Formation of a dinitrosyl iron complex by NorA, a nitric oxide-binding di-iron protein from Ralstonia eutropha H16.
In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Diferrous NorA binds two molecules of NO maximally. Unexpectedly, binding of NO to the diferrous NorA required an external reductant. Two different NorA-NO species could be resolved. A minor species (up to 20%) showed an S = (1/2) EPR signal with g( perpendicular) = 2.041, and g( parallel) = 2.018, typical of a paramagnetic dinitrosyl iron complex. The major species was EPR-silent, showing characteristic signals at 420 nm and 750 nm in the optical spectrum. This species is proposed to represent a novel dinitrosyl iron complex of the form Fe(2+)-[NO](2)(2-), i.e. NO is bound as NO(-). The NO binding capacity of NorA in conjunction with its high cytoplasmic concentration (20 mum) suggests that NorA regulates transcription by lowering the free cytoplasmic concentration of NO.
Strube
Katja
K
Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany.
de Vries
Simon
S
Cramm
Rainer
R
D001426:Bacterial Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D020118:Cupriavidus necator / Q000502:physiology*; D006422:Hemerythrin / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D009876:Operon / Q000502:physiology; D010088:Oxidoreductases / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology
D001426:Bacterial Proteins; D002352:Carrier Proteins; D006422:Hemerythrin; D014157:Transcription Factors; D009569:Nitric Oxide; D007501:Iron; D010088:Oxidoreductases; C034747:nitric-oxide reductase