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

09 September 2019, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

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 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.

Keywords

metalloenzyme
[FeFe] hydrogenase
reaction mechanism
vibrational spectroscopy
EPR spectroscopy

Supplementary materials

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Meszaros & Ceccaldi et al ESI 2019
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