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J. Biol. Chem., Vol. 282, Issue 29, 21301-21307, July 20, 2007

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Critical Role of the Heme Axial Ligand, Met⁹⁵, in Locking Catalysis of the Phosphodiesterase from Escherichia coli (Ec DOS) toward Cyclic diGMP^*

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

Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O₂ or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg⁹⁷ in the heme distal side in Ec DOS interacts with the O₂ molecule and Met⁹⁵ serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O₂ complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O₂. In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O₂, CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met⁹⁵ coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met⁹⁵ caused by the binding of the external ligands or mutations at Met⁹⁵ releases the catalytic lock and activates catalysis.

Received for publication, March 5, 2007 , and in revised form, May 9, 2007.

^* This work was supported in part by the Grand-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental data and Figs. S1–S7.

¹ To whom correspondence should be addressed: 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan. Tel.: 81-22-217-5604/5605/5606; Fax: 81-22-217-5604/5605/5390; E-mail: shimizu{at}tagen.tohoku.ac.jp.

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