The oxygen-resistant [FeFe]-hydrogenase CbA5H harbors an unknown radical signal |
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Authors: | Melanie Heghmanns,Andreas Rutz,Yury Kutin,Vera Engelbrecht,Martin Winkler,Thomas Happe,Mü ge Kasanmascheff |
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Affiliation: | TU Dortmund University, Department of Chemistry and Chemical Biology, Otto-Hahn-Straße 6, 44227 Dortmund Germany.; Ruhr University Bochum, Faculty of Biology and Biotechnology, Photobiotechnology, Universitätsstr. 150, 44801 Bochum Germany.; Technical University of Munich Campus Straubing for Biotechnology and Sustainability, Professorship for Electrobiotechnology, Uferstrasse 53, 94315 Straubing Germany |
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Abstract: | [FeFe]-hydrogenases catalyze the reversible conversion of molecular hydrogen into protons and electrons with remarkable efficiency. However, their industrial applications are limited by their oxygen sensitivity. Recently, it was shown that the [FeFe]-hydrogenase from Clostridium beijerinckii (CbA5H) is oxygen-resistant and can be reactivated after oxygen exposure. In this work, we used multifrequency continuous wave and pulsed electron paramagnetic resonance (EPR) spectroscopy to characterize the active center of CbA5H, the H-cluster. Under oxidizing conditions, the spectra were dominated by an additional and unprecedented radical species. The generation of this radical signal depends on the presence of an intact H-cluster and a complete proton transfer pathway including the bridging azadithiolate ligand. Selective 57Fe enrichment combined with isotope-sensitive electron-nuclear double resonance (ENDOR) spectroscopy revealed a spin density distribution that resembles an H-cluster state. Overall, we uncovered a radical species in CbA5H that is potentially involved in the redox sensing of CbA5H.Electron paramagnetic resonance spectroscopy revealed an unprecedented radical species in the oxygen-resistant [FeFe]-hydrogenase CbA5H. Analysis of the isotope-sensitive data suggests that it is related to the active site, the H-cluster. |
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