Pioneering Method Sheds Light on Enzyme Mechanisms
Hydrogen-producing enzymes, known as [FeFe]-hydrogenases, are nature’s blueprint for efficient hydrogen conversion. Found in bacteria like Desulfovibrio desulfuricans, these enzymes operate at near-perfect efficiency with minimal energy loss. A key mystery, however, has been how their iron-sulfur ([FeS]) clusters manage electron transfers during this process, in particular, whether the reduction of the clusters is proton-coupled during the catalytic cycle.
In a recent communication published in the Journal of the American Chemical Society, researchers from the MPI CEC and Nankai University developed a novel electrode system to decode this elusive mechanism, revealing critical insights for sustainable energy applications.
Nanostructured Electrodes Capture Elusive Signals
Using nanostructured electrodes functionalized with indium tin oxide (ITO), the team, and in particular visiting student and CSC scholar Yanxin Gao, who executed the electrode measurements at MPI CEC, successfully detected the "non-turnover" electrochemical signals of all three [4Fe4S] clusters in [FeFe]-hydrogenase for the first time. This breakthrough technique allowed precise measurement of the clusters’ reduction potentials, essentially, their readiness to accept electrons. The ITO platform enabled stable enzyme binding and high-sensitivity detection, overcoming limitations of previous methods. By analyzing these signals, the researchers lead by Olaf Rüdiger, group leader at the MPI CEC, quantified enzyme concentrations on the electrode and confirmed that electron transfer occurs efficiently between the clusters and the electrode surface.
pH Dependence Points to Indirect Proton Control
A central debate has revolved around whether protons directly bind to the [4Fe4S] clusters during catalysis. Surprisingly, the study revealed only a moderate pH dependence (~30 mV per pH unit) in the clusters’ reduction potentials. This weak response suggests that amino acids in the enzyme’s second coordination sphere fine-tune the clusters’ behavior through electrostatic effects. This delicate "redox tuning" optimizes the enzyme’s ability to operate close to the thermodynamic ideal across a wide pH range. That’s a key feature for its remarkable efficiency.
Real-Time Enzyme Activation and Future Impact
The team also demonstrated rapid in situ maturation of the enzyme on the electrode, where the inactive "apo-enzyme" incorporated its catalytic subcluster 50 times faster than in solution. This nanoconfinement effect, facilitated by the ITO electrode, enabled direct measurement of the enzyme’s turnover frequency (up to 227 reactions per second).
These findings provide a powerful toolkit for studying metalloenzymes and designing bio-inspired catalysts. As MPI CEC researchers Serena DeBeer and Olaf Rüdiger emphasize, “Understanding how nature minimizes energy loss in hydrogen conversion brings us closer to scalable green energy solutions.”
The full study is openly accessible under CC-BY 4.0 via the American Chemical Society.
Original Paper:
Decoding the Elusive Redox Properties of [FeS] Clusters in [FeFe]-Hydrogenase on a Nanostructured Electrode. Yanxin Gao, Lei Wan, Serena DeBeer, Liyun Zhang, and Olaf Rüdiger. Journal of the American Chemical Society, J. Am. Chem. Soc. 2025, XXXX, XXX, XXX-XXX DOI: 10.1021/jacs.5c12671 https://pubs.acs.org/doi/10.1021/jacs.5c12671