Completely identified the proton path


New discoveries open the way for the chemical reproduction of hydrogen-producing enzymes.

The question of how certain algae enzymes carry out the high rate of transfer of protons to the production of hydrogen had already been the object of speculation in the past. Dr. Martin Winkler, Dr. Jifu Duan, Professor Eckhard Hofmann and Professor Thomas Happe of the Ruhr-Universit├Ąt Bochum (RUB), together with colleagues from the Freie Universit├Ąt Berlin, traced the path of the protons to the active center of [FeFe]-hydrogenases. Their findings may allow scientists to create stable chemical reproductions of efficient yet fragile biocatalysts. The researchers published their report in Nature Communications on November 9, 2018.

Unique efficiency due to transfer path

In its catalytic center, the hydrogenases make molecular hydrogen (H2) from two protons and two electrons. They extract the protons required for this process from the surrounding water and transfer them – via a transport chain – to their catalytic core. The exact path of the proton through the hydrogenase had not yet been understood. "This transfer path is a puzzle piece, crucial to understanding the cofactor and protein interaction, which is why biocatalysts are much more efficient than complex chemical-producing hydrogen," explains Dr. Martin Winkler, a of the authors of this study. of RUB's photobiotechnology research group.

Structures of enzymatic variants decoded

In order to find out which of the building blocks of hydrogenase are involved in the transfer of protons, the researchers replaced them individually. They each substituted for an amino acid with a similar function or for a dysfunctional amino acid. Thus, 22 variants of two different hydrogens were created. Subsequently, the researchers compared these variants in relation to different aspects, including their spectroscopic properties and their enzymatic activity. "The molecular structures of twelve protein variants, which were resolved using X-ray structure analysis, were particularly informative," says Winkler.

Amino acids without function deactivate hydrogenase

Depending on where and how the researchers switched to hydrogenase, hydrogen production has become less efficient or stopped altogether. "So we find out why some variants are seriously undermined in terms of enzyme activity and why other people are hardly hampered – against all expectations," says Martin Winkler.

The closer to the catalytic center the substituted amino acids were located, the less able the hydrogenase was to compensate for such modifications. If the building blocks without function were incorporated into sensitive locations, the production of hydrogen would be deactivated. "The state thus generated resembles a supersaturation due to the proton voltage, where the protons, like hydrogen, are simultaneously introduced into the hydrogenase," explains Martin Winkler. "In the course of our project, we were able for the first time to stabilize and analyze this highly transient state we had already encountered in experiments."

Valuable reference information

This study made it possible to assign the individual amino acid functions to the proton transfer pathway to the enzymatic group of [FeFe] hydrogenases. "In addition, it provides valuable insights into the molecular mechanism of proton transfer by active redox proteins and the structural requirements thereof," concludes Thomas Happe.


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