Spectroscopic Investigations under in vivo Conditions Reveal the Complex Metal Hydride Chemistry of [FeFe]-hydrogenase

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 in vivo 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 as well as Fourier-transform infrared spectroscopy. Through a combination of gas treatments, pH titrations and isotope editing, we were able to corroborate the physiological relevance of a number of proposed catalytic intermediates, including reactive iron-hydride species. We demonstrate the formation of the so-called hydride state in vivo. Moreover, two previously uncharacterized redox species are reported herein, illustrating the complex metal hydride chemistry of [FeFe]-hydrogenase.