A study of NO trafficking from dinitrosyl-iron complexes to the recombinant E. coli transcriptional factor SoxR.
SoxR is a transcriptional factor in Escherichia coli that induces the expression of SoxS to initiate the production of enzymes in response to oxidative stress. In addition to superoxide, SoxR is also sensitive to cellular NO to produce a protein-bound dinitrosyl-iron complex (DNIC) with a characteristic electron paramagnetic resonance (EPR) signal at g(av)=2.03. Toward developing a strategy for NO sensing based on this property of SoxR, we have overexpressed and purified the recombinant His-tagged SoxR protein. Upon treatment of the purified protein under anaerobic conditions with (1) NO solution, (2) S-nitrosothiol (RSNO), and (3) chemically synthesized low molecular weight DNICs (LMW-DNICs), we have observed enhancement of the EPR signal at g(av)=2.03 from the protein-bound DNICs over time, reflecting the redistribution of NO from the NO solution, RSNO and LMW-DNICs to the SoxR. We have exploited this NO exchange to investigate the kinetics and mechanisms of release and delivery of NO from various LMW-DNICs to an isopropyl-beta-D-thiogalactopyranoside-dependent SoxR expressed in E. coli cells. These experiments revealed that the NO from RSNO and LMW-DNICs could cross the biological membrane and enter the cytoplasm of the cell to form the SoxR protein-bound DNIC complex. For comparison, we have also studied the direct NO transfer from the LMW-DNICs to the SoxR protein in buffer. The NO transfer was found to be rapid. From the kinetic data derived, we showed that LMW-DNICs with bidentate thiolate ligands displayed greater stability in aqueous solution but exhibited more facile NO delivery to cytoplasmic SoxR in whole cells.
Lo
Feng-Chun
FC
Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan.
Chen
Chang-Li
CL
Lee
Chien-Ming
CM
Tsai
Ming-Che
MC
Lu
Tsai-Te
TT
Liaw
Wen-Feng
WF
Yu
Steve S-F
SS
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005290:Ferric Compounds / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008968:Molecular Conformation; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D009603:Nitroso Compounds / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D005290:Ferric Compounds; D009603:Nitroso Compounds; D011994:Recombinant Proteins; D014157:Transcription Factors; C070724:SoxR protein, Bacteria; D009569:Nitric Oxide