Direct oxidation of the [2Fe-2S] cluster in SoxR protein by superoxide: distinct differential sensitivity to superoxide-mediated signal transduction.
The [2Fe-2S] transcription factor SoxR is activated by reversible one-electron oxidation of its [2Fe-2S] cluster, leading to enhanced production of various antioxidant proteins through induction of the soxRS regulon in Escherichia coli. Recently, there has been considerable debate about whether superoxide (O(2)(•)) activates SoxR directly. To elucidate the underlying activation mechanism, we investigated SoxR interaction with O(2)(•) using pulse radiolysis. Radiolytically generated hydrated electrons reduced the oxidized form of the [2Fe-2S] cluster of SoxR within 2 μs. A subsequent increase in absorption in the visible region corresponding to reoxidation of the [2Fe-2S] cluster was observed on a time scale of milliseconds. Addition of human copper/zinc superoxide dismutase inhibited this delayed oxidation in a concentration-dependent fashion (I(50) = 1.0 μm), indicating that O(2)(•) oxidized the reduced form of SoxR directly. The second-order rate constant of this process was estimated to be 5 × 10(8) m(-1) s(-1). A similar result was observed after pulse radiolysis of Pseudomonas aeruginosa SoxR. However, superoxide dismutase inhibited the oxidation of reduced SoxR much more effectively in P. aeruginosa, even at a lower concentration (I(50) = 80 nm), indicating that the soxRS response is much more sensitive to O(2)(•) in E. coli than in P. aeruginosa. These results suggest that SoxR proteins play a distinct regulatory role in the activation of O(2)(•).
Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
Mayu
M
Fujikawa
Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan. Electronic address: kobayasi@sanken.osaka-u.ac.jp.
Kazuo
K
Kobayashi
Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
Takahiro
T
Kozawa
D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D006801:Humans; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D015398:Signal Transduction; D013482:Superoxide Dismutase / Q000737:chemistry*; D013481:Superoxides / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry*
D001426:Bacterial Proteins; D014157:Transcription Factors; D013481:Superoxides; C070724:SoxR protein, Bacteria; D013482:Superoxide Dismutase