assessed. In D. gigas, neelaredoxin is transcribed as a monocistronic mRNA of 500 bases as revealed by Northern analysis. Putative promoter elements resembling sigma(70) recognition sequences were identified. Neelaredoxin is abundantly and constitutively expressed, and its expression is not further induced during treatment with O(2) or H(2)O(2). The neelaredoxin gene was cloned by PCR and expressed in E. coli, and the protein was purified to homogeneity. The recombinant neelaredoxin has spectroscopic properties identical to those observed for the native one. Mutations of Cys-115, one of the iron ligands, show that this ligand is essential for the activity of neelaredoxin. In an attempt to elucidate the function of neelaredoxin within the cell, it was expressed in an E. coli mutant deficient in cytoplasmic superoxide dismutases (sodA sodB). Neelaredoxin suppresses the deleterious effects produced by superoxide, indicating that it is involved in oxygen detoxification in the anaerobe D. gigas.
Silva
G
G
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras, Portugal.
LeGall
J
J
Xavier
A V
AV
Teixeira
M
M
Rodrigues-Pousada
C
C
D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial; D003901:Desulfovibrio / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli; D016166:Free Radical Scavengers / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D033862:Iron-Binding Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009876:Operon; D018384:Oxidative Stress; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000502:physiology
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D016166:Free Radical Scavengers; D033862:Iron-Binding Proteins; D011993:Recombinant Fusion Proteins; D010088:Oxidoreductases; C095500:SodA protein, Bacteria; C087504:SodB protein, Bacteria; D013482:Superoxide Dismutase; C414179:nlr protein, Desulfovibrio gigas; D010100:Oxygen