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J. Biol. Chem., Vol. 281, Issue 16, 11271-11278, April 21, 2006

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Evidence for Displacements of the C-helix by CO Ligation and DNA Binding to CooA Revealed by UV Resonance Raman Spectroscopy^*

Minoru Kubo, Sayaka Inagaki, Shiro Yoshioka, Takeshi Uchida, Yasuhisa Mizutani^¶, Shigetoshi Aono, and Teizo Kitagawa¹

From the Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki 444-8787, Japan, the Department of Structural Molecule Science, The Graduate University for Advanced Studies, Okazaki 444-8585, Japan, and the ^¶Molecular Photoscience Research Center, Kobe University, Kobe 657-8501, Japan

The UV and visible resonance Raman spectra are reported for CooA from Rhodospirillum rubrum, which is a transcriptional regulator activated by growth in a CO atmosphere. CO binding to heme in its sensor domain causes rearrangement of its DNA-binding domain, allowing binding of DNA with a specific sequence. The sensor and DNA-binding domains are linked by a hinge region that follows a long C-helix. UV resonance Raman bands arising from Trp-110 in the C-helix revealed local movement around Trp-110 upon CO binding. The indole side chain of Trp-110, which is exposed to solvent in the CO-free ferrous state, becomes buried in the CO-bound state with a slight change in its orientation but maintains a hydrogen bond with a water molecule at the indole nitrogen. This is the first experimental data supporting a previously proposed model involving displacement of the C-helix and heme sliding. The UV resonance Raman spectra for the CooA-DNA complex indicated that binding of DNA to CooA induces a further displacement of the C-helix in the same direction during transition to the complete active conformation. The Fe-CO and C-O stretching bands showed frequency shifts upon DNA binding, but the Fe-His stretching band did not. Moreover, CO-geminate recombination was more efficient in the DNA-bound state. These results suggest that the C-helix displacement in the DNA-bound form causes the CO binding pocket to narrow and become more negative.

Received for publication, December 13, 2005 , and in revised form, January 23, 2006.

^* This work was supported by grant-in-aid 14001004 from the Ministry of Culture, Education, Sports, Science, and Technology of Japan (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.

¹ To whom correspondence should be addressed: 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan. Tel.: 81-564-59-5225; Fax: 81-564-59-5229; E-mail: teizo{at}ims.ac.jp.

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