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J. Biol. Chem., Vol. 283, Issue 11, 6942-6949, March 14, 2008

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Protein Conformation Changes of HemAT-Bs upon Ligand Binding Probed by Ultraviolet Resonance Raman Spectroscopy^*

From the Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan

HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O₂ sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector (O₂) from other gas molecules (CO and NO), although all of them bind to a heme. To monitor the conformational changes in the protein moiety upon binding of different ligands, we have investigated ultraviolet resonance Raman (UVRR) spectra of the ligand-free and O₂-, CO-, and NO-bound forms of full-length HemAT-Bs and several mutants (Y70F, H86A, T95A, and Y133F) and found that Tyr⁷⁰ in the heme distal side and Tyr¹³³ and Trp¹³² from the G-helix in the heme proximal side undergo environmental changes upon ligand binding. In addition, the UVRR results confirmed our previous model, which suggested that Thr⁹⁵ forms a hydrogen bond with heme-bound O₂, but Tyr⁷⁰ does not. It is deduced from this study that hydrogen bonds between Thr⁹⁵ and heme-bound O₂ and between His⁸⁶ and heme 6-propionate communicate the heme structural changes to the protein moiety upon O₂ binding but not upon CO and NO binding. Accordingly, the present UVRR results suggest that O₂ binding to heme causes displacement of the G-helix, which would be important for transduction of the conformational changes from the sensor domain to the signaling domain.

Received for publication, November 9, 2007 , and in revised form, December 20, 2007.

^* This study was supported by Japan Society for the Promotion of Science fellowships (to S. F. E-M. and Y. G.); Grant-in-aid for Basic Scientific Research 19350089 from the Japan Society for the Promotion of Science (to T. K.); Grant-in-aid for Scientific Research (B) 19370059 from the Japan Society for the Promotion of Science (to S. A.); and Grant-in-aid for Scientific Research on Priority Areas 19045031 from the Ministry of Education, Culture, Sports, Science, and Technology (to S. A.). 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–S4.

¹ Present address: Dept. of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461.

² To whom correspondence should be addressed: Toyota Physical and Chemical Research Inst., Nagakute, Aichi 480-1192, Japan. Tel.: 81-80-1620-8159; Fax: 81-561-63-6302; E-mail: teizo{at}ims.ac.jp.

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				S. F. El-Mashtoly, S. Nakashima, A. Tanaka, T. Shimizu, and T. Kitagawa 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 J. Biol. Chem., July 4, 2008; 283(27): 19000 - 19010. [Abstract] [Full Text] [PDF]