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

 
: Duan, Jifu; Senger, Moritz; Esselborn, Julian; Engelbrecht, Vera; Wittkamp, Florian; Apfel, Ulf-Peter; Hofmann, Eckhard; Stripp, Sven Timo; Happe, Thomas; Winkler, Martin

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Volltext ()

Nature Communications 9 (2018), Art. 4726, 11 S.
ISSN: 2041-1723
Englisch
Zeitschriftenaufsatz, Elektronische Publikation
Fraunhofer UMSICHT Oberhausen ()
X-ray crystallography; enzyme mechanisms

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.

: http://publica.fraunhofer.de/dokumente/N-519651.html