Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Comparative Analysis of CbHydA1 Homologues: Insights into O2 Tolerance in [FeFe] Hydrogenases (#524)

Rinat Khundoker 1 , Alexey Silakov 1
  1. Chemistry, Pennsylvania State University, State College, PA, United States

[FeFe] hydrogenases are complex metalloenzymes that can carry out the bidirectional reaction of hydrogen oxidation and proton reduction at a high catalytic turnover rate. Most [FeFe] hydrogenases studied so far are obligatory anaerobes. The discovery of O2-tolerant [FeFe] hydrogenase from Clostridium beijerinckii (CbHydA1) opens the possibility of finding other O2-tolerant hydrogenases.2-4

This work explores the hypothesis that O2 tolerance in CbHydA1 is due to a specific amino acid sequence in the H-cluster binding region. We hypothesize that it is possible to predict the function and phenotype of uncharacterized enzymes using Sequence Similarity Network (SSN) analysis. Our sequence similarity network analysis using custom algorithm shows that O2 tolerant CbHydA1 and known O2-intolerant species can be confidently separated into different regions of the map. Intriguingly, CbHydA1 then belongs to the largest cluster in the network (3057 out of 9774 unique sequences analyzed). This sub-network can then be further fractioned at higher similarity score cutoffs into smaller subgroups. To delineate O2-sensitivity of these smaller subgroups of highly similar homologues, we chose representatives from larger subgroups and performed an array of experiments such as activity assays, Fourier Transform Infrared spectroscopy (FTIR) and protein film voltammetry experiments. This work identified several O2-tolerant and O2-intolerant clusters of CbHydA1 homologues providing invaluable information about the genetic signatures of this desired property of the [FeFe] hydrogenase and demonstrates the complex structure-function relationships that are at play in this subclass of enzymes.1

  1. Khundoker, R.; Majer, S. H.; Silakov, A. in preparation
  2. Rumbaugh, T. D.; Gorka, M. J.; Baker, C. S.; Golbeck, J. H.; Silakov, A. Light-Induced H2 Generation in a Photosystem I-O2-Tolerant [FeFe] Hydrogenase Nanoconstruct. Proc Natl Acad Sci U S A 2024, 121 (34). https://doi.org/10.1073/pnas.2400267121.
  3. Corrigan, P. S.; Majer, S. H.; Silakov, A. Evidence of Atypical Structural Flexibility of the Active Site Surrounding of an [FeFe] Hydrogenase from Clostridium Beijerinkii. J Am Chem Soc 2023, 145 (20), 11033–11044. https://doi.org/10.1021/jacs.2c13458.
  4. Corrigan, P. S.; Tirsch, J. L.; Silakov, A. Investigation of the Unusual Ability of the [FeFe] Hydrogenase from Clostridium Beijerinckii to Access an O2-Protected State. J Am Chem Soc 2020, 142 (28), 12409–12419. https://doi.org/10.1021/jacs.0c04964.