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

Nat Commun. 2018 Nov 9;9(1):4726. doi: 10.1038/s41467-018-07140-x.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Hydrogen / metabolism
  • Hydrogen Bonding
  • Hydrogen-Ion Concentration
  • Hydrogenase / chemistry*
  • Hydrogenase / metabolism
  • Iron-Sulfur Proteins / chemistry*
  • Iron-Sulfur Proteins / metabolism
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Protons*
  • Spectrophotometry, Infrared


  • Iron-Sulfur Proteins
  • Mutant Proteins
  • Protons
  • Hydrogen
  • iron hydrogenase
  • Hydrogenase