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J. Biol. Chem., Vol. 277, Issue 26, 23821-23827, June 28, 2002

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Characterization of a Direct Oxygen Sensor Heme Protein from Escherichia coli
EFFECTS OF THE HEME REDOX STATES AND MUTATIONS AT THE HEME-BINDING SITE ON CATALYSIS AND STRUCTURE^*

Yukie Sasakura, Satoshi Hirata, Shunpei Sugiyama, Shingo Suzuki, Sue Taguchi, Miki Watanabe, Toshitaka Matsui, Ikuko Sagami, and Toru Shimizu

From the Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai 980-8577, Japan

A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia 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 -helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min¹ 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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