Spectroscopic and Kinetic Properties of the Molybdenum-containing, NAD+-dependent Formate Dehydrogenase From Ralstonia Eutropha

J Biol Chem. 2016 Jan 15;291(3):1162-74. doi: 10.1074/jbc.M115.688457. Epub 2015 Nov 9.


We have examined the rapid reaction kinetics and spectroscopic properties of the molybdenum-containing, NAD(+)-dependent FdsABG formate dehydrogenase from Ralstonia eutropha. We confirm previous steady-state studies of the enzyme and extend its characterization to a rapid kinetic study of the reductive half-reaction (the reaction of formate with oxidized enzyme). We have also characterized the electron paramagnetic resonance signal of the molybdenum center in its Mo(V) state and demonstrated the direct transfer of the substrate Cα hydrogen to the molybdenum center in the course of the reaction. Varying temperature, microwave power, and level of enzyme reduction, we are able to clearly identify the electron paramagnetic resonance signals for four of the iron/sulfur clusters of the enzyme and find suggestive evidence for two others; we observe a magnetic interaction between the molybdenum center and one of the iron/sulfur centers, permitting assignment of this signal to a specific iron/sulfur cluster in the enzyme. In light of recent advances in our understanding of the structure of the molybdenum center, we propose a reaction mechanism involving direct hydride transfer from formate to a molybdenum-sulfur group of the molybdenum center.

Keywords: electron paramagnetic resonance (EPR); enzyme catalysis; enzyme kinetics; enzyme mechanism; iron/sulfur protein; molybdenum.

Publication types

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

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Biocatalysis
  • Cold Temperature
  • Cupriavidus necator / enzymology*
  • Electron Spin Resonance Spectroscopy
  • Flavin Mononucleotide
  • Formate Dehydrogenases / chemistry
  • Formate Dehydrogenases / isolation & purification
  • Formate Dehydrogenases / metabolism*
  • Formates / chemistry
  • Formates / metabolism
  • Hydrogen-Ion Concentration
  • Iron-Sulfur Proteins / chemistry
  • Iron-Sulfur Proteins / metabolism*
  • Kinetics
  • Metalloproteins / chemistry
  • Metalloproteins / metabolism*
  • Models, Molecular*
  • Molybdenum / chemistry*
  • NAD / chemistry
  • NAD / metabolism
  • Oxidation-Reduction
  • Protein Conformation
  • Protein Subunits / chemistry
  • Protein Subunits / isolation & purification
  • Protein Subunits / metabolism
  • Spectrophotometry


  • Bacterial Proteins
  • Formates
  • Iron-Sulfur Proteins
  • Metalloproteins
  • Protein Subunits
  • NAD
  • formic acid
  • Flavin Mononucleotide
  • Molybdenum
  • Formate Dehydrogenases

Associated data

  • PDB/1AA6
  • PDB/1FDO
  • PDB/3IAM