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

Eurobic9, 2-6 September, 2008, Wrocław, Poland
O26. Kinetics of Gas Diffusion in Hydrogenase: Experimental Approaches
F. Leroux a , B. Burlat a , S. Dementin a , L. Cournac b , A. Volbeda c , B. Guigliarelli e ,
P. Bertrand a , J. Fontecilla-Camps c , M. Rousset a , C. Léger a
a BIP, CNRS, 31 ch. J. Aiguier, 13009, Marseille, France
e-mail: leger@ibsm.cnrs-mrs.fr
b LBBBM, CEA,, 13108, St Paul-lez-Durance, France
c LCCP, CEA, 41 rue Jules Horowitz, 38027, Grenoble, France
Hydrogenases, which catalyze H2 to H + conversion as part of the bioenergetic metabolism of many
microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described using
crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion
kinetics is unexplored.
Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses
protein film voltammetry [1-3] to resolve the kinetics of binding and release of the competitive inhibitor CO;
the other is based on interpreting the yield in the isotope exchange assay.
We study structurally-characterized mutants of a NiFe hydrogenase, and we show that two mutations, which
significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO
and H2 toward and from the active site by up to two orders of magnitude. This proves the existence of a
functional channel which matches the hydrophobic cavity found in the crystal. However, the changes in
diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the X-ray
structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of sidechain
dynamics in determining these [4].
References:
[1] C. Léger at al. J. Am. Chem. Soc. 126, 12162 (2004)
[2] C. Baffert et al. Angewandte Chemie Int. Ed. 47, 2052 (2008)
[3] C. Léger et al. Chemical Reviews. In press http://dx.doi.org/10.1021/cr0680742 (2008)
[4] F. Leroux et al. Submitted (2008)
_____________________________________________________________________
113