HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 4, 2344-2352, January 25, 2008

This Article Full Text Full Text (PDF) All Versions of this Article: 283/4/2344    most recent M708123200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Yamashita, T. Articles by Vos, M. H. PubMed PubMed Citation Articles by Yamashita, T. Articles by Vos, M. H.

Ligand Dynamics and Early Signaling Events in the Heme Domain of the Sensor Protein Dos from Escherichia coli^*

Taku Yamashita¹, Latifa Bouzhir-Sima, Jean-Christophe Lambry, Ursula Liebl, and Marten H. Vos²

From the Laboratoire d'Optique et Biosciences, CNRS, Ecole Polytechnique, F-91128 Palaiseau and INSERM U696, F-91128 Palaiseau, France

In the heme-based sensor Dos from Escherichia coli, the ferrous heme is coordinated by His-77 and Met-95. The latter residue is replaced upon oxygen binding or oxidation of the heme. Here we investigate the early signaling processes upon dissociation of the distal ligand using ultrafast spectroscopy and site-directed mutagenesis. Geminate CO rebinding to the heme domain DosH appears insensitive to replacement of Met-95, in agreement with the notion that this residue is oriented out of the heme pocket in the presence of external ligands. A uniquely slow 35-ps phase in rebinding of the flexible methionine side chain after dissociation from ferrous DosH is completely abolished in rebinding of the more rigid histidine side chain in the M95H mutant protein, where only the 7-ps phase, common to all 6-coordinate heme proteins, is observed. Temperature-dependence studies indicate that all rebinding of internal and external ligands is essentially barrierless, but that CfigsO escape from the heme pocket is an activated process. Solvent viscosity studies combined with molecular dynamics simulations show that there are two configurations in the ferrous 6-coordinate protein, involving two isomers of the Met-95 side chain, of which the structural changes extend to the solvent-exposed backbone, which is part of the flexible FG loop. One of these configurations has considerable motional freedom in the Met-95-dissociated state. We suggest that this configuration corresponds to an early signaling intermediate state, is responsible for the slow rebinding, and allows small ligands in the protein to efficiently compete for binding with the heme.

Received for publication, October 1, 2007 , and in revised form, November 8, 2007.

^* 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 Recipient of an EC Marie Curie Incoming International Fellowship.

² To whom correspondence should be addressed. Tel.: 33-169-335-066; Fax: 33-169-335-084; E-mail: marten.vos{at}polytechnique.edu.