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Published online on April 19, 2007, 10.1073/pnas.0700445104
PNAS | May 1, 2007 | vol. 104 | no. 18 | 7408-7413


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BIOLOGICAL SCIENCES / BIOPHYSICS
Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy

Sergei G. Kruglik{dagger},{ddagger},§, Audrius Jasaitis{dagger},{ddagger}, Klara Hola{dagger},{ddagger}, Taku Yamashita{dagger},{ddagger}, Ursula Liebl{dagger},{ddagger}, Jean-Louis Martin{dagger},{ddagger}, and Marten H. Vos{dagger},{ddagger},¶

{dagger}Laboratoire d'Optique et Biosciences, Centre National de la Recherche, Ecole Polytechnique, 91128 Palaiseau Cedex, France; {ddagger}Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 696, 91128 Palaiseau, France; and §Laboratoire de Biophysique Moléculaire, Cellulaire, et Tissulaire, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7033, University Pierre & Marie Curie, Genopole Campus 1, Batiment Genavenir 8, 5 Rue Henri Desbrueres, 91030 Evry, France

Edited by William A. Eaton, National Institutes of Health, Bethesda, MD, and approved March 14, 2007 (received for review January 17, 2007)

Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR3) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR3 measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron–ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron–histidine band around 217 cm–1 as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the "oxygen cage" properties of this oxygen sensor protein.

heme protein | molecular dynamics | ultrafast spectroscopy | vibrational spectroscopy

Author contributions: S.G.K., U.L., J.-L.M., and M.H.V. designed research; S.G.K., A.J., K.H., and T.Y. performed research; K.H. and U.L. contributed new reagents/analytic tools; S.G.K. analyzed data; and S.G.K. and M.H.V. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0700445104/DC1.

To whom correspondence should be addressed at the {dagger} address. E-mail: marten.vos{at}polytechnique.edu

© 2007 by The National Academy of Sciences of the USA


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