DOI: 10.1073/pnas.0803689105. Experimental approaches to kinetics of gas diffusion in hydrogenase

Experimental approaches to kinetics of gas diffusion in hydrogenase

10.1073/pnas.0803689105

Crossref journal-article

Proceedings of the National Academy of Sciences

Proceedings of the National Academy of Sciences (341)

Abstract

Hydrogenases, which catalyze H 2 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 by 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 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 H 2 toward and from the active site by up to 2 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 side-chain dynamics in determining these.

Bibliography

Leroux, F., Dementin, S., Burlat, B., Cournac, L., Volbeda, A., Champ, S., Martin, L., Guigliarelli, B., Bertrand, P., Fontecilla-Camps, J., Rousset, M., & LÃ©ger, C. (2008). Experimental approaches to kinetics of gas diffusion in hydrogenase. Proceedings of the National Academy of Sciences, 105(32), 11188â11193.

Authors 12

Fanny Leroux (first)
Sébastien Dementin (additional)
Bénédicte Burlat (additional)
Laurent Cournac (additional)
Anne Volbeda (additional)
Stéphanie Champ (additional)
Lydie Martin (additional)
Bruno Guigliarelli (additional)
Patrick Bertrand (additional)
Juan Fontecilla-Camps (additional)
Marc Rousset (additional)
Christophe Léger (additional)

References 34 Referenced 147

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10.1073/pnas.0402242101
10.1080/10409230391036766
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10.1016/j.jsb.2007.01.016
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10.1107/S0907444903006516
W Kabsch International Tables for Crystallography, eds MG Rossmann, E Arnold (Kluwer, Dordrecht, The Netherlands) Vol F, 218–225 (2001). / International Tables for Crystallography by Kabsch W (2001)
10.1107/S0907444996012255

Dates

Type	When
Created	17 years ago (Aug. 6, 2008, 9:35 p.m.)
Deposited	3 years, 4 months ago (April 12, 2022, 4:36 p.m.)
Indexed	2 months, 1 week ago (June 21, 2025, 11:43 a.m.)
Issued	17 years ago (Aug. 12, 2008)
Published	17 years ago (Aug. 12, 2008)
Published Online	17 years ago (Aug. 12, 2008)
Published Print	17 years ago (Aug. 12, 2008)

Funders 0

None

BibTeX

@article{Leroux_2008, title={Experimental approaches to kinetics of gas diffusion in hydrogenase}, volume={105}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.0803689105}, DOI={10.1073/pnas.0803689105}, number={32}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Leroux, Fanny and Dementin, Sébastien and Burlat, Bénédicte and Cournac, Laurent and Volbeda, Anne and Champ, Stéphanie and Martin, Lydie and Guigliarelli, Bruno and Bertrand, Patrick and Fontecilla-Camps, Juan and Rousset, Marc and Léger, Christophe}, year={2008}, month=aug, pages={11188–11193} }

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