The question of how some algae enzymes achieve a high rate of proton transfer to hydrogen production has been speculated in the past. Dr. Martin Winkler, Dr. Jifu Duan, Professor Eckhard Hofmann and Professor Thomas Happe from the Ruhr-Universität Bochum (RUB), along with colleagues from the Freie Universität Berlin, traced the proton path to the active center [FeFe]-hydrogenazy. Their findings may enable scientists to create stable chemical reproductions of such efficient and delicate biocatalysts. Scientists have published their report in the journal Nature Communications from November 9, 2018.
Unique performance thanks to the transfer path
At their catalytic center, hydrogens produce molecular hydrogen (H2) from two protons and two electrons. They extract the protons required for this process from the surrounding water and transport them – via the transport chain – to their catalytic core. The exact proton route through the hydrogenase has not been understood yet. "This transfer path is a piece of puzzle, key to understanding the interaction of cofactor and protein, which is why biocatalysts are much more efficient than hydrogen-producing chemical complexes," explains Dr. Martin Winkler, one of the authors of this research from the Photobiotechnology research group. RUB.
The structures of the enzyme variants have been decoded
To find out which of the hydrogenase building blocks is involved in the transfer of protons, scientists replaced them individually. They replaced each with an amino acid with a similar function or a dysfunctional amino acid. In this way, 22 variants of two different hydrogenases were formed. The researchers then compared these variants for various aspects, including their spectroscopic properties and enzymatic activity. "Molecular structures of the twelve protein variants that have been solved by X-ray structure analysis have proved to be particularly instructive," says Winkler.
Amino acids without function disable hydrogenazy
Depending on where and how scientists changed hydrogenase, hydrogen production became less efficient or completely stopped. "In this connection, we have determined why some variants are seriously weakened in terms of enzymatic activity and why others are not disturbed at all – against all expectations," says Martin Winkler.
The closer the amino acids were to the catalytic center, the less it was able to compensate for these modifications. If the building blocks without functions have been placed in critical locations, the production of hydrogen has been closed. "The state thus generated is similar to saturation caused by proton stress, in which protons and hydrogen are simultaneously introduced into hydrogenazy" – explains Martin Winkler. "During our project for the first time, we were able to stabilize and analyze this very transitional state that we have already encountered during the experiments."
Valuable basic information
This study made it possible to assign the function of individual amino acids to the proton transfer pathway for the enzyme group [FeFe] hydrogenase. "In addition, it provides valuable information on the molecular mechanism of proton transfer through redox activating proteins and their structural requirements," concludes Thomas Happe.
The project was financed by the Volkswagen Foundation, the China Scholarship Council and the German research foundation under the patronage of the Resolv Cluster of Excellence (EXC1069).
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Materials provided by Ruhr-University Bochum. Original written by Meike Drießen; translated by Donata Zuber. Note: The content can be edited due to style and length.