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Papers In Press, published online ahead of print April 22, 2002
J. Biol. Chem, 10.1074/jbc.M202738200
Submitted on March 21, 2002
Revised on April 16, 2002
Accepted on April 22, 2002
Inst. Mult. Res. Adv. Mat., Tohoku University, Sendai, Miyagi 980-8577
Corresponding Author: shimizu{at}tagen.tohoku.ac.jp
A protein containig a heme-binding PAS domain from E. coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low-spin ferric heme complex. Its a-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV vs. SHE. Mutation of His77 of the isolated PAS domain abolished heme binding, whereas mutation of His83 did not, suggesting that His77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min-1 with cAMP, which was optimal at pH 8.5 in the presence of Mg2+ and was strongly inhibited by CO, NO and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.
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