In the continuous quest to harness renewable energy and develop more sustainable biochemical processes, hydrogenases—enzymes that catalyze hydrogen production and uptake—have emerged as pivotal players. Among these, [FeFe]-hydrogenases are renowned for their remarkable catalytic efficiency in hydrogen evolution. However, their practical application remains hampered by a critical vulnerability: extreme sensitivity to oxygen. This sensitivity causes rapid enzyme inactivation under aerobic conditions, limiting their viability for industrial and biotechnological endeavors. A groundbreaking study led by Ruhr University Bochum researchers has unveiled a variant of [FeFe]-hydrogenase derived from the thermophilic bacterium Thermosediminibacter oceani, exhibiting unprecedented resistance to oxygen, alongside outstanding thermostability.
Thermophilic organisms, thriving in high-temperature environments, often evolve proteins with enhanced stability that retain functionality under conditions that denature their mesophilic counterparts. The research team utilized advanced bioinformatics approaches to mine microbial genomes, focusing on thermophilic bacteria that could encode oxygen-tolerant hydrogenases. Their exploration identified Thermosediminibacter oceani, which thrives optimally around 70°C and carries a gene encoding a distinctive Group B [FeFe]-hydrogenase with promising structural features suggestive of robustness and stability.
The team successfully cloned, expressed, and purified this novel enzyme variant, which they subjected to an array of biochemical and biophysical assessments. Remarkably, the enzyme not only retained catalytic function at elevated temperatures but also survived continuous exposure to atmospheric oxygen for several days without irreversible inactivation. This resilience contrasts starkly with most characterized [FeFe]-hydrogenases, which lose activity within minutes of oxygen exposure. Such aerobic endurance redefines the potential of [FeFe]-hydrogenases in practical applications such as biohydrogen production and biofuel cells.
To unravel the molecular basis of this extraordinary oxygen tolerance, the researchers employed a multidisciplinary toolkit combining hydrogen production assays, advanced spectroscopic techniques, site-directed mutagenesis, machine learning–guided structure predictions, and molecular dynamics simulations. These tools allowed them to dissect the enzyme’s architecture and dynamics at atomistic resolution, elucidating how structural elements contribute to functional stability under oxidative stress.
One of the most striking findings was the identification of a sulfur-containing amino acid residue in close proximity to the enzyme’s catalytic H-cluster—a unique active site complex containing iron and sulfur atoms. This amino acid appears to play a crucial protective role by mitigating oxidative damage. It likely acts as a sacrificial site or contributes to redox buffering, preventing the catalytic center from deleterious interactions with molecular oxygen. Alterations at this residue via mutational analysis led to a marked decrease in oxygen tolerance, confirming its functional importance.
Further analysis revealed a cluster of hydrophobic amino acids forming a dynamic microenvironment surrounding the catalytic site. These residues modulate protein folding and dynamics, effectively creating a barrier that restricts oxygen access to the sensitive active site. Molecular dynamics simulations highlighted that these hydrophobic patches facilitate conformational changes that transiently shield the enzyme, dynamically balancing substrate access with oxygen exclusion. This sophisticated mechanism of oxygen resistance is unprecedented among [FeFe]-hydrogenases characterized to date.
These insights present an exciting avenue to engineer other [FeFe]-hydrogenases towards enhanced oxygen tolerance. The integration of these naturally evolved structural motifs and dynamics into less stable enzyme variants could yield robust bio-catalysts suitable for industrial hydrogen production under ambient conditions. Such advancements hold promise for developing sustainable bioenergy technologies that can operate efficiently in real-world environments where oxygen presence is unavoidable.
Beyond practical implications, this study enriches our fundamental understanding of enzyme adaptation in extreme environments. The fusion of bioinformatics, experimental biochemistry, and computational modeling demonstrated here exemplifies the power of interdisciplinary research to solve longstanding challenges in enzymology and renewable energy. It underscores the latent potential hidden in extremophilic microorganisms, which serve as reservoirs of novel biochemical innovations waiting to be harnessed.
The Ruhr University Bochum researchers, including lead investigators Professors Thomas Happe and Lars Schäfer, emphasize that this enzyme’s discovery marks a significant milestone in the hydrogenase field. Subhasri Ghosh, the first author, notes that understanding and replicating such natural oxygen resistance mechanisms could revolutionize hydrogenase-based technologies. Future research will undoubtedly focus on optimizing these enzymes for scalable biohydrogen production and integrating them into bioelectrochemical systems.
With the global energy landscape urgently seeking clean and renewable alternatives, developments such as this bring us closer to realizing efficient biocatalytic hydrogen production. By overcoming oxygen sensitivity—a major bottleneck—the path toward sustainable, cost-effective hydrogen fuel generation becomes clearer. The thermophilic [FeFe]-hydrogenase from Thermosediminibacter oceani thus represents not just a scientific curiosity but a beacon guiding next-generation bioenergy solutions.
This study, published in the Journal of the American Chemical Society, sets a new benchmark for oxygen-stable hydrogenases. It invites the broader scientific community to explore thermophilic biodiversity for enzymes with industrial relevance. Moreover, it highlights the critical importance of protein dynamics and microenvironment engineering in enzyme function, offering fresh perspectives for protein engineers and synthetic biologists alike.
As research progresses, integrating machine learning with experimental approaches will accelerate the identification and optimization of such oxygen-tolerant enzymes. This synergy stands to transform the landscape of enzymatic biohydrogen production, enabling cleaner energy technologies that align with environmental sustainability goals. The Ruhr University Bochum team’s insights propel this vision forward, showcasing nature-inspired solutions to some of the most pressing energy challenges of our time.
Subject of Research: Cells
Article Title: Protein Dynamics Affect O2-Stability of Group B [FeFe]-Hydrogenase from Thermosediminibacter oceani
News Publication Date: 23-Apr-2025
Web References: http://dx.doi.org/10.1021/jacs.4c18483
Image Credits: © RUB, Marquard
Keywords
[FeFe]-hydrogenase, oxygen stability, thermostability, Thermosediminibacter oceani, biohydrogen production, protein dynamics, enzyme engineering, extremophiles, molecular dynamics, site-directed mutagenesis, catalytic H-cluster, bioinformatics
