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  4. Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases
 
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2018
Journal Article
Title

Crystallographic and spectroscopic assignment of the proton transfer pathway in [FeFe]-hydrogenases

Abstract
The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H2 their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H2O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.
Author(s)
Duan, Jifu
Ruhr-Universität Bochum  
Senger, Moritz
Freie Universität Berlin
Esselborn, Julian
Ruhr-Universität Bochum  
Engelbrecht, Vera
Ruhr-Universität Bochum  
Wittkamp, Florian
Ruhr-Universität Bochum  
Apfel, Ulf-Peter  
Fraunhofer-Institut für Umwelt-, Sicherheits- und Energietechnik UMSICHT  
Hofmann, Eckhard
Ruhr-Universität Bochum  
Stripp, Sven Timo
Freie Universität Berlin
Happe, Thomas
Ruhr-Universität Bochum  
Winkler, Martin
Ruhr-Universität Bochum  
Journal
Nature Communications  
Open Access
DOI
10.1038/s41467-018-07140-x
Additional full text version
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Language
English
Fraunhofer-Institut für Umwelt-, Sicherheits- und Energietechnik UMSICHT  
Keyword(s)
  • X-ray crystallography

  • enzyme mechanisms

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