Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Defining natural diversity in [FeFe]-hydrogenase active site microenvironments and its effect on the biophysical properties of catalytic states and enzyme reactivity.  (#485)

Effie Kisgeropoulos 1 , Kate Stroeva 1 , Michael Ratzloff 1 , Jacob Artz 1 , Sarah Hasan 1 , David Mulder 1 , Paul King 1 2
  1. Biosciences Center, National Renewable Energy Laboratory, Golden, CO, United States
  2. Renewable and Sustainable Energy Institute (RASEI), University of Colorado, Boulder, Boulder, CO, United States

The [FeFe]-hydrogenase enzymes are bidirectional catalysts which both evolve and oxidize H2 – coupled to the oxidation-reduction of electron carrier molecules – to function in energy metabolism and microbial pathogenicity.1-2 The active site cofactor, or H-cluster, of these hydrogenases is comprised of a [4Fe-4S] cubane subsite linked by a cysteine thiolate to a bridged organometallic diiron subsite.3-4 While the H-cluster is identical across [FeFe]-hydrogenases natural variation is present in the active site microenvironment. This diversity is hypothesized to play an important role in tuning the biophysical properties of the cofactor and its catalytic states, ultimately modulating enzyme reactivity.5-6 During catalysis the H-cluster subsites cycle through sequential redox changes, initiated from the catalytic “resting state” of Hox ([4Fe-4S]2+-[FeII-FeI]) and passing through 2 e- reduced states of Hsred(H+) ([4Fe-4S]+-[FeI-FeI]) and Hhyd ([4Fe-4S]+-[FeII-FeII]-H-), with the hydride as the canonically stabilized 2e- state. Through analysis of distinct biophysical properties observed in [FeFe]-hydrogenases II and III of Clostridium pasteurianum (CpII and CpIII, respectively) we have shown that both enzymes represent exceptions to these paradigms while also spanning an impressive 105 range of catalytic bias.5,7 Utilizing structural modeling, and EPR and FTIR spectroscopies in combination with spectral simulations, we have (1) characterized the properties of the H-cluster in reduced CpIII to reveal a distinct set of reduced intermediate states, (2) determined the hydride intermediate in CpII is destabilized in favor of the fully reduced Hsred(H+) state, and (3) modeled structural determinants surrounding the H-cluster to identify key site-specific variations in CpII and CpIII. The collective results support hypotheses on the role of H-cluster tuning by the microenvironment, i.e., enabling differential stabilization of reduced states in the different hydrogenases, as a basis for the observed unique mechanistic plasticity and diversity in catalytic bias.

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