Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase |
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Authors: | Lívia S Mszros Pierre Ceccaldi Marco Lorenzi Holly J Redman Emanuel Pfitzner Joachim Heberle Moritz Senger Sven T Stripp Gustav Berggren |
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Institution: | Molecular Biomimetics, Dept. of Chemistry – Ångström Laboratory, Uppsala University, Lägerhyddsvägen 1, SE-75120 Uppsala Sweden.; Institute of Experimental Physics, Experimental Molecular Biophysics, Freie Universität Berlin, Arnimallee 14, Berlin DE-14195 Germany, |
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Abstract: | Hydrogenases are among the fastest H2 evolving catalysts known to date and have been extensively studied under in vitro conditions. Here, we report the first mechanistic investigation of an FeFe]-hydrogenase under whole-cell conditions. Functional FeFe]-hydrogenase from the green alga Chlamydomonas reinhardtii is generated in genetically modified Escherichia coli cells by addition of a synthetic cofactor to the growth medium. The assembly and reactivity of the resulting semi-synthetic enzyme was monitored using whole-cell electron paramagnetic resonance and Fourier-transform Infrared difference spectroscopy as well as scattering scanning near-field optical microscopy. Through a combination of gas treatments, pH titrations, and isotope editing we were able to corroborate the formation of a number of proposed catalytic intermediates in living cells, supporting their physiological relevance. Moreover, a previously incompletely characterized catalytic intermediate is reported herein, attributed to the formation of a protonated metal hydride species.The mechanism of hydrogen gas formation by FeFe] hydrogenase is probed under whole cell conditions, revealing the formation of reactive metal hydride species under physiologically relevant conditions. |
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