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J. Biol. Chem., Vol. 283, Issue 27, 19000-19010, July 4, 2008

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Roles of Arg-97 and Phe-113 in Regulation of Distal Ligand Binding to Heme in the Sensor Domain of Ec DOS Protein

RESONANCE RAMAN AND MUTATION STUDY^*

Samir F. El-Mashtoly¹, Satoru Nakashima, Atsunari Tanaka^¶2, Toru Shimizu^¶, and Teizo Kitagawa³

From the Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, the Department of Life Science, Graduate School of Science, Hyogo Prefecture University, Kamigoricho, Akougun 678-1201, and the ^¶Institute of Multidisciplinary Research for Advanced Material, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577, Japan

The direct oxygen sensor protein isolated from Escherichia coli (Ec DOS) is a heme-based signal transducer protein responsible for phosphodiesterase (PDE) activity. Binding of O₂, CO, or NO to a reduced heme significantly enhances the PDE activity toward 3',5'-cyclic diguanylic acid. We report stationary and time-resolved resonance Raman spectra of the wild-type and several mutants (Glu-93 Ile, Met-95 Ala, Arg-97 Ile, Arg-97 Ala, Arg-97 Glu, Phe-113 Leu, and Phe-113 Thr) of the heme-containing PAS domain of Ec DOS. For the CO- and NO-bound forms, both the hydrogen-bonded and non-hydrogen-bonded conformations were found, and in the former Arg-97 forms a hydrogen bond with the heme-bound external ligand. The resonance Raman results revealed significant interactions of Arg-97 and Phe-113 with a ligand bound to the sixth coordination site of the heme and profound structural changes in the heme propionates upon dissociation of CO. Mutation of Phe-113 perturbed the PDE activities, and the mutation of Arg-97 and Phe-113 significantly influenced the transient binding of Met-95 to the heme upon photodissociation of CO. This suggests that the electrostatic interaction of Arg-97 and steric interaction of Phe-113 are crucial for regulating the competitive recombination of Met-95 and CO to the heme. On the basis of these results, we propose a model for the role of the heme propionates in communicating the heme structural changes to the protein moiety.

Received for publication, February 15, 2008 , and in revised form, April 28, 2008.

^* This work was supported by a Japan Society for the Promotion of Science (JSPS) fellowship (to S. F. E.-M.) and by the JSPS (Grant-in-Aid for Basic Scientific Research 19350089 to T. K.). 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 Figs. S1-S9.

¹ Present address: Dept. of Chemistry, Faculty of Science, King Khalid University, Abha 9004, Saudi Arabia.

² Present address: The Wistar Institute, Philadelphia, PA 19104.

³ Present address: Toyota Physical & Chemical Research Institute, Nagakute, Aichigun, Aichi 480-1192, Japan. To whom correspondence should be addressed: Tel.: 81-80-1620-8159; Fax: 81-561-63-6302; E-mail: teizo{at}ims.ac.jp.

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