ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P110. Oxygen Sensibility of Ni-Fe Hydrogenases with Modified Gas
Channels
F. Leroux a , S. Dementin a , B. Burlat a , A. Volbeda b , J. Fontecilla-Camps b , M. Rousset a ,
P. Bertrand a , B. Guigliarelli a , Ch. Léger a
a
Laboratoire de Bioénergétique et Ingénierie des, CNRS - Université de Provence, 31 chemin Jospeh Aiguier,
13402, Marseille Cedex 20, France,
b
Laboratoire de Cristallographie et Cristallogenès, CEA, Grenoble, France
e-mail: fanny.leroux@ibsm.cnrs-mrs.fr
Hydrogenases catalyze the conversion between H2 and H+ as part of the bioenergetic metabolism of most
bacteria. The main obstacle for their use as catalysts in biofuel cells is the fact that they are inhibited by
molecular oxygen [1]. The active site of the so-called Ni-Fe hydrogenase is buried in the protein. Structural and
molecular dynamic studies suggest that a hydrophobic channel guides hydrogen and inhibitors towards the active
site and recent results suggest that the shape of this tunnel may determine the sensitivity to oxygen [2].
We introduce a quantitative experimental method for probing the rates of intra-molecular diffusion within
hydrogenases based on using Protein Film Voltammetry, a technique where enzymes molecules are adsorbed on
an electrode, a potential is applied and the resulting current is proportional to enzyme's activity [3-4]. We use it
to resolve the kinetics of binding and release of the competitive inhibitor CO and of the reaction with O2 [5-6].
We study how the structure of the channel affects the diffusion of CO and the rate of O2 inhibition in several
mutants whose structures have been determined. We will show that certain mutations slow down gas diffusion
[7] and affect the sensitivity of the enzyme. However, CO always reacts with the enzyme much faster than O2
does, this implies that intramolecular transport does not limit the rate of oxygen inhibition, at least in the WT
enzyme.
References:
[1] De Lacey, Fernandez, Rousset, Cammack, Chem. Rev. 2007, 107 (10), 4304-30.
[2] Buhrke, Lenz, Krauss, Friedrich, J. Biol. Chem. 2005, 23791.
[3] Léger, Elliott, Hoke, Jeuken, Jones, Armstrong, Biochemistry, 2003, 42, 8653.
[4] Léger, Bertrand, Chem Rev, in press (july 2008).
[5] Léger, Dementin, Bertrand, Rousset, Guigliarelli, J. Am. Chem. Soc., 126, 2004, 38.
[6] Baffert, Demuez, Cournac, Burlat, Guigliarelli, Bertrand, Girbal, Léger, Angew. Chem. Int. Ed. 47, 2008,
2052.
[7] Leroux, Dementin, Burlat, Cournac, Volbeda, Champ, Martin, Guigliarelli, Bertrand, Fontecilla-Camps,
Rousset, Léger, submitted.
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