Vibrational spectroscopic studies on [NiFe] hydrogenases: insights into structure and function

Molecular hydrogen has significant potential as an energy carrier in sustainable industrial production aiming to eliminate carbon dioxide emission. This potential, however, relies on the development of electrolyzers for energy storage through water splitting and fuel cell technologies for converting H2 and O2 back into electricity, using cheap, abundant, and efficient catalysts. In nature, H2 cycling is orchestrated by metalloenzymes known as hydrogenases, which operate with remarkable catalytic rates using exclusively nickel and/or iron metals. The discovery of hydrogenases has been a breakthrough towards novel catalytic strategies to replace noble metals like platinum. Additionally, hydrogenases have also been employed in H2-dependent biotechnological applications such as NAD(P)H-cofactor regeneration systems and biosensors. Despite these achievements, these enzymes also display a few drawbacks limiting their application. First, they are not easy to produce as their maturation depends on a complex biosynthetic machinery. Secondly, most hydrogenases are extremely sensitive to oxygen, which inhibits the catalytic sites. Thirdly, as hydrogenases are biomolecules with large molecular weights, the achievement of high catalyst densities at electrode surfaces is often problematic. To overcome these drawbacks, a profound understanding of the catalytic mechanism and the maturation of these enzymes is required, thereby defining the objective of this work which employs a combination of infrared (IR) spectroscopic techniques. In the first part, the catalytic subunit HoxG of the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator, lacking the [Fe-S] cluster-containing small subunit HoxK, has been subjected to detailed spectroscopic investigations. This project revealed so far unknown maturation intermediates of the stepwise assembled [NiFe] cofactor. Among them, the metal-free (preHoxGΔFeNi) and Ni-depleted (preHoxGΔNi) large subunit intermediates offer novel possibilities to introduce different active site metals aiming to develop in the future chemzymes with alternative catalytic functions. Subsequently, the HoxG subunit containing a fully equipped NiFe(CN)2CO cofactor was analyzed in detail in solution and subsequently also immobilized on surfaces using surface-enhanced infrared spectroscopy to understand its supramolecular arrangement, stability and (redox) reactivity. Experimental results were complemented by theoretical calculations by the group of Prof. Mroginski to achieve a comprehensive insight regarding orientation of the immobilized proteins and their distance from the electrode surface. In the second part, the soluble NAD+-reducing [NiFe]-hydrogenase from Hydrogenophilus thermoluteolus and the membrane-bound [NiFe]-hydrogenase from Cupriavidus necator were used as model enzymes for IR spectroscopic investigations targeting the elucidation of certain catalytic and (oxygen-) inhibited intermediates. Both enzymes were investigated in a broad temperature range, focusing on the effect of temperature and light induced perturbations of the enrichment of certain redox species. One of the new discoveries was a light triggered conversion of the fully reduced state of the active site, Nia-SR, to the one-electron oxidized Nia-L. The light-driven Nia-SR → Nia-L reaction represents a photochemical shortcut of the catalytic cycle and may be a milestone for the manipulation of hydrogenases with light. Finally, we additionally resolved the unexpected IR spectral contributions of protonated cysteine residues during the conversion of certain hydrogenase redox states. These findings, backed up by biochemical and computational data performed in collaboration with other researchers highlight the importance of careful interpretation of IR signals.

Author dc:creator

• Karafoulidi-Retsou, Chara

Advisors dc:contributor.advisor

• Hildebrandt, Peter
• Zebger, Ingo

Licence dc:rights.uri

• https://creativecommons.org/licenses/by-nc-sa/4.0/

Language dc:language.iso

en

Identifier URI

https://doi.org/10.14279/depositonce-20145

OAI identifier oai:identifier

oai:depositonce.tu-berlin.de:11303/21345