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Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase.

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.

SUBMITTER: Meszaros LS

PROVIDER: S-EPMC8159234 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase.

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