Single-Amino Acid Modifications Reveal Additional Controls on the Proton Pathway of [FeFe]-Hydrogenase

Biochemistry. 2016 Jun 7;55(22):3165-73. doi: 10.1021/acs.biochem.5b01044. Epub 2016 May 27.


The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an ∼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate ∼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acids / chemistry*
  • Amino Acids / genetics
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Clostridium / enzymology*
  • Hydrogenase / chemistry
  • Hydrogenase / genetics
  • Hydrogenase / metabolism*
  • Ion Transport
  • Iron / metabolism
  • Iron-Sulfur Proteins / chemistry
  • Iron-Sulfur Proteins / genetics
  • Iron-Sulfur Proteins / metabolism*
  • Molecular Dynamics Simulation
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism*
  • Mutation / genetics*
  • Protons*


  • Amino Acids
  • Bacterial Proteins
  • Iron-Sulfur Proteins
  • Mutant Proteins
  • Protons
  • Iron
  • iron hydrogenase
  • Hydrogenase