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How nature optimizes hydrogen-producing biocatalysts

Oxygen can destroy hydrogen-producing enzymes. Researchers from Bochum and Osaka have discovered how an extraordinary protein survives in the presence of oxygen.

[FeFe]-hydrogenases are the most efficient hydrogen (H₂)-producing biocatalysts. However, most of their active centers, where H₂ is produced, are destroyed by oxygen (O₂), making it very difficult to used them in large-scale H₂ production. An exception, [FeFe]-hydrogenase CbA5H, from the bacterium Clostridium beijerinckii, survives in the presence of oxygen.

An international collaboration led by the Photobiotechnology group at Ruhr University Bochum, Germany, and the protein crystallography group in Osaka University, Japan, headed by Professor Thomas Happe and Professor Genji Kurisu respectively, has revealed the molecular mechanism behind this in an article in the Proceedings of the National Academy of Sciences.

CryoEM provides a complete picture of CbA5H

Three years ago, a partial structure of CbA5H solved in the presence of O₂ showed that the active center is shielded by a nearby sulfur-containing group, which allowed us to only make a hypothesis about its O₂-protection mechanism. However, this does not reveal how it produces hydrogen.

By collaborating with the Japanese group, the Bochum-based researchers have now obtained a complete structure of CbA5H under anoxic conditions using cryoEM. The new structure shows the shielding sulfur-containing group is detached to the active center, therefore representing the hydrogen producing state of CbA5H.

"Comparing both structures obtained under anoxic and aerobic conditions respectively allows us to conclude its working and O₂ protection principles," says Jifu Duan.

Zn²⁺-mediated dimerization increases the stability of CbA5H

In addition to acquiring an understanding of the workings of CbA5H and its O₂ protection principles, the complete structure also provides important information in another aspect: CbA5H forms a homodimer (i.e., two CbA5H molecules are assembled together to form the minimal functional unit) and the homodimer is retained by a Zn²⁺-binding motif.

It is further demonstrated that the Zn²⁺-dependent homodimer is significantly more stable than the monomeric form when the Zn²⁺ was removed by a genetic modification.

"Taken all together, the study provides very good understandings in working mechanism, O₂-protection strategy and dimerization-dependent stability, which would help in discovering new O₂-resistant [FeFe]-hydrogenases," concludes Happe.