Properties of the Iron-Sulfur Cluster Electron Transfer Relay in an [FeFe]-Hydrogenase That Is Tuned for H2 Oxidation Catalysis: Article No. 107292

Effie Kisgeropoulos, Jacob Artz, Mathew Blahut, John Peters, Paul King, David Mulder

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[FeFe]-hydrogenases catalyze the reversible oxidation of H2 from electrons and protons at an organometallic active site cofactor named the H-cluster. In addition to the H-cluster, most [FeFe]-hydrogenases possess accessory FeS cluster (F-cluster) relays that function in mediating electron transfer with catalysis. There is significant variation in the structural properties of F-cluster relays among the [FeFe]-hydrogenases; however, it is unknown how this variation relates to the electronic and thermodynamic properties, and thus the electron transfer properties, of enzymes. Clostridium pasteurianum [FeFe]-hydrogenase II (CpII) exhibits a large catalytic bias for H2 oxidation (compared to H2 production), making it a notable system for examining if F-cluster properties contribute to the overall function and efficiency of the enzyme. By applying a combination of multifrequency and potentiometric electron paramagnetic resonance, we resolved two electron paramagnetic resonance signals with distinct power- and temperature-dependent properties at g = 2.058 1.931 1.891 (F2.058) and g = 2.061 1.920 1.887 (F2.061), with assigned midpoint potentials of -140 +- 18 mV and -406 +- 12 mV versus normal hydrogen electrode, respectively. Spectral analysis revealed features consistent with spin-spin coupling between the two [4Fe-4S] F-clusters, and possible functional models are discussed that account for the contribution of coupling to the electron transfer landscape. The results signify the interplay of electronic coupling and free energy properties and parameters of the FeS clusters to the electron transfer mechanism through the relay and provide new insight as to how relays functionally complement the catalytic directionality of active sites to achieve highly efficient catalysis.
Original languageAmerican English
Number of pages13
JournalJournal of Biological Chemistry
Issue number6
StatePublished - 2024

NREL Publication Number

  • NREL/JA-2700-88822


  • catalysis
  • directional
  • electron paramagnetic resonance (EPR)
  • electron transfer
  • electronic coupling
  • enzyme mechanism
  • hydrogenase
  • iron-sulfur protein
  • oxidation-reduction (redox)
  • thermodynamics


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