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J. Biol. Chem., Vol. 280, Issue 15, 15279-15288, April 15, 2005

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Role of Arginine 220 in the Oxygen Sensor FixL from Bradyrhizobium japonicum^*

Véronique Balland, Latifa Bouzhir-Sima, Laurent Kiger¶, Michael C. Marden¶, Marten H. Vos, Ursula Liebl, and Tony A. Mattioli||

From the Laboratoire de Biophysique du Stress Oxydant, SBE/DBJC and CNRS URA 2096, CEA/Saclay, 91191 Gif-sur-Yvette cedex, France, the Laboratoire d'Optique et Biosciences, INSERM U451, CNRS UMR 7645, Ecole Polytechnique-ENSTA, 91128 Palaiseau, France, and ^¶INSERM Unité 473, 78, rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France

In the heme-based oxygen sensor protein FixL, conformational changes induced by oxygen binding to the heme sensor domain regulate the activity of a neighboring histidine kinase, eventually restricting expression of specific genes to hypoxic conditions. The conserved arginine 220 residue is suggested to play a key role in the signal transduction mechanism. To obtain detailed insights into the role of this residue, we replaced Arg²²⁰ by histidine (R220H), glutamine (R220Q), glutamate (R220E), and isoleucine (R220I) in the heme domain FixLH from Bradyrhizobium japonicum. These mutations resulted in dramatic changes in the O₂ affinity with K_d values in the order R220I < R220Q < wild type < R220H. For the R220H and R220Q mutants, residue 220 interacts with the bound O₂ or CO ligands, as seen by resonance Raman spectroscopy. For the oxy-adducts, this H-bond modifies the acidity of the O₂ ligand, and its strength is correlated with the back-bonding-sensitive ₄ frequency, the k_off value for O₂ dissociation, and heme core-size conformational changes. This effect is especially strong for the wild-type protein where Arg²²⁰ is, in addition, positively charged. These observations strongly suggest that neither strong ligand fixation nor the displacement of residue 220 into the heme distal pocket are solely responsible for the reported heme conformational changes associated with kinase activity regulation, but that a significant decrease of the heme ^* electron density because of strong back-bonding toward the oxygen ligand also plays a key role.

Received for publication, December 10, 2004 , and in revised form, January 25, 2005.

^* This work was supported in part by the Regional Council of the Ile-de-France (to T. A. M.). 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 Materials.

^|| To whom correspondence should be addressed: Service de Bioénergétique, Département de Biologie Joliot-Curie, CEA Saclay, F-91191, Gif-sur-Yvette Cedex, France. Tel.: 33-169-08-41-66; Fax: 33-169-08-87-17; E-mail: tony.mattioli{at}cea.fr.

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