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J. Biol. Chem., Vol. 281, Issue 39, 29165-29173, September 29, 2006

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Heme Displacement Mechanism of CooA Activation

MUTATIONAL AND RAMAN SPECTROSCOPIC EVIDENCE^*

Mohammed Ibrahim, Robert L. Kerby, Mrinalini Puranik¹, Ingar H. Wasbotten², Hwan Youn, Gary P. Roberts, and Thomas G. Spiro³

From the Department of Chemistry, Princeton University, Princeton, New Jersey 08544 and Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

The heme-containing protein CooA of Rhodospirillum rubrum regulates the expression of genes involved in CO oxidation. CooA binds its target DNA sequence in response to CO binding to its heme. Activity measurements and resonance Raman (RR) spectra are reported for CooA variants that bind DNA even in the absence of CO, those in which the wild-type residues at the 121-126 positions, TSCMRT, are replaced by the residues AYLLRL or RYLLRL, and also for variants that bind DNA poorly in the presence of CO, such as L120S and L120F. The Fe-C and C-O stretching resonance Raman (RR) frequencies of all CooAs examined deviate from the expected back-bonding correlation in a manner indicating weakening of the Fe-His-77 proximal ligand bond, and the extent of weakening correlates positively with DNA binding activity. The (A/R) YLLRL variants have detectable populations of a 5-coordinate heme resulting from partial dissociation of the endogenous distal ligand, Pro-2. Selective excitation of this population reveals downshifted Fe-His-77-stretching RR bands, confirming the proximal bond weakening. These results support our previous hypothesis that the conformational change required for DNA binding is initiated by displacement of the heme into an adjacent hydrophobic cavity once CO displaces the Pro-2 ligand. Examination of the crystal structure reveals a physical basis for these results, and a mechanism is proposed to link heme displacement to conformational change.

Received for publication, June 9, 2006 , and in revised form, July 17, 2006.

^* This work was supported by National Institutes of Health Grants GM33576 (to T. G. S.) and GM53228 (to G. P. R.). 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 Present address: The National Centre for Biological Sciences, Bangalore, 560065, India.

² Present address: Department of Chemistry, University of Tromsø, Tromsø 9037, Norway.

³ To whom correspondence should be addressed. Tel.: 609-258-3907; Fax: 609-258-0348; E-mail: spiro{at}princeton.edu.

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